Artículos publicados por el Departamento de Histología y Embriología

  • Mol Neurobiol. 2022 Apr 6. doi: 10.1007/s12035-022-02798-3. Online ahead of print.


    Iron deficiency anemia is a prevalent health problem among pregnant women and infants, particularly in the developing countries that causes brain development deficits and poor cognitive outcomes. Since tissue iron depletion may impair myelination and trigger cellular hypoxic signaling affecting blood vessels, we studied myelination and the neurovascular unit (NVU) in infant rats born to mothers fed with an iron deficient (ID) or control diet from embryonic day 5 till weaning. Blood samples and brains of rat pups at postnatal day (PND) 14 and 30 were analyzed. PND 14 ID rats had severe microcytic hypochromic anemia that was almost reversed at PND 30 although hypomyelination and astrocyte immature phenotype in the corpus callosum were significant at that age. In CA1 hippocampal region, PND 14 and PND 30 ID rats showed significant reduced expression of the receptor β of the platelet-derived growth factor localized in pericytes and associated to aquaporin 4 (AQP4) immunopositive capillaries. Shorter AQP4 + capillaries and reduced AQP4 expression were also evidenced in PND 14 and PND 30 ID rats. In addition, pericyte membrane permeability through large-pore channels was transiently increased in ID rats at PND 14 but not at PND 30, while the blood-brain barrier permeability was not affected. Remarkably, transient increased pericyte permeability found in PND 14 ID rats was not directly related to iron depletion, suggesting the involvement of other iron deficiency anemia-induced mechanisms. In summary, severe ID during gestation and lactation produces persistent hypomyelination and significantly affects hippocampal pericytes and astrocytes in the NVU which may trigger impaired neurovascular function.

    PMID:35381889 | DOI:10.1007/s12035-022-02798-3

  • Biomolecules. 2022 Mar 16;12(3):456. doi: 10.3390/biom12030456.


    Myelination of the peripheral nervous system requires Schwann cells (SC) differentiation into the myelinating phenotype. The peripheral myelin protein-22 (PMP22) is an integral membrane glycoprotein, expressed in SC. It was initially described as a growth arrest-specific (gas3) gene product, up-regulated by serum starvation. PMP22 mutations were pathognomonic for human hereditary peripheral neuropathies, including the Charcot-Marie-Tooth disease (CMT). Trembler-J (TrJ) is a heterozygous mouse model carrying the same pmp22 point mutation as a CMT1E variant. Mutations in lamina genes have been related to a type of peripheral (CMT2B1) or central (autosomal dominant leukodystrophy) neuropathy. We explore the presence of PMP22 and Lamin B1 in Wt and TrJ SC nuclei of sciatic nerves and the colocalization of PMP22 concerning the silent heterochromatin (HC: DAPI-dark counterstaining), the transcriptionally active euchromatin (EC), and the nuclear lamina (H3K4m3 and Lamin B1 immunostaining, respectively). The results revealed that the number of TrJ SC nuclei in sciatic nerves was greater, and the SC volumes were smaller than those of Wt. The myelin protein PMP22 and Lamin B1 were detected in Wt and TrJ SC nuclei and predominantly in peripheral nuclear regions. The level of PMP22 was higher, and those of Lamin B1 lower in TrJ than in Wt mice. The level of PMP22 was higher, and those of Lamin B1 lower in TrJ than in Wt mice. PMP22 colocalized more with Lamin B1 and with the transcriptionally competent EC, than the silent HC with differences between Wt and TrJ genotypes. The results are discussed regarding the probable nuclear role of PMP22 and the relationship with TrJ neuropathy.

    PMID:35327648 | DOI:10.3390/biom12030456

  • Sci Rep. 2022 Mar 15;12(1):4439. doi: 10.1038/s41598-022-06470-7.


    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive death of motor neurons and muscle atrophy, with defective neuron-glia interplay and emergence of aberrant glial phenotypes having a role in disease pathology. Here, we have studied if the pigment violacein with several reported protective/antiproliferative properties may control highly neurotoxic astrocytes (AbAs) obtained from spinal cord cultures of symptomatic hSOD1G93A rats, and if it could be neuroprotective in this ALS experimental model. At concentrations lower than those reported as protective, violacein selectively killed aberrant astrocytes. Treatment of hSOD1G93A rats with doses equivalent to the concentrations that killed AbAs caused a marginally significant delay in survival, partially preserved the body weight and soleus muscle mass and improved the integrity of the neuromuscular junction. Reduced motor neuron death and glial reactivity was also found and likely related to decreased inflammation and matrix metalloproteinase-2 and -9. Thus, in spite that new experimental designs aimed at extending the lifespan of hSOD1G93A rats are needed, improvements observed upon violacein treatment suggest a significant therapeutic potential that deserves further studies.

    PMID:35292673 | DOI:10.1038/s41598-022-06470-7

  • J Biol Chem. 2022 Feb 9:101711. doi: 10.1016/j.jbc.2022.101711. Online ahead of print.


    Acute and chronic inflammation are key homeostatic events in health and disease. Sirtuins, a family of NAD-dependent protein deacylases, play a pivotal role in the regulation of these inflammatory responses. Indeed, sirtuins have anti-inflammatory effects through a myriad of signalling cascades, including histone deacetylation and gene silencing, p65/RelA deacetylation and inactivation, and NLRP3 inflammasome inhibition. Nevertheless, recent findings show that sirtuins, specifically SIRT6, are also necessary for mounting an active inflammatory response in macrophages. SIRT6 has been shown to positively regulate tumor necrosis factor (TNF)-ɑ secretion by demiristoylating pro-TNFɑ in the cytoplasm. However, how SIRT6, a nuclear, chromatin binding protein, fulfills this function in the cytoplasm is currently unknown. Herein, we show by western blot and immunofluorescence, that in macrophages and fibroblasts there is a sub-population of SIRT6 that is highly unstable and is quickly degraded via the proteasome. Upon LPS stimulation in Raw 264.7, bone marrow and peritoneal macrophages, this population of SIRT6 is rapidly stabilized and localizes in the cytoplasm, specifically in the vicinity of the endoplasmic reticulum (ER), promoting TNFɑ secretion. Further, we also found that acute SIRT6 inhibition dampens TNFɑ secretion both in vitro and in vivo, decreasing LPS-induced septic shock. Finally, we tested SIRT6 relevance in systemic inflammation using an obesity-induced chronic inflammatory in vivo model, where TNFɑ plays a key role, and we show that short term genetic deletion of SIRT6 in macrophages of obese mice ameliorated systemic inflammation and hyperglycemia, suggesting that SIRT6 plays and active role in inflammation-mediated glucose intolerance during obesity.

    PMID:35150745 | DOI:10.1016/j.jbc.2022.101711

  • Bio Protoc. 2021 Nov 20;11(22):e4222. doi: 10.21769/BioProtoc.4222. eCollection 2021 Nov 20.


    This protocol describes a method for high-resolution confocal imaging of pericytes with the far-red fluorophore TO-PROTM-3 Iodide 642/661 in cerebral slices of murine. Identification of pericytes with TO-PRO-3 is a short time-consuming, high cost-effective and robust technique to label pericytes with no need for immunostaining or generation of reporter mice. Since the TO-PRO-3 stain resists immunofluorescence, and lacks spectral overlap, the probe is well suited for multiple labelling. Our procedures also combine TO-PRO-3-staining of pericytes with fluorescent markers for astrocytes and vessels in brain slices. These approaches should enable the assessment of pericyte biology in gliovascular unit.

    PMID:34909443 | PMC:PMC8635853 | DOI:10.21769/BioProtoc.4222

  • Redox Biol. 2021 Nov 1;48:102176. doi: 10.1016/j.redox.2021.102176. Online ahead of print.


    To fertilize an oocyte, sperm must undergo several biochemical and functional changes known as capacitation. A key event in capacitation is calcium influx through the cation channel of sperm (CatSper). However, the molecular mechanisms of capacitation downstream of this calcium influx are not completely understood. Capacitation is also associated with an increase in mitochondrial oxygen consumption, and several lines of evidence indicate that regulated calcium entry into mitochondria increases the efficiency of oxidative respiration. Thus, we hypothesized that calcium influx through CatSper during capacitation increases mitochondrial calcium concentration and mitochondrial efficiency and thereby contributes to sperm hyperactivation and fertilization capacity. To test this hypothesis, we used high-resolution respirometry to measure mouse sperm mitochondrial activity. We also measured mitochondrial membrane potential, ATP/ADP exchange during capacitation, and mitochondrial calcium concentration in sperm from wild-type and CatSper knockout mice. We show that the increase in mitochondrial activity in capacitated wild-type sperm parallels the increase in mitochondrial calcium concentration. This effect is blunted in sperm from CatSper knockout mice. Importantly, these mechanisms are needed for optimal hyperactivation and fertilization in wild-type mice, as confirmed by using mitochondrial inhibitors. Thus, we describe a novel mechanism of sperm capacitation. This work contributes to our understanding of the role of mitochondria in sperm physiology and opens the possibility of new molecular targets for fertility treatments and male contraception.

    PMID:34753004 | PMC:PMC8585656 | DOI:10.1016/j.redox.2021.102176

  • Biomed Res Int. 2021 Oct 21;2021:2641068. doi: 10.1155/2021/2641068. eCollection 2021.


    Gramicidin is a thoroughly studied cation ionophore widely used to experimentally manipulate the plasma membrane potential (PMP). In addition, it has been established that the drug, due to its hydrophobic nature, is capable of affecting the organization of membrane lipids. We have previously shown that modifications in the plasma membrane potential of epithelial cells in culture determine reorganizations of the cytoskeleton. To elucidate the molecular mechanisms involved, we explored the effects of PMP depolarization on some putative signaling intermediates. In the course of these studies, we came across some results that could not be interpreted in terms of the properties of gramicidin as an ionic channel. The purpose of the present work is to communicate these results and, in general, to draw attention to the fact that gramicidin effects can be misleadingly attributed to its ionic or electrical properties. In addition, this work also contributes with some novel findings of the modifications provoked on the signaling intermediates by PMP depolarization and hyperpolarization.

    PMID:34722759 | PMC:PMC8553451 | DOI:10.1155/2021/2641068

  • Mol Reprod Dev. 2021 Nov;88(11):758-770. doi: 10.1002/mrd.23545. Epub 2021 Oct 25.


    Ovarian surface epithelium (OSE) is a cell monolayer surrounding the ovary; it is involved in the regulation of the ovulatory process and the genesis of ovarian carcinoma. However, intercellular messengers regulating signaling events, like proliferation in the OSE, have not been completely described. Purines have emerged as novel intercellular messengers in the ovary, in which expression of purinergic receptors has been reported in different cell types. In the present work, we described the functional expression of P2Y2 receptor (P2Y2R), a purinergic receptor widely associated with cell proliferation, in the OSE. The expression of P2Y2R by immunofluorescence and RT-PCR, and its functionality by Ca2+ recording was demonstrated in primary cultured OSE. Functional expression of P2Y2R was also exhibited in situ, by recording of intracellular Ca2+ release and detection of ERK phosphorylation after injection of a selective agonist into the ovarian bursa. Furthermore, P2Y2R activation with UTPγS, in situ, induced cell proliferation at 24 h, whereas continuous stimulation of P2Y2R during a complete estrous cycle significantly modified the size distribution of the follicular population. This is the first evidence of the functional expression of purinergic P2Y2R in the OSE and opens new perspectives on the roles played by purines in ovarian physiology.

    PMID:34694051 | DOI:10.1002/mrd.23545

  • Biochim Biophys Acta Mol Basis Dis. 2022 Jan 1;1868(1):166285. doi: 10.1016/j.bbadis.2021.166285. Epub 2021 Oct 6.


    During pregnancy, a series of physiological changes are determined at the molecular, cellular and macroscopic level that make the mother and fetus more susceptible to certain viral and bacterial infections, especially the infections in this and the companion review. Particular situations increase susceptibility to infection in neonates. The enhanced susceptibility to certain infections increases the risk of developing particular diseases that can progress to become morbidly severe. For example, during the current pandemic caused by the SARS-CoV-2 virus, epidemiological studies have established that pregnant women with COVID-19 disease are more likely to be hospitalized. However, the risk for intensive care unit admission and mechanical ventilation is not increased compared with nonpregnant women. Although much remains unknown with this particular infection, the elevated risk of progression during pregnancy towards more severe manifestations of COVID-19 disease is not associated with an increased risk of death. In addition, the epidemiological data available in neonates suggest that their risk of acquiring COVID-19 is low compared with infants (<12 months of age). However, they might be at higher risk for progression to severe COVID-19 disease compared with older children. The data on clinical presentation and disease severity among neonates are limited and based on case reports and small case series. It is well documented the importance of the Zika virus infection as the main cause of several congenital anomalies and birth defects such as microcephaly, and also adverse pregnancy outcomes. Mycoplasma infections also increase adverse pregnancy outcomes. This review will focus on the molecular, pathophysiological and biophysical characteristics of the mother/placental-fetal/neonatal interactions and the possible mechanisms of these pathogens (SARS-CoV-2, ZIKV, and Mycoplasmas) for promoting disease at this level.

    PMID:34624499 | PMC:PMC8492386 | DOI:10.1016/j.bbadis.2021.166285

  • Brain Behav Immun Health. 2020 Dec 14;11:100191. doi: 10.1016/j.bbih.2020.100191. eCollection 2021 Feb.


    Generalized Anxiety Disorder (GAD) presents a high prevalence in the population, leading to distress and disability. Immune system alterations have been associated with anxiety-related behaviors in rodents and GAD patients. CD300f immune receptors are highly expressed in microglia and participate not only in the modulation of immune responses but also in pruning and reshaping synapses. It was recently demonstrated that CD300f might be influential in the pathogenesis of depression in a sex-dependent manner. Here, we evaluated the role of CD300f immune receptor in anxiety, using CD300f knockout mice (CD300f-/-) and patients with GAD. We observed that male CD300f-/- mice had numerous behavioral changes associated with a low-anxiety phenotype, including increased open field central locomotion and rearing behaviors, more exploration in the open arms of the elevated plus-maze test, and decreased latency to eat in the novelty suppressed feeding test. In a cross-sectional population-based study, including 1111 subjects, we evaluated a common single-nucleotide polymorphism rs2034310 (C/T) in the cytoplasmatic tail of CD300f gene in individuals with GAD. Notably, we observed that the T allele of the rs2034310 polymorphism conferred protection against GAD in men, even after adjusting for confounding variables. Overall, our data demonstrate that CD300f immune receptors are involved in the modulation of pathological anxiety behaviors in a sex-dependent manner. The biological basis of these sex differences is still poorly understood, but it may provide significant clues regarding the neuropathophysiological mechanisms of GAD and can pave the way for future specific pharmacological interventions.

    PMID:34589728 | PMC:PMC8474181 | DOI:10.1016/j.bbih.2020.100191

  • J Vis Exp. 2021 Aug 14;(174). doi: 10.3791/62620.


    The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

    PMID:34459813 | DOI:10.3791/62620

  • Gen Comp Endocrinol. 2021 Nov 1;313:113886. doi: 10.1016/j.ygcen.2021.113886. Epub 2021 Aug 16.


    The vasopressin-vasotocin (AVP-AVT) and oxytocin-mesotocin-isotocin (OT-MT-IT) families of nonapeptides are of great importance in shaping context-dependent modulations of a conserved and yet highly plastic network of brain areas involved in social behavior: the social behavior network. The nonapeptide systems of teleost fish are highly conserved and share a common general organization. In this study, we first describe the presence of IT cells and projections in the brain of an electric fish, Gymnotus omarorum. Second, we confirm that IT neuron types and distribution in the preoptic area (POA) follow the same general pattern previously described in other teleost species. Third, we show that although IT and AVT neurons occur intermingled within the POA of G. omarorum and can be classified into the same subgroups, they present subtle but remarkable differences in size, number, and location. Finally, we show that unlike AVT, IT has no effect on basal electric signaling, reinforcing the specificity in the actions that each one of these nonapeptides has on social behavior and communication.

    PMID:34411583 | DOI:10.1016/j.ygcen.2021.113886

  • Neuroimmunomodulation. 2021;28(4):204-212. doi: 10.1159/000516926. Epub 2021 Jun 25.


    ALS is a human neurodegenerative disorder that induces a progressive paralysis of voluntary muscles due to motor neuron loss. The causes are unknown, and there is no curative treatment available. Mitochondrial dysfunction is a hallmark of ALS pathology; however, it is currently unknown whether it is a cause or a consequence of disease progression. Recent evidence indicates that glial mitochondrial function changes to cope with energy demands and critically influences neuronal death and disease progression. Aberrant glial cells detected in the spinal cord of diseased animals are characterized by increased proliferation rate and reduced mitochondrial bioenergetics. These features can be compared with cancer cell behavior of adapting to nutrient microenvironment by altering energy metabolism, a concept known as metabolic reprogramming. We focus on data that suggest that aberrant glial cells in ALS undergo metabolic reprogramming and profound changes in glial mitochondrial activity, which are associated with motor neuron death in ALS. This review article emphasizes on the association between metabolic reprogramming and glial reactivity, bringing new paradigms from the area of cancer research into neurodegenerative diseases. Targeting glial mitochondrial function and metabolic reprogramming may result in promising therapeutic strategies for ALS.

    PMID:34175843 | DOI:10.1159/000516926

  • Basic Clin Androl. 2021 May 6;31(1):8. doi: 10.1186/s12610-021-00128-6.


    BACKGROUND: Over the last years, there has been an increasing concern about a global decline in men's fertility. Specifically, some evidence indicates that sperm quality has decreased over the last years. However, reports showing the changes in sperm quality with time are inconsistent. Part of the contradictions between studies is attributed to geographical differences. Surprisingly, few studies include data from South American countries, creating a bias in the conclusions. This study aims to determine how sperm quality has evolved over the past 30 years in Uruguay. For this purpose, 317 medical records from allegedly healthy sperm donor candidates, aged between 18 and 36 years old, who voluntarily requested to be considered as sperm donors between 1988 and 2019, were analyzed. The studied variables were the following sperm parameters: semen volume, sperm cell concentration, total sperm number, progressive motility, vitality, and sperm morphology. A correlative statistical analysis was performed between seminal parameter values and the year data were collected.

    RESULTS: We found a statistically significant decrease in sperm concentration and normal sperm morphology during the studied period. There was no decrease in vitality, seminal volume, and total progressive motility. Semen parameters were not associated with tobacco, drugs, or alcohol consumption.

    CONCLUSIONS: We conclude that the sperm quality of donor candidates in Uruguay decreased during this period. Further studies should be carried out to verify the occurrence of this phenomenon in the general population and find its possible causes.

    PMID:33952196 | PMC:PMC8101031 | DOI:10.1186/s12610-021-00128-6

  • Reprod Sci. 2021 Oct;28(10):2799-2806. doi: 10.1007/s43032-021-00559-6. Epub 2021 Apr 6.


    Androgens are relevant in order to achieve a normal growth and maturation of the follicle and oocyte, since both excess and absence of androgens may affect the correct ovarian function. The current study analyzes the impact of neonatal androgenization in the first ovulation and oocyte maturation in response to exogenous gonadotrophin stimulation. Neonatal rats were daily treated with testosterone, dihydrotestosterone, or vehicle during follicle assembly period (days 1 to 5). At juvenile period, rats were stimulated sequentially with PMSG and hCG. Ovulation, ovarian histology, hormonal milieu, morphological characteristics of meiotic spindle, and in vitro fertilization rate in oocytes were analyzed. Our data shows that oocytes from androgenized rats displayed a major proportion of aberrant spindles and altered meiotic advance that control animals. These alterations were accompanied with an increase in both fertilization rate and aberrant embryos after 48 h of culture. Our findings showed a direct impact of neonatal androgens on oocyte development; their effects may be recognized at adulthood, supporting the idea of a programming effect exerted by neonatal androgens. These results could be relevant to explain the low fertility rate seen in polycystic ovary syndrome patients after in vitro fertilization procedures.

    PMID:33825168 | DOI:10.1007/s43032-021-00559-6

  • J Exp Biol. 2021 May 1;224(9):jeb242242. doi: 10.1242/jeb.242242. Epub 2021 May 4.


    Early sensory relay circuits in the vertebrate medulla often adopt a cerebellum-like organization specialized for comparing primary afferent inputs with central expectations. These circuits usually have a dual output, carried by center ON and center OFF neurons responding in opposite ways to the same stimulus at the center of their receptive fields. Here, we show in the electrosensory lateral line lobe of Gymnotiform weakly electric fish that basilar pyramidal neurons, representing 'ON' cells, and non-basilar pyramidal neurons, representing 'OFF' cells, have different intrinsic electrophysiological properties. We used classical anatomical techniques and electrophysiological in vitro recordings to compare these neurons. Basilar neurons are silent at rest, have a high threshold to intracellular stimulation, delayed responses to steady-state depolarization and low pass responsiveness to membrane voltage variations. They respond to low-intensity depolarizing stimuli with large, isolated spikes. As stimulus intensity increases, the spikes are followed by a depolarizing after-potential from which phase-locked spikes often arise. Non-basilar neurons show a pacemaker-like spiking activity, smoothly modulated in frequency by slow variations of stimulus intensity. Spike-frequency adaptation provides a memory of their recent firing, facilitating non-basilar response to stimulus transients. Considering anatomical and functional dimensions, we conclude that basilar and non-basilar pyramidal neurons are clear-cut, different anatomo-functional phenotypes. We propose that, in addition to their role in contrast processing, basilar pyramidal neurons encode sustained global stimuli such as those elicited by large or distant objects while non-basilar pyramidal neurons respond to transient stimuli due to movement of objects with a textured surface.

    PMID:33707195 | PMC:PMC8132969 | DOI:10.1242/jeb.242242

  • J Neurochem. 2021 May;157(4):1377-1391. doi: 10.1111/jnc.15193. Epub 2020 Oct 14.


    Perivascular pericytes are key regulators of the blood-brain barrier, vascular development, and cerebral blood flow. Deciphering pericyte roles in health and disease requires cellular tracking; yet, pericyte identification remains challenging. A previous study reported that the far-red fluorophore TO-PRO-3 (642/661), usually employed as a nuclear dye in fixed tissue, was selectively captured by live pericytes from the subventricular zone. Herein, we validated TO-PRO-3 as a specific pericyte tracer in the nervous system (NS). Living pericytes from ex vivo murine hippocampus, cortex, spinal cord, and retina robustly incorporated TO-PRO-3. Classical pericyte immunomarkers such as chondroitin sulphate proteoglycan neuron-glial antigen 2 (NG2) and platelet-derived growth factor receptor beta antigen (PDGFrβ) and the new pericyte dye NeuroTrace 500/525 confirmed cellular specificity of dye uptake. The TO-PRO-3 signal enabled quantification of pericytes density and morphometry; likewise, TO-PRO-3 labeling allowed visualization of pericytes associated with other components of the neurovascular unit. A subset of TO-PRO-3 stained cells expressed the contractile protein α-SMA, indicative of their ability to control the capillary diameter. Uptake of TO-PRO-3 was independent of connexin/pannexin channels but was highly sensitive to temperature and showed saturation, suggesting that a yet unidentified protein-mediated active transport sustained dye incorporation. We conclude that TO-PRO-3 labeling provides a reliable and simple tool for the bioimaging of pericytes in the murine NS microvasculature.

    PMID:32974913 | DOI:10.1111/jnc.15193

  • Pain. 2020 Dec;161(12):2786-2797. doi: 10.1097/j.pain.0000000000001992.


    Glial reactivity in the dorsal horn of the spinal cord is a hallmark in most chronic pain conditions. Neuroinflammation-associated reactive glia, in particular astrocytes, have been shown to exhibit reduced mitochondrial respiratory function. Here, we studied the mitochondrial function at the lumbar spinal cord tissue from complete Freund's adjuvant-induced inflammatory pain rat and chronic constriction injury mouse models by high-resolution respirometry. A significant decrease in mitochondrial bioenergetic parameters at the injury-related spinal cord level coincided with highest astrocytosis. Oral administration of dichloroacetate (DCA) significantly increased mitochondrial respiratory function by inhibiting pyruvate dehydrogenase kinase and decreased glial fibrillary acidic protein and Iba-1 immunoreactivity in spinal cord. Importantly, DCA treatment significantly reduced the ipsilateral pain-related behavior without affecting contralateral sensitivity in both pain models. Our results indicate that mitochondrial metabolic modulation with DCA may offer an alternative therapeutic strategy to alleviate chronic and persistent inflammatory pain.

    PMID:32658145 | DOI:10.1097/j.pain.0000000000001992

  • Neurotherapeutics. 2020 Oct;17(4):2089. doi: 10.1007/s13311-020-00872-z.


    PMID:32436178 | PMC:PMC7851254 | DOI:10.1007/s13311-020-00872-z

  • Sci Rep. 2020 Mar 25;10(1):5479. doi: 10.1038/s41598-020-62163-z.


    Perineuronal nets (PNNs) are aggregations of extracellular matrix associated with specific neuronal populations in the central nervous system, suggested to play key roles in neural development, synaptogenesis and experience-dependent synaptic plasticity. Pregnancy and lactation are characterized by a dramatic increase in neuroplasticity. However, dynamic changes in the extracellular matrix associated with maternal circuits have been mostly overlooked. We analyzed the structure of PNNs in an essential nucleus of the maternal circuit, the medial preoptic area (mPOA), during the reproductive cycle of rats, using the Wisteria floribunda (WFA) label. PNNs associated to neurons in the mPOA start to assemble halfway through gestation and become highly organized prior to parturition, fading through the postpartum period. This high expression of PNNs during pregnancy appears to be mediated by the influence of estrogen, progesterone and prolactin, since a hormonal simulated-gestation treatment induced the expression of PNNs in ovariectomized females. We found that PNNs associated neurons in the mPOA express estrogen receptor α and progesterone receptors, supporting a putative role of reproductive hormones in the signaling mechanisms that trigger the assembly of PNNs in the mPOA. This is the first report of PNNs presence and remodeling in mPOA during adulthood induced by physiological variables.

    PMID:32214157 | PMC:PMC7096482 | DOI:10.1038/s41598-020-62163-z

  • Proc Natl Acad Sci U S A. 2020 Mar 24;117(12):6651-6662. doi: 10.1073/pnas.1911816117. Epub 2020 Mar 9.


    A role for microglia in neuropsychiatric diseases, including major depressive disorder (MDD), has been postulated. Regulation of microglial phenotype by immune receptors has become a central topic in many neurological conditions. We explored preclinical and clinical evidence for the role of the CD300f immune receptor in the fine regulation of microglial phenotype and its contribution to MDD. We found that a prevalent nonsynonymous single-nucleotide polymorphism (C/T, rs2034310) of the human CD300f receptor cytoplasmic tail inhibits the protein kinase C phosphorylation of a threonine and is associated with protection against MDD, mainly in women. Interestingly, CD300f-/- mice displayed several characteristic MDD traits such as augmented microglial numbers, increased interleukin 6 and interleukin 1 receptor antagonist messenger RNA, alterations in synaptic strength, and noradrenaline-dependent and persistent depressive-like and anhedonic behaviors in females. This behavioral phenotype could be potentiated inducing the lipopolysaccharide depression model. RNA sequencing and biochemical studies revealed an association with impaired microglial metabolic fitness. In conclusion, we report a clear association that links the function of the CD300f immune receptor with MDD in humans, depressive-like and anhedonic behaviors in female mice, and altered microglial metabolic reprogramming.

    PMID:32152116 | PMC:PMC7104369 | DOI:10.1073/pnas.1911816117

  • Gene Ther. 2020 Feb;27(1-2):1. doi: 10.1038/s41434-020-0129-9. Epub 2020 Feb 25.


    PMID:32099107 | DOI:10.1038/s41434-020-0129-9

  • Sci Rep. 2019 Oct 7;9(1):14381. doi: 10.1038/s41598-019-50789-7.


    The protein Deleted in Breast Cancer-1 is a regulator of several transcription factors and epigenetic regulators, including HDAC3, Rev-erb-alpha, PARP1 and SIRT1. It is well known that DBC1 regulates its targets, including SIRT1, by protein-protein interaction. However, little is known about how DBC1 biological activity is regulated. In this work, we show that in quiescent cells DBC1 is proteolytically cleaved, producing a protein (DN-DBC1) that misses the S1-like domain and no longer binds to SIRT1. DN-DBC1 is also found in vivo in mouse and human tissues. Interestingly, DN-DBC1 is cleared once quiescent cells re-enter to the cell cycle. Using a model of liver regeneration after partial hepatectomy, we found that DN-DBC1 is down-regulated in vivo during regeneration. In fact, WT mice show a decrease in SIRT1 activity during liver regeneration, coincidentally with DN-DBC1 downregulation and the appearance of full length DBC1. This effect on SIRT1 activity was not observed in DBC1 KO mice. Finally, we found that DBC1 KO mice have altered cell cycle progression and liver regeneration after partial hepatectomy, suggesting that DBC1/DN-DBC1 transitions play a role in normal cell cycle progression in vivo after cells leave quiescence. We propose that quiescent cells express DN-DBC1, which either replaces or coexist with the full-length protein, and that restoring of DBC1 is required for normal cell cycle progression in vitro and in vivo. Our results describe for the first time in vivo a naturally occurring form of DBC1, which does not bind SIRT1 and is dynamically regulated, thus contributing to redefine the knowledge about its function.

    PMID:31591441 | PMC:PMC6779753 | DOI:10.1038/s41598-019-50789-7

  • J Cell Physiol. 2020 Mar;235(3):2947-2962. doi: 10.1002/jcp.29200. Epub 2019 Sep 18.


    In previous work, we reported that plasma membrane potential depolarization (PMPD) provokes cortical F-actin remodeling in bovine corneal endothelial (BCE) cells in culture, which eventually leads to the appearance of intercellular gaps. In kidney epithelial cells it has been shown that PMPD determines an extracellular-signal-regulated kinase (ERK)/Rho-dependent increase in diphosphorylated myosin light chain (ppMLC). The present study investigated the signaling pathways involved in the response of BCE cells to PMPD. Differently to renal epithelial cells, we observed that PMPD leads to a decrease in monophosphorylated MLC (pMLC) without affecting diphosphorylated MLC. Also, that the pMLC reduction is a consequence of cyclic adenosine 3',5'-monophosphate (cAMP)/protein kinase A (PKA) activation. In addition, we found evidence that the cAMP increase mostly depends on soluble adenylyl cyclase activity. Inhibition of this enzyme reduces the effect of PMPD on the cAMP rise, F-actin remodeling, and pMLC decrease. No changes in phosho-ERK were observed, although we could determine that RhoA undergoes activation. Our results suggested that active RhoA is not involved in the intercellular gap formation. Overall, the findings of this study support the view that, differently to renal epithelial cells, in BCE cells PMPD determines cytoskeletal reorganization via activation of the cAMP/PKA pathway.

    PMID:31535377 | DOI:10.1002/jcp.29200

  • Front Integr Neurosci. 2019 Aug 13;13:37. doi: 10.3389/fnint.2019.00037. eCollection 2019.


    Social behavior exhibits a wide diversity among vertebrates though it is controlled by a conserved neural network, the social behavior network (SBN). The activity of the SBN is shaped by hypothalamic nonapeptides of the vasopressin-oxytocin family. The weakly electric fish Brachyhypopomus gauderio emits social electrical signals during courtship. Three types of vasotocin (AVT) cells occur in the preoptic area (POA), one of the SBN nodes. In this study, we aimed to test if POA neurons of the nucleus preopticus ventricularis anterior (PPa) and posterior (PPp), and in particular AVT+ cells, were activated by social stimuli using a 2-day behavioral protocol. During the first night, male-female dyads were recorded to identify courting males. During the second night, these males were divided in two experimental conditions: isolated and social (male with a female). Both AVT cells and the cellular activation of the POA neurons (measured by FOS) were identified. We found that the PPa of social males showed more FOS+ cells than the PPa of isolated males, and that the PPa had more AVT+ cells in social males than in isolated males. The double-immunolabeling for AVT and FOS indicated the activation of AVT+ neurons. No significant differences in the activation of AVT+ cells were found between conditions, but a clear association was observed between the number of AVT+ cells and certain behavioral traits. In addition, a different activation of AVT+ cell-types was observed for social vs. isolated males. We conclude that the POA of B. gauderio exhibits changes induced by social stimuli in reproductive context, involving an increase in AVT production and a different profile activation among AVT+ cell populations.

    PMID:31456670 | PMC:PMC6700327 | DOI:10.3389/fnint.2019.00037

  • Biochem J. 2019 Sep 10;476(17):2463-2486. doi: 10.1042/BCJ20190405.


    Cellular senescence is an endpoint of chemotherapy, and targeted therapies in melanoma and the senescence-associated secretory phenotype (SASP) can affect tumor growth and microenvironment, influencing treatment outcomes. Metabolic interventions can modulate the SASP, and an enhanced mitochondrial energy metabolism supports resistance to therapy in melanoma cells. Herein, we assessed the mitochondrial function of therapy-induced senescent melanoma cells obtained after exposing the cells to temozolomide (TMZ), a methylating chemotherapeutic agent. Senescence induction in melanoma was accompanied by a substantial increase in mitochondrial basal, ATP-linked, and maximum respiration rates and in coupling efficiency, spare respiratory capacity, and respiratory control ratio. Further examinations revealed an increase in mitochondrial mass and length. Alterations in mitochondrial function and morphology were confirmed in isolated senescent cells, obtained by cell-size sorting. An increase in mitofusin 1 and 2 (MFN1 and 2) expression and levels was observed in senescent cells, pointing to alterations in mitochondrial fusion. Silencing mitofusin expression with short hairpin RNA (shRNA) prevented the increase in mitochondrial length, oxygen consumption rate and secretion of interleukin 6 (IL-6), a component of the SASP, in melanoma senescent cells. Our results represent the first in-depth study of mitochondrial function in therapy-induced senescence in melanoma. They indicate that senescence increases mitochondrial mass, length and energy metabolism; and highlight mitochondria as potential pharmacological targets to modulate senescence and the SASP.

    PMID:31431479 | PMC:PMC6735661 | DOI:10.1042/BCJ20190405

  • Mol Neurobiol. 2019 Nov;56(11):7694-7707. doi: 10.1007/s12035-019-1620-4. Epub 2019 May 18.


    Glutaric acidemia I (GA-I) is an inherited neurometabolic childhood disease characterized by bilateral striatal neurodegeneration upon brain accumulation of millimolar concentrations of glutaric acid (GA) and related metabolites. Vascular dysfunction, including abnormal cerebral blood flow and blood-brain barrier damage, is an early pathological feature in GA-I, although the affected cellular targets and underlying mechanisms remain unknown. In the present study, we have assessed the effects of GA on capillary pericyte contractility in cerebral cortical slices and pericyte cultures, as well as on the survival, proliferation, and migration of cultured pericytes. GA induced a significant reduction in capillary diameter at distances up to ~ 10 μm from the center of pericyte somata. However, GA did not affect the contractility of cultured pericytes, suggesting that the response elicited in slices may involve GA evoking pericyte contraction by acting on other cellular components of the neurovascular unit. Moreover, GA indirectly inhibited migration of cultured pericytes, an effect that was dependent on soluble glial factors since it was observed upon application of conditioned media from GA-treated astrocytes (CM-GA), but not upon direct GA addition to the medium. Remarkably, CM-GA showed increased expression of cytokines and growth factors that might mediate the effects of increased GA levels not only on pericyte migration but also on vascular permeability and angiogenesis. These data suggest that some effects elicited by GA might be produced by altering astrocyte-pericyte communication, rather than directly acting on pericytes. Importantly, GA-evoked alteration of capillary pericyte contractility may account for the reduced cerebral blood flow observed in GA-I patients.

    PMID:31104295 | DOI:10.1007/s12035-019-1620-4

  • BMC Med Genomics. 2019 Apr 1;12(1):53. doi: 10.1186/s12920-019-0498-3.


    BACKGROUND: Preterm birth (PTB), defined as infant delivery before 37 weeks of completed gestation, results from the interaction of both genetic and environmental components and constitutes a complex multifactorial syndrome. Transcriptome analysis of PTB has proven challenging because of the multiple causes of PTB and the numerous maternal and fetal gestational tissues that must interact to facilitate parturition. The transcriptome of the chorioamnion membranes at the site of rupture in PTB and term fetuses may reflect the molecular pathways of preterm labor.

    METHODS: In this work, chorioamnion membranes from severe preterm and term fetuses were analyzed using RNA sequencing. Functional annotations and pathway analysis of differentially expressed genes were performed with the GAGE and GOSeq packages. A subset of differentially expressed genes in PTB was validated in a larger cohort using qRT-PCR and by comparing our results with genes and pathways previously reported in the literature.

    RESULTS: A total of 270 genes were differentially expressed (DE): 252 were upregulated and 18 were down-regulated in severe preterm births relative to term births. Inflammatory and immunological pathways were upregulated in PTB. Both types of pathways were previously suggested to lead to PTB. Pathways that were not previously reported in PTB, such as the hemopoietic pathway, appeared upregulated in preterm membranes. A group of 18 downregulated genes discriminated between term and severe preterm cases. These genes potentially characterize a severe preterm transcriptome pattern and therefore are candidate genes for understanding the syndrome. Some of the downregulated genes are involved in the nervous system, morphogenesis (WNT1, DLX5, PAPPA2) and ion channel complexes (KCNJ16, KCNB1), making them good candidates as biomarkers of PTB.

    CONCLUSIONS: The identification of this DE gene pattern will help with the development of a multi-gene disease classifier. These markers were generated in an admixed South American population in which PTB has a high incidence. Since the genetic background may differentially impact different populations, it is necessary to include populations such as those from South America and Africa, which are usually excluded from high-throughput approaches. These classifiers should be compared to those in other populations to obtain a global landscape of PTB.

    PMID:30935390 | PMC:PMC6444860 | DOI:10.1186/s12920-019-0498-3

  • Gene Ther. 2020 Feb;27(1-2):96-103. doi: 10.1038/s41434-019-0073-8. Epub 2019 Mar 29.


    Traumatic brain injury (TBI) is a complex and progressive brain injury with no approved treatments that needs both short- and long-term therapeutic strategies to cope with the variety of physiopathological mechanisms involved. In particular, neuroinflammation is a key process modulating TBI outcome, and the potentiation of these mechanisms by pro-inflammatory gene therapy vectors could contribute to the injury progression. Here, we evaluate in the controlled cortical impact model of TBI, the safety of integrative-deficient lentiviral vectors (IDLVs) or the non-viral HNRK recombinant modular protein/DNA nanovector. These two promising vectors display different tropisms, transduction efficiencies, short- or long-term transduction or inflammatory activation profile. We show that the brain intraparenchymal injection of these vectors overexpressing green fluorescent protein after a CCI is not neurotoxic, and interestingly, can decrease the short-term sensory neurological deficits, and diminish the brain tissue loss at 90 days post lesion (dpl). Moreover, only IDLVs were able to mitigate the memory deficits elicited by a CCI. These vectors did not alter the microglial or astroglial reactivity at 90 dpl, suggesting that they do not potentiate the on-going neuroinflammation. Taken together, these data suggest that both types of vectors could be interesting tools for the design of gene therapy strategies targeting immediate or long-term neuropathological mechanisms of TBI.

    PMID:30926962 | DOI:10.1038/s41434-019-0073-8

  • J Mol Histol. 2019 Jun;50(3):189-202. doi: 10.1007/s10735-019-09817-z. Epub 2019 Mar 25.


    The cilia and flagella of eukaryotic cells serve many functions, exhibiting remarkable conservation of both structure and molecular composition in widely divergent eukaryotic organisms. SPAG6 and SPAG16 are the homologous in the mice to Chlamydomonas reinhardtii PF16 and PF20. Both proteins are associated with the axonemal central apparatus and are essential for ciliary and flagellar motility in mammals. Recent data derived from high-throughput studies revealed expression of these genes in tissues that do not contain motile cilia. However, the distribution of SPAG6 and SPAG16 in ciliated and non-ciliated tissues is not completely understood. In this work, we performed a quantitative analysis of the expression of Spag6 and Spag16 genes in parallel with the immune-localization of the proteins in several tissues of adult mice. Expression of mRNA was higher in the testis and tissues bearing motile cilia than in the other analyzed tissues. Both proteins were present in ciliated and non-ciliated tissues. In the testis, SPAG6 was detected in spermatogonia, spermatocytes, and in the sperm flagella whereas SPAG16 was found in spermatocytes and in the sperm flagella. In addition, both proteins were detected in the cytoplasm of cells from the brain, spinal cord, and ovary. A small isoform of SPAG16 was localized in the nucleus of germ cells and some neurons. In a parallel set of experiments, we overexpressed EGFP-SPAG6 in cultured cells and observed that the protein co-localized with a subset of acetylated cytoplasmic microtubules. A role of these proteins stabilizing the cytoplasmic microtubules of eukaryotic cells is discussed.

    PMID:30911868 | DOI:10.1007/s10735-019-09817-z

  • Front Pharmacol. 2019 Mar 5;10:193. doi: 10.3389/fphar.2019.00193. eCollection 2019.


    Ibogaine is an atypical psychedelic alkaloid, which has been subject of research due to its reported ability to attenuate drug-seeking behavior. Recent work has suggested that ibogaine effects on alcohol self-administration in rats are related to the release of Glial cell Derived Neurotrophic Factor (GDNF) in the Ventral Tegmental Area (VTA), a mesencephalic region which hosts the soma of dopaminergic neurons. Although previous reports have shown ibogaine's ability to induce GDNF expression in rat midbrain, there are no studies addressing its effect on the expression of GDNF and other neurotrophic factors (NFs) such as Brain Derived Neurotrophic Factor (BDNF) or Nerve Growth Factor (NGF) in distinct brain regions containing dopaminergic neurons. In this work, we examined the effect of ibogaine acute administration on the expression of these NFs in the VTA, Prefrontal Cortex (PFC), Nucleus Accumbens (NAcc) and the Substantia Nigra (SN). Rats were i.p. treated with ibogaine 20 mg/kg (I20), 40 mg/kg (I40) or vehicle, and NFs expression was analyzed after 3 and 24 h. At 24 h an increase of the expression of the NFs transcripts was observed in a site and dose dependent manner. Only for I40, GDNF was selectively upregulated in the VTA and SN. Both doses elicited a large increase in the expression of BDNF transcripts in the NAcc, SN and PFC, while in the VTA a significant effect was found only for I40. Finally, NGF mRNA was upregulated in all regions after I40, while I20 showed a selective upregulation in PFC and VTA. Regarding protein levels, an increase of GDNF was observed in the VTA only for I40 but no significant increase for BDNF was found in all the studied areas. Interestingly, an increase of proBDNF was detected in the NAcc for both doses. These results show for the first time a selective increase of GDNF specifically in the VTA for I40 but not for I20 after 24 h of administration, which agrees with the effective dose found in previous self-administration studies in rodents. Further research is needed to understand the contribution of these changes to ibogaine's ability to attenuate drug-seeking behavior.

    PMID:30890941 | PMC:PMC6411846 | DOI:10.3389/fphar.2019.00193

  • Integr Comp Biol. 2019 Nov 1;59(5):1176-1189. doi: 10.1093/icb/icz007.


    Quantifying how the environment shapes host immune defense is important for understanding which wild populations may be more susceptible or resistant to pathogens. Spatial variation in parasite risk, food and predator abundance, and abiotic conditions can each affect immunity, and these factors can also manifest at both local and biogeographic scales. Yet identifying predictors and the spatial scale of their effects is limited by the rarity of studies that measure immunity across many populations of broadly distributed species. We analyzed leukocyte profiles from 39 wild populations of the common vampire bat (Desmodus rotundus) across its wide geographic range throughout the Neotropics. White blood cell differentials varied spatially, with proportions of neutrophils and lymphocytes varying up to six-fold across sites. Leukocyte profiles were spatially autocorrelated at small and very large distances, suggesting that local environment and large-scale biogeographic factors influence cellular immunity. Generalized additive models showed that bat populations closer to the northern and southern limits of the species range had more neutrophils, monocytes, and basophils, but fewer lymphocytes and eosinophils, than bats sampled at the core of their distribution. Habitats with access to more livestock also showed similar patterns in leukocyte profiles, but large-scale patterns were partly confounded by time between capture and sampling across sites. Our findings suggest that populations at the edge of their range experience physiologically limiting conditions that predict higher chronic stress and greater investment in cellular innate immunity. High food abundance in livestock-dense habitats may exacerbate such conditions by increasing bat density or diet homogenization, although future spatially and temporally coordinated field studies with common protocols are needed to limit sampling artifacts. Systematically assessing immune function and response over space will elucidate how environmental conditions influence traits relevant to epidemiology and help predict disease risks with anthropogenic disturbance, land conversion, and climate change.

    PMID:30873523 | PMC:PMC6907035 | DOI:10.1093/icb/icz007

  • Mol Reprod Dev. 2019 Mar;86(3):308-318. doi: 10.1002/mrd.23106. Epub 2019 Jan 17.


    Extracellular purines through specific receptors have been recognized as new regulators of ovarian function. It is known that P2Y2 receptor activity induces theca cell proliferation, we hypothesized that purinergic signaling controls the changes related to hyperthecosis in polycystic ovarian syndrome (PCOS). The aim of this study was to analyze the expression of UTP-sensitive P2Y receptors and their role in theca cells (TC) proliferation in experimentally-induced PCOS (EI-PCOS). In primary cultures of TC from intact rats, all the transcripts of P2Y receptors were detected by polymerase chain reaction; in these cells, UTP (10 μM) induced extracellular signal-regulated kinases (ERK) phosphorylation. Rats with EI-PCOS showed a reduced expression of P2Y2R in TC whereas P2Y4R did not change. By analyzing ERK phosphorylation, it was determined that P2Y2R is the most relevant receptor in TC. UTP promoted cell proliferation in TC from control but not from EI-PCOS rats. The in silico analysis of P2yr2 promoter indicated the presence of androgen response elements; the stimulation of TC primary cultures with testosterone promoted a significant reduction in the expression of the P2yr2 transcript. We concluded that P2Y2R participates in controlling the proliferative rate of TCs from healthy ovaries, but this regulation is lost during EI-PCOS.

    PMID:30624816 | DOI:10.1002/mrd.23106

  • J Mol Histol. 2018 Dec;49(6):631-637. doi: 10.1007/s10735-018-9800-5. Epub 2018 Oct 9.


    This study analyzes the effects of neonatal androgenization on follicular growth and first ovulation in response to gonadotrophins, using a model of exogenous stimulation or the use of subcutaneous ovary grafts in castrated animals to replace the hypothalamus-pituitary signal. Neonatal rats (days 1-5) were treated with testosterone, dihydrotestosterone or vehicle. At juvenile period, rats were stimulated with PMSG, hCG (alone or combined) or used as ovarian donors to be grafted on castrated adult female rats. Ovulation and ovarian histology were analyzed in both groups. Animals treated with vehicle or dihydrotestosterone stimulated with gonadotrophins (pharmacological or by using an ovary graft) ovulated, showing a normal histological morphology whereas rats exposed to testosterone and injected with the same doses of gonadotrophins did not it. In this group, ovulation was reached using a higher dose of hCG. Ovaries in the testosterone group were characterized by the presence of follicles with atretic appearance and a larger size than those observed in control or dihydrotestosterone groups. A similar appearance was observed in testosterone ovary grafts although luteinization and some corpora lutea were also identified. Our findings suggest that neonatal exposure to aromatizable androgens induces a more drastic signalling on the ovarian tissue that those driven by non-aromatizable androgens in response to gonadotrophins.

    PMID:30302594 | DOI:10.1007/s10735-018-9800-5

  • Biochem J. 2018 Oct 11;475(19):3153-3169. doi: 10.1042/BCJ20180297.


    Synucleinopathies are a group of neurodegenerative disorders characterized by the presence of aggregated and fibrillar forms of alpha-synuclein (α-syn). Here, we analyze the effect of different species of α-syn, including monomeric, oligomeric and fibrillar forms of the protein, on rat astrocytes. Astrocytes treated with these distinct forms of α-syn showed an increase in long and thin processes and glial fibrillary acidic protein expression, indicating cell activation, high levels of intracellular oxidants and increased expression of cytokines. Moreover, astrocytes incubated with the different species induced hippocampal neuronal death in co-culture, and cytotoxicity was particularly enhanced by exposure to fibrillar α-syn. Further exploration of the mechanisms behind astrocyte activation and cytotoxicity revealed differences between the assessed α-syn species. Only oligomers induced mitochondrial dysfunction in astrocytes and significantly increased extracellular hydrogen peroxide production by these cells. Besides, TNF-α and IL-1β (interleukin 1β) expression presented different kinetics and levels depending on which species induced the response. Our data suggest that α-syn species (monomeric, oligomeric and fibrillar) induce astrocyte activation that can lead to neuronal death. Nevertheless, the tested α-syn species act through different preferential mechanisms and potency. All together these results help to understand the effect of α-syn species on astrocyte function and their potential impact on the pathogenesis of Parkinson's disease and related α-synucleinopathies.

    PMID:30185433 | DOI:10.1042/BCJ20180297

  • Neurotherapeutics. 2019 Jan;16(1):203-215. doi: 10.1007/s13311-018-0659-7.


    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron (MN) degeneration and gliosis. Neonatal astrocytes obtained from the SOD1G93A rat model of ALS exhibit mitochondrial dysfunction and neurotoxicity that can be reduced by dichloroacetate (DCA), a metabolic modulator that has been used in humans, and shows beneficial effects on disease outcome in SOD1G93A mice. Aberrant glial cells (AbGC) isolated from the spinal cords of adult paralytic SOD1G93A rats exhibit highly proliferative and neurotoxic properties and may contribute to disease progression. Here we analyze the mitochondrial activity of AbGC and whether metabolic modulation would modify their phenotypic profile. Our studies revealed fragmented mitochondria and lower respiratory control ratio in AbGC compared to neonatal SOD1G93A and nontransgenic rat astrocytes. DCA (5 mM) exposure improved AbGC mitochondrial function, reduced their proliferative rate, and importantly, decreased their toxicity to MNs. Furthermore, oral DCA administration (100 mg/kg, 10 days) to symptomatic SOD1G93A rats reduced MN degeneration, gliosis, and the number of GFAP/S100β double-labeled hypertrophic glial cells in the spinal cord. DCA treatment of AbGC reduced extracellular lactate levels indicating that the main recognized DCA action, targeting the pyruvate dehydrogenase kinase/pyruvate dehydrogenase complex, may underlie our findings. Our results show that AbGC metabolic phenotype is related to their toxicity to MNs and indicate that its modulation can reduce glial mediated pathology in the spinal cord. Together with previous findings, these results further support glial metabolic modulation as a valid therapeutic strategy in ALS.

    PMID:30159850 | PMC:PMC6361051 | DOI:10.1007/s13311-018-0659-7

  • Brain Behav Immun. 2018 Oct;73:416-426. doi: 10.1016/j.bbi.2018.06.002. Epub 2018 Jun 2.


    The interaction between CD200 and its receptor CD200R1 is among the central regulators of microglia and macrophage phenotype. However, it remains to be established whether, in the context of a traumatic CNS injury, CD200R1 act as a negative regulator of these particular innate immune cells, and if the exogenous delivery of CD200 may ameliorate neurological deficits. In the present study, we first evaluated whether preventing the local interaction between the pair CD200-CD200R1, by using a selective blocking antibody against CD200R1, has a role on functional and inflammatory outcome after contusion-induced spinal cord injury (SCI) in mice. The injection of the αCD200R1, but not control IgG1, into the lesioned spinal cord immediately after the SCI worsened locomotor performance and exacerbated neuronal loss and demyelination. At the neuroimmunological level, we observed that microglial cells and macrophages showed increased levels of iNOS and Ly6C upon CD200R1 blockade, indicating that the disruption of CD200R1 drove these cells towards a more pro-inflammatory phenotype. Moreover, although CD200R1 blockade had no effect in the initial infiltration of neutrophils into the lesioned spinal cord, it significantly impaired their clearance, which is a key sign of excessive inflammation. Interestingly, intraparenchymal injection of recombinant CD200-His immediately after the injury induced neuroprotection and robust and long-lasting locomotor recovery. In conclusion, this study reveals that interaction of CD200-CD200R1 plays a crucial role in limiting inflammation and lesion progression after SCI, and that boosting the stimulation of this pathway may constitute a new therapeutic approach.

    PMID:29870752 | DOI:10.1016/j.bbi.2018.06.002

  • Front Mol Neurosci. 2018 Apr 30;11:131. doi: 10.3389/fnmol.2018.00131. eCollection 2018.


    The lack of current treatments for amyotrophic lateral sclerosis (ALS) highlights the need of a comprehensive understanding of the biological mechanisms of the disease. A consistent neuropathological feature of ALS is the extensive inflammation around motor neurons and axonal degeneration, evidenced by accumulation of reactive astrocytes and activated microglia. Final products of inflammatory processes may be detected as a screening tool to identify treatment response. Herein, we focus on (a) detection of arachidonic acid (AA) metabolization products by lipoxygenase (LOX) and prostaglandin endoperoxide H synthase in SOD1G93A mice and (b) evaluate its response to the electrophilic nitro-oleic acid (NO2-OA). Regarding LOX-derived products, a significant increase in 12-hydroxyeicosatetraenoic acid (12-HETE) levels was detected in SOD1G93A mice both in plasma and brain whereas no changes were observed in age-matched non-Tg mice at the onset of motor symptoms (90 days-old). In addition, 15-hydroxyeicosatetraenoic acid (15-HETE) levels were greater in SOD1G93A brains compared to non-Tg. Prostaglandin levels were also increased at day 90 in plasma from SOD1G93A compared to non-Tg being similar in both types of animals at later stages of the disease. Administration of NO2-OA 16 mg/kg, subcutaneously (s/c) three times a week to SOD1G93A female mice, lowered the observed increase in brain 12-HETE levels compared to the non-nitrated fatty acid condition, and modified many others inflammatory markers. In addition, NO2-OA significantly improved grip strength and rotarod performance compared to vehicle or OA treated animals. These beneficial effects were associated with increased hemeoxygenase 1 (HO-1) expression in the spinal cord of treated mice co-localized with reactive astrocytes. Furthermore, significant levels of NO2-OA were detected in brain and spinal cord from NO2-OA -treated mice indicating that nitro-fatty acids (NFA) cross brain-blood barrier and reach the central nervous system to induce neuroprotective actions. In summary, we demonstrate that LOX-derived oxidation products correlate with disease progression. Overall, we are proposing that key inflammatory mediators of AA-derived pathways may be useful as novel footprints of ALS onset and progression as well as NO2-OA as a promising therapeutic compound.

    PMID:29760648 | PMC:PMC5936757 | DOI:10.3389/fnmol.2018.00131

  • Curr Protoc Immunol. 2018 Feb 21;120:24.1.1-24.1.25. doi: 10.1002/cpim.42.


    Traumatic brain injury (TBI) is a leading cause of death and disability and is a risk factor for the later development of neuropsychiatric disorders and neurodegenerative diseases. Many models of TBI have been developed, but their further refinement and a more detailed long-term follow-up is needed. We have used the Thy1-YFP-H transgenic mouse line and the parallel rod floor test to produce an unbiased and robust method for the evaluation of the multiple effects of a validated model of controlled cortical injury. This approach reveals short- and long-term progressive changes, including compromised biphasic motor function up to 85 days post-lesion, which correlates with neuronal atrophy, dendrite and spine loss, and long-term axonal pathology evidenced by axon spheroids and fragmentation. Here we present methods for inducing a controlled cortical injury in the Thy1-YFP-H transgenic mouse line and for evaluating the resulting deficits in the parallel rod floor test. This technique constitutes a new, unbiased, and robust method for the evaluation of motor and behavioral alterations after TBI. © 2018 by John Wiley & Sons, Inc.

    PMID:29512144 | DOI:10.1002/cpim.42

  • Sci Rep. 2018 Feb 14;8(1):3019. doi: 10.1038/s41598-018-21329-6.


    Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, polydactyly, renal disease and mental retardation. CCDC28B is a BBS-associated protein that we have previously shown plays a role in cilia length regulation whereby its depletion results in shortened cilia both in cells and Danio rerio (zebrafish). At least part of that role is achieved by its interaction with the mTORC2 component SIN1, but the mechanistic details of this interaction and/or additional functions that CCDC28B might play in the context of cilia remain poorly understood. Here we uncover a novel interaction between CCDC28B and the kinesin 1 molecular motor that is relevant to cilia. CCDC28B interacts with kinesin light chain 1 (KLC1) and the heavy chain KIF5B. Notably, depletion of these kinesin 1 components results in abnormally elongated cilia. Furthermore, through genetic interaction studies we demonstrate that kinesin 1 regulates ciliogenesis through CCDC28B. We show that kinesin 1 regulates the subcellular distribution of CCDC28B, unexpectedly, inhibiting its nuclear accumulation, and a ccdc28b mutant missing a nuclear localization motif fails to rescue the phenotype in zebrafish morphant embryos. Therefore, we uncover a previously unknown role of kinesin 1 in cilia length regulation that relies on the BBS related protein CCDC28B.

    PMID:29445114 | PMC:PMC5813027 | DOI:10.1038/s41598-018-21329-6

  • J Vis Exp. 2017 Dec 30;(130):55854. doi: 10.3791/55854.


    Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation. With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing. The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

    PMID:29364201 | PMC:PMC5908410 | DOI:10.3791/55854

  • Neuroimmunomodulation. 2017;24(3):143-153. doi: 10.1159/000480639. Epub 2017 Nov 1.


    OBJECTIVE: We aimed to determine the potential of aberrant glial cells (AbAs) isolated from the spinal cord of adult SOD1G93A symptomatic rats to induce gliosis and neuronal damage following focal transplantation into the lumbar spinal cord of wild-type rats.

    METHODS: AbAs were obtained from the spinal cords of SOD1G93A symptomatic rats. One hundred thousand cells were injected using a glass micropipette into the lumbar spinal cords (L3-L5) of syngeneic wild-type adult rats. Equal volumes of culture medium or wild-type neonatal microglia were used as controls. Seven days after transplantation, immunohistochemistry analysis was carried out using astrocytic and microglia cell markers. Transplanted SOD1G93A AbAs were recognized by specific antibodies to human SOD1 (hSOD1) or misfolded human SOD1.

    RESULTS: Seven days after transplantation, AbAs were mainly detected in the medial region of the lumbar ventral horn as a well-limited cell cluster formed at the site of injection by their immunoreactivity to either misfolded SOD1 or normally folded hSOD1. Compared with controls, transplanted AbAs were surrounded by marked microgliosis and reactive astrocytes. Marked microgliosis was observed to extend bilaterally up to the cervical cord. Motor neurons close to AbA transplants were surrounded by activated glial cells and displayed ubiquitin aggregation.

    CONCLUSIONS: AbAs bearing mutant SOD1G93A have the potential to induce neuroinflammation along the spinal cord and incipient damage to the motor neurons. The emergence of AbAs during amyotrophic lateral sclerosis pathogenesis may therefore be a mechanism to boost neuroinflammation and spread motor neuron damage along the neuroaxis.

    PMID:29131016 | DOI:10.1159/000480639

  • Sci Rep. 2017 Oct 19;7(1):13544. doi: 10.1038/s41598-017-12881-8.


    Herein, we have used bioinformatics tools to predict five clusters defining ligand-binding sites on the extracellular domain of human CD300b receptor, presumably involved in the formation of both homodimers and heterodimers with other CD300 family members. Site-directed mutagenesis revealed residues glutamic acid 28 and glutamine 29 in cluster 5 to be necessary for the formation of CD300b complexes. Surprisingly, the disruption of cluster 2 and 4 reconstituted the binding capability lost by the mutation of residues glutamic acid 28 to alanine, glutamine 29 to alanine (E28A-Q29G). We identified a missense mutation arginine 33 to glutamine (R33Q) in CD300f by direct sequencing of exon 2 in peripheral blood samples from 50 patients with multiple sclerosis (MS). Levels of expression of CD300f were almost undetectable on monocytes from the patient bearing the R33Q mutation compared with healthy individuals. Whereas R33Q mutation had no effect in the formation of CD300f complexes, the inhibition of protein synthesis with cycloheximide indicated that CD300f R33Q is less stable than native CD300f. Finally, we report that the levels of expression of CD300f on the surface of classical and intermediate monocytes from MS patients are significantly lower when compared to the same cell populations in healthy individuals.

    PMID:29051512 | PMC:PMC5648872 | DOI:10.1038/s41598-017-12881-8

  • Front Neurosci. 2017 Aug 17;11:437. doi: 10.3389/fnins.2017.00437. eCollection 2017.


    Adult neurogenesis, an essential mechanism of brain plasticity, enables brain development along postnatal life, constant addition of new neurons, neuronal turnover, and/or regeneration. It is amply distributed but negatively modulated during development and along evolution. Widespread cell proliferation, high neurogenic, and regenerative capacities are considered characteristics of teleost brains during adulthood. These anamniotes are promising models to depict factors that modulate cell proliferation, migration, and neurogenesis, and might be intervened to promote brain plasticity in mammals. Nevertheless, the migration path of derived cells to their final destination was not studied in various teleosts, including most weakly electric fish. In this group adult brain morphology is attributed to sensory specialization, involving the concerted evolution of peripheral electroreceptors and electric organs, encompassed by the evolution of neural networks involved in electrosensory information processing. In wave type gymnotids adult brain morphology is proposed to result from lifelong region specific cell proliferation and neurogenesis. Consistently, pulse type weakly electric gymnotids and mormyrids show widespread distribution of proliferation zones that persists in adulthood, but their neurogenic potential is still unknown. Here we studied the migration process and differentiation of newborn cells into the neuronal phenotype in the pulse type gymnotid Gymnotus omarorum. Pulse labeling of S-phase cells with 5-Chloro-2'-deoxyuridine thymidine followed by 1 to 180 day survivals evidenced long distance migration of newborn cells from the rostralmost telencephalic ventricle to the olfactory bulb, and between layers of all cerebellar divisions. Shorter migration appeared in the tectum opticum and torus semicircularis. In many brain regions, derived cells expressed early neuronal markers doublecortin (chase: 1-30 days) and HuC/HuD (chase: 7-180 days). Some newborn cells expressed the mature neuronal marker tyrosine hydroxylase in the subpallium (chase: 90 days) and olfactory bulb (chase: 180 days), indicating the acquisition of a mature neuronal phenotype. Long term CldU labeled newborn cells of the granular layer of the corpus cerebelli were also retrogradely labeled "in vivo," suggesting their insertion into the neural networks. These findings evidence the neurogenic capacity of telencephalic, mesencephalic, and rhombencephalic brain proliferation zones in G. omarorum, supporting the phylogenetic conserved feature of adult neurogenesis and its functional significance.

    PMID:28860962 | PMC:PMC5562682 | DOI:10.3389/fnins.2017.00437

  • Brain Res. 2017 Oct 15;1673:11-22. doi: 10.1016/j.brainres.2017.08.003. Epub 2017 Aug 8.


    Our previous studies demonstrated that Austrolebias charrua annual fish is an excellent model to study adult brain cell proliferation and neurogenesis due to the presence of active and fast neurogenesis in several regions during its short lifespan. Our main goal was to identify and localize the cells that compose the neurogenic areas throughout the Austrolebias brain. To do this, we used two thymidine halogenated analogs to detect cell proliferation at different survival times: 5-chloro-2'-deoxyuridine (CldU) at 1day and 5-iodo-2'-deoxyuridine (IdU) at 30days. Three types of proliferating cells were identified: I - transient amplifying or fast cycling cells that uptake CldU; II - stem cells or slow cycling cells, that were labeled with both CldU and IdU and did not migrate; and III - migrant cells that uptake IdU. Mapping and 3D-reconstruction of labeled nuclei showed that type I and type II cells were preferentially found close to ventricle walls. Type III cells appeared widespread and migrating in tangential and radial routes. Use of proliferation markers together with Vimentin or Nestin evidenced that type II cells are the putative stem cells that are located at the ventricular lumen. Double label cells with IdU+ and NeuN or HuC/D allowed us identify migrant neurons. Quantitation of labeled nuclei indicates that the proportion of putative stem cells is around 10% in all regions of the brain. This percentage of stem cells suggests the existence of a constant brain cell population in Austrolebias charrua that seems functional to the maintainance of adult neurogenesis.

    PMID:28797690 | DOI:10.1016/j.brainres.2017.08.003

  • BMC Med Genet. 2017 Jul 26;18(1):78. doi: 10.1186/s12881-017-0438-z.


    BACKGROUND: Infertility affects 15% of human couples, with men being responsible in approximately 50% of cases. Moreover, the aetiology of male factor infertility is poorly understood. The majority of male factor infertility remains idiopathic and potentially genetic in origin. The association of the Y chromosome and mitochondrial haplogroups with male infertility has been previously reported. This association differs between studied populations and their geographical distributions. These effects have been only rarely analysed in mixed populations, such as South Americans.

    METHODS: In this study, we analysed the contributions of the Y chromosome and mitochondrial haplogroups to male infertility in a mixed population. A case control study was conducted. Regular PCR and high-resolutionmelting- real-time PCR were performed to type haplogroups from fertile and infertile men. The sperm parameters from infertile men were compared in each haplogroup by logistic regression analysis and ANOVA.

    RESULTS: The genotyping confirmed the known admixture characteristic of the Uruguayan population. The European paternal contribution was higher than the maternal contribution in both fertile and infertile men. Neither maternal nor paternal ancestry presented differences between the cases and controls. Men belonging to the Y chromosome haplogroup F(xK) more frequently presented with an abnormal sperm morphology than men from other haplogroups. The sperm parameters were not associated with the mitochondrial haplogroups.

    CONCLUSIONS: The data presented in this study showed an association between male infertility and ancestry in the Uruguayan population. Specifically, abnormal sperm morphology was associated with the Y chromosome haplogroup F(xK). Since the Y chromosome lacks recombination, these data suggest that some genes that determine sperm morphology might be inherited in blocks with the region that determines specific haplogroups. However, the possible association between the Y chromosome haplogroup F(xK) and sperm morphology requires further confirmatory testing. Data linking infertility with ancestry are needed to establish the possible causes of infertility and define male populations susceptible to infertility. Whether the admixed characteristics of the Uruguayan population exert any pressure on male fertility potential must be further investigated.

    PMID:28747152 | PMC:PMC5530489 | DOI:10.1186/s12881-017-0438-z

  • Brain Behav Immun. 2017 Aug;64:367-383. doi: 10.1016/j.bbi.2017.03.002. Epub 2017 Mar 2.


    Over the past three decades, an intricate interaction between immune activation, release of pro-inflammatory cytokines and changes in brain circuits related to mood and behavior has been described. Despite extensive efforts, questions regarding when inflammation becomes detrimental or how we can target the immune system to develop new therapeutic strategies for the treatment of psychiatric disorders remain unresolved. In this context, novel aspects of the neuroinflammatory process activated in response to stressful challenges have recently been documented in major depressive disorder (MDD). The Nod-like receptor pyrin containing 3 inflammasome (NLRP3) is an intracellular multiprotein complex responsible for a number of innate immune processes associated with infection, inflammation and autoimmunity. Recent data have demonstrated that NLRP3 activation appears to bridge the gap between immune activation and metabolic danger signals or stress exposure, which are key factors in the pathogenesis of psychiatric disorders. In this review, we discuss both preclinical and clinical evidence that links the assembly of the NLRP3 complex and the subsequent proteolysis and release of the pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18) in chronic stress models and patients with MDD. Importantly, we also focus on the therapeutic potential of targeting the NLRP3 inflammasome complex to improve stress resilience and depressive symptoms.

    PMID:28263786 | DOI:10.1016/j.bbi.2017.03.002

  • Tissue Cell. 2017 Apr;49(2 Pt B):257-269. doi: 10.1016/j.tice.2017.02.003. Epub 2017 Feb 16.


    Hypothalamic nonapeptides (arginin vasotocin-vasopressin, oxytocin-isotocin) are known to modulate social behaviors across vertebrates. The neuroanatomical conservation of nonapeptide systems enables the use of novel vertebrate model species to identify general strategies of their functional mechanisms. We present a detailed immunohistochemical description of vasotocin (AVT) cell populations and their projections in two species of weakly electric fish with different social structure, Gymnotus omarorum and Brachyhypopomus gauderio. Strong behavioral, pharmacological, and electrophysiological evidence support that AVT modulation of electric behavior differs between the gregarious B. gauderio and the solitary G. omarorum. This functional diversity does not necessarily depend on anatomical differences of AVT neurons. To test this, we focus on interspecific comparisons of the AVT system in basal non-breeding males along the brain. G. omarorum and B. gauderio showed similar AVT somata sizes and comparable distributions of AVT somata and fibers. Interestingly, AVT fibers project to areas related to the control of social behavior and electromotor displays in both species. We found that no gross anatomical differences in the organization of the AVT system account for functional differences between species, which rather shall depend on the pattern of activation of neurons embedded in the same basic anatomical organization of the AVT system.

    PMID:28242105 | DOI:10.1016/j.tice.2017.02.003

  • Curr Alzheimer Res. 2017;14(7):778-783. doi: 10.2174/1567205014666170202121709.


    BACKGROUND: Astrocytes contribute to neuroinflammation that accompanies neurodegenerative disorders such as Alzheimer's disease (AD). In this sense, the toxicity of these diseases might be attenuated through the modulation of astrocytic inflammatory responses. Recently, the CD300f immunoreceptor was described as a new member of the CD300 immunoreceptor family, showing promising modulatory properties.

    OBJECTIVE: Here, we investigated whether overexpression of hCD300f (the human isoform of CD300f) in astrocytes protects hippocampal neurons against the degeneration induced by amyloid-beta (Aβ) oligomer.

    METHOD: Astrocyte monolayers were transfected with hCD300f before seeding the hippocampal neurons, and then the co-culture was exposed to Aβ1-42 oligomers (5 μM, 48h).

    RESULTS: hCD300f expression significantly abrogated the neuronal loss elicited by Aβ. This effect was dependent on neuron-astrocyte cell-cell interactions since no protection was observed using conditioned media from transfected astrocytes. Astrocyte modulation was dependent on the cytoplasmic signaling tail of hCD300f. Furthermore hCD300f expression did not affect the ability of astrocytes to uptake Aβ1- 42 oligomers by endocytosis, which discards the possibility that increased Aβ1-42 clearance could mediate neuroprotection by hCD300f.

    CONCLUSION: These results suggest that the astrocyte-directed expression of the hCD300f immune receptor can be a neuroprotective strategy in AD disease.

    PMID:28155597 | DOI:10.2174/1567205014666170202121709

  • J Physiol Paris. 2016 Oct;110(3 Pt B):245-258. doi: 10.1016/j.jphysparis.2016.11.007. Epub 2016 Nov 23.


    The anatomical organization of African Mormyrids' brain is a clear example of departure from the average brain morphotype in teleosts, probably related to functional specialization associated to electrosensory processing and sensory-motor coordination. The brain of Mormyrids is characterized by a well-developed rhombencephalic electrosensory lobe interconnected with relatively large mesencephalic torus semicircularis and optic tectum, and a huge and complex cerebellum. This unique morphology might imply cell addition from extraventricular proliferation zones up to late developmental stages. Here we studied the ontogeny of these brain regions in Mormyrus rume proboscirostris from embryonic to adult stages by classical histological techniques and 3D reconstruction, and analyzed the spatial-temporal distribution of proliferating cells, using pulse type BrdU labeling. Brain morphogenesis and maturation progressed in rostral-caudal direction, from 4day old free embryos, through larvae, to juveniles whose brain almost attained adult morphological complexity. The change in the relative size of the telencephalon, and mesencephalic and rhombencephalic brain regions suggest a developmental shift in the relative importance of visual and electrosensory modalities. In free embryos, proliferating cells densely populated the lining of the ventricular system. During development, ventricular proliferating cells decreased in density and extension of distribution, constituting ventricular proliferation zones. The first recognizable one was found at the optic tectum of free embryos. Several extraventricular proliferation zones were found in the cerebellar divisions of larvae, persisting along life. Adult M. rume proboscirostris showed scarce ventricular but profuse cerebellar proliferation zones, particularly at the subpial layer of the valvula cerebelli, similar to lagomorphs. This might indicate that adult cerebellar proliferation is a conserved vertebrate feature.

    PMID:27888101 | DOI:10.1016/j.jphysparis.2016.11.007

  • PLoS One. 2016 Aug 31;11(8):e0162033. doi: 10.1371/journal.pone.0162033. eCollection 2016.


    Gli2 is the primary transcriptional activator of Hedgehog signalling in mammals. Upon stimulation of the pathway, Gli2 moves into the cilium before reaching the nucleus. However, the mechanisms underlying its entry into the cilium are not completely understood. Since several similarities have been reported between nuclear and ciliary import, we investigated if the nuclear import machinery participates in Gli2 ciliary entry. Here we show that while two conserved classical nuclear localization signals mediate Gli2 nuclear localization via importin (Imp)-α/β1, these sequences are not required for Gli2 ciliary import. However, blocking Imp-mediated transport through overexpression of GTP-locked Ran reduced the percentage of Gli2 positive cilia, an effect that was not explained by increased CRM1-dependent export of Gli2 from the cilium. We explored the participation of Imp-β2 in Gli2 ciliary traffic and observed that this transporter is involved in moving Gli2 into the cilium, as has been described for other ciliary proteins. In addition, our data indicate that Imp-β2 might also collaborate in Gli2 nuclear entry. How does Imp-β2 determine the final destination of a protein that can localize to two distinct subcellular compartments remains an open question. Therefore, our data shows that the nuclear-cytoplasmic shuttling machinery plays a critical role mediating the subcellular distribution of Gli2 and the activation of the pathway, but distinct importins likely play a differential role mediating its ciliary and nuclear translocation.

    PMID:27579771 | PMC:PMC5007031 | DOI:10.1371/journal.pone.0162033

  • J Mol Histol. 2016 Oct;47(5):491-501. doi: 10.1007/s10735-016-9692-1. Epub 2016 Aug 19.


    The effects of neonatal testosterone or dihydrotestosterone exposure on ovarian follicular dynamics were analysed at prepubertal, pubertal or adult age in Wistar rats. Both androgens induced a transitory increase on follicular endowment that was partially corrected at puberty. At adult age testosterone prevented ovulation, without significant modifications on follicular dynamics. An increased number of cystic structures were observed from puberty to adult age. However, ovaries of rats treated with dihydrotestosterone showed follicles with evident morphological alterations in granulosa and thecal layers although several corpora lutea were observed. A significant increase in preantral follicles and few cystic structures were detected at advanced adulthood. The size of cyst increased with age. No immunohistochemical changes on growth factors or enzymes related to steroidogenesis in growing follicles were obvious in any group. In both androgenized groups, cysts shared immunohistochemical characteristics exhibited by preovulatory follicles but they were unable to ovulate spontaneously. Our results provide an insight into the role of different androgens in female reproductive system development, indicating a direct effect of dihydrotestosterone on ovarian tissues whereas a central effect would be the main feature of neonatal testosterone exposure. Heterogeneous clinical manifestations seen in pathologies such as polycystic ovary syndrome among women could be associated with subtle hormonal changes during follicular population development.

    PMID:27541036 | DOI:10.1007/s10735-016-9692-1

  • J Neuroinflammation. 2016 Jul 11;13(1):177. doi: 10.1186/s12974-016-0620-9.


    BACKGROUND: In the SOD1(G93A) mutant rat model of amyotrophic lateral sclerosis (ALS), neuronal death and rapid paralysis progression are associated with the emergence of activated aberrant glial cells that proliferate in the degenerating spinal cord. Whether pharmacological downregulation of such aberrant glial cells will decrease motor neuron death and prolong survival is unknown. We hypothesized that proliferation of aberrant glial cells is dependent on kinase receptor activation, and therefore, the tyrosine kinase inhibitor masitinib (AB1010) could potentially control neuroinflammation in the rat model of ALS.

    METHODS: The cellular effects of pharmacological inhibition of tyrosine kinases with masitinib were analyzed in cell cultures of microglia isolated from aged symptomatic SOD1(G93A) rats. To determine whether masitinib prevented the appearance of aberrant glial cells or modified post-paralysis survival, the drug was orally administered at 30 mg/kg/day starting after paralysis onset.

    RESULTS: We found that masitinib selectively inhibited the tyrosine kinase receptor colony-stimulating factor 1R (CSF-1R) at nanomolar concentrations. In microglia cultures from symptomatic SOD1(G93A) spinal cords, masitinib prevented CSF-induced proliferation, cell migration, and the expression of inflammatory mediators. Oral administration of masitinib to SOD1(G93A) rats starting after paralysis onset decreased the number of aberrant glial cells, microgliosis, and motor neuron pathology in the degenerating spinal cord, relative to vehicle-treated rats. Masitinib treatment initiated 7 days after paralysis onset prolonged post-paralysis survival by 40 %.

    CONCLUSIONS: These data show that masitinib is capable of controlling microgliosis and the emergence/expansion of aberrant glial cells, thus providing a strong biological rationale for its use to control neuroinflammation in ALS. Remarkably, masitinib significantly prolonged survival when delivered after paralysis onset, an unprecedented effect in preclinical models of ALS, and therefore appears well-suited for treating ALS.

    PMID:27400786 | PMC:PMC4940876 | DOI:10.1186/s12974-016-0620-9

  • Surg Radiol Anat. 2009 Sep;31 Suppl 1:95-229. doi: 10.1007/BF03371486.


    PMID:27392492 | DOI:10.1007/BF03371486

  • J Econ Entomol. 2016 Aug;109(4):1951-6. doi: 10.1093/jee/tow133. Epub 2016 Jun 21.


    The horn fly, Haematobia irritans (L.), is a blood-sucking ectoparasite that is responsible for sizeable economic losses in livestock. The salivary gland products facilitate blood intake. Taking advantage of the identification of novel H. irritans salivary antigens (Hematobin, HTB and Irritans 5, IT5), we investigated the parasite loads, H. irritans blood intake, and antibody response of naturally infected bovines during the fly season. Fly loads and fly hemoglobin content fluctuated during the trial. Each time horn fly loads exceeded 200 flies per cattle, a reduction in horn fly blood intake was observed three weeks later. All of the cattle elicited an antibody response against HTB and IT5 that declined once the fly season was over. Cattle anti-IT5 titers were positively correlated with parasite loads and negatively correlated with fly blood intake. These results suggest that the natural changes in the H. irritans blood intake observed in this study were associated with a natural host response against horn fly salivary antigens.

    PMID:27329632 | DOI:10.1093/jee/tow133

  • Eur J Obstet Gynecol Reprod Biol. 2016 Jul;202:20-5. doi: 10.1016/j.ejogrb.2016.04.030. Epub 2016 Apr 29.


    CONTEXT: Preterm birth (PTB) is a complex disease in which medical, social, cultural, and hereditary factors contribute to the pathogenesis of this adverse event. Interactions between genes and environmental factors may complicate our understanding of the relative influence of both effects on PTB. To overcome this, we combined data obtained from a cohort of newborns and their mothers with multiplex analysis of inflammatory-related genes and several environmental risk factors of PTB to describe the environmental-genetic influence on PTB.

    OBJECTIVE: The study aimed to investigate the association between maternal and fetal genetic variations in genes related to the inflammation pathway with PTB and to assess the interaction between environmental factors with these variations.

    STUDY DESIGN: We conducted a case-control study at the Pereira Rossell Hospital Center, Montevideo, Uruguay. The study included 143 mother-offspring dyads who delivered at preterm (gestational age<37 weeks) and 108 mother-offspring dyads who delivered at term. We used real-time PCR followed by a high-resolution melting analysis to simultaneously identify gene variations involved in inflammatory pathways in the context of environmental variables. The genes analyzed were: Toll-like receptor 4 (TLR4), Interleukin 6 (IL6), Interleukin 1 beta (IL1B) and Interleukin 12 receptor beta (IL12RB).

    RESULTS: We detected a significant interaction between IL1B rs16944 polymorphism in maternal samples and IL6 rs1800795 polymorphism in newborns, emphasizing the role of the interaction of maternal and fetal genomes in PTB. In addition, smoke exposure and premature rupture of membranes (PROM) were significantly different between the premature group and controls. IL1B and IL6 polymorphisms in mothers were significantly associated with PTB when controlling for smoke exposure. TLR4 polymorphism and PROM were significantly associated with PTB when controlling for PROM, but only in the case of severe PTB.

    CONCLUSIONS: Interactions between maternal and fetal genomes may influence the timing of birth. By incorporating environmental data, we revealed genetic associations with PTB, a finding not found when we analyzed genetic data alone. Our results stress the importance of studying the effect of genotype interactions between mothers and children in the context of environmental factors because they substantially contribute to phenotype variability.

    PMID:27156152 | DOI:10.1016/j.ejogrb.2016.04.030

  • Free Radic Biol Med. 2016 Jun;95:112-20. doi: 10.1016/j.freeradbiomed.2016.03.013. Epub 2016 Mar 22.


    Nitro-fatty acids (NO2-FA) are electrophilic signaling mediators formed in tissues during inflammation, which are able to induce pleiotropic cytoprotective and antioxidant pathways including up regulation of Nuclear factor erythroid 2-related factor 2 (Nrf2) responsive genes. Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons associated to an inflammatory process that usually aggravates the disease progression. In ALS animal models, the activation of the transcription factor Nrf2 in astrocytes confers protection to neighboring neurons. It is currently unknown whether NO2-FA can exert protective activity in ALS through Nrf2 activation. Herein we demonstrate that nitro-arachidonic acid (NO2-AA) or nitro-oleic acid (NO2-OA) administrated to astrocytes expressing the ALS-linked hSOD1(G93A) induce antioxidant phase II enzyme expression through Nrf2 activation concomitant with increasing intracellular glutathione levels. Furthermore, treatment of hSOD1(G93A)-expressing astrocytes with NO2-FA prevented their toxicity to motor neurons. Transfection of siRNA targeted to Nrf2 mRNA supported the involvement of Nrf2 activation in NO2-FA-mediated protective effects. Our results show for the first time that NO2-FA induce a potent Nrf2-dependent antioxidant response in astrocytes capable of preventing motor neurons death in a culture model of ALS.

    PMID:27012417 | PMC:PMC4867302 | DOI:10.1016/j.freeradbiomed.2016.03.013

  • Mol Biochem Parasitol. 2016 Mar-Apr;206(1-2):13-9. doi: 10.1016/j.molbiopara.2016.03.002. Epub 2016 Mar 11.


    The trace element selenium is found in polypeptides as selenocysteine, the 21(st) amino acid that is co-translationally inserted into proteins at a UGA codon. In proteins, selenocysteine usually plays a role as an efficient redox catalyst. Trypanosomatids previously examined harbor a full set of genes encoding the machinery needed for selenocysteine biosynthesis and incorporation into three selenoproteins: SelK, SelT and, the parasite-specific, Seltryp. We investigated the selenoproteome of kinetoplastid species in recently sequenced genomes and assessed the in vivo relevance of selenoproteins for African trypanosomes. Database mining revealed that SelK, SelT and Seltryp genes are present in most kinetoplastids, including the free-living species Bodo saltans, and Seltryp was lost in the subgenus Viannia from the New World Leishmania. Homology and sinteny with bacterial sulfur dioxygenases and sulfur transferases suggest a putative role for Seltryp in sulfur metabolism. A Trypanosoma brucei selenocysteine synthase (SepSecS) null-mutant, in which selenoprotein synthesis is abolished, displayed similar sensitivity to oxidative stress induced by a short-term exposure to high concentrations of methylglyoxal or H2O2 to that of the parental wild-type cell line. Importantly, the infectivity of the SepSecS knockout cell line was not impaired when tested in a mouse infection model and compensatory effects via up-regulation of proteins involved in thiol-redox metabolism were not observed. Collectively, our data show that selenoproteins are not required for survival of African trypanosomes in a mammalian host and exclude a role for selenoproteins in parasite antioxidant defense and/or virulence. On this basis, selenoproteins can be disregarded as drug target candidates.

    PMID:26975431 | DOI:10.1016/j.molbiopara.2016.03.002

  • Gene Expr Patterns. 2015 Sep-Nov;19(1-2):120-8. doi: 10.1016/j.gep.2015.10.002. Epub 2015 Oct 23.


    Patched-related (Ptr) encodes a protein with 12 potential transmembrane domains and a sterol-sensing domain that is closely related in predicted topology and domain organization to Patched, the canonical receptor of the Hedgehog pathway. Here we describe the production of an antibody specific for Drosophila Ptr and analyse its spatial and temporal distribution in the embryo. We find that at early developmental stages Ptr is predominantly localized at cell periphery but later on it becomes strongly and almost exclusively expressed in hemocytes. Interestingly Ptr null mutant embryos died without hatching. Our findings suggest that Ptr plays an essential function in Drosophila development, perhaps as a new receptor of embryonic hemocytes.

    PMID:26506022 | DOI:10.1016/j.gep.2015.10.002

  • Biol Reprod. 2015 Nov;93(5):119. doi: 10.1095/biolreprod.115.130989. Epub 2015 Oct 7.


    Infertility affects about 15% of couples of reproductive age. The male factor is involved in nearly 50% of infertility cases. Defective human sperm function has been associated with evidence of high levels of reactive oxygen species (ROS) and a resultant loss of fertilizing potential in vivo and in vitro. Analogous to what has been observed in somatic cells, mitochondria are likely the major sources of ROS in sperm cells. In this study, we analyzed mitochondrial function using high-resolution respirometry, ROS production, and footprints of oxidative and nitrative stress processes in intact human sperm cells. We showed that mitochondrial dysfunction (measured through the respiratory control ratio) was correlated with a decrease in human sperm motility. The samples analyzed presented nitro-oxidative modifications of proteins, such as protein 3-nitrotyrosine, that were observed mainly in the mid-piece (where mitochondria are localized) and in the sperm head. Semen samples presenting lower percentage of motile sperm showed higher amounts of nitro-oxidative protein modifications than those with larger quantities of motile sperm. When spermatozoa were exposed to inhibitors of the respiratory mitochondrial function, in the presence of a nitric oxide flux, sperm produced potent nitro-oxidative species (i.e., peroxynitrite). This effect was observed in more than 90% of intact living sperm cells and in sperm mitochondrial fractions. These data suggest that dysfunctional mitochondria in sperm cells produce oxidants that may contribute to male infertility. These data provide the rationale for testing the potential of compounds that improve sperm mitochondrial function to treat male infertility.

    PMID:26447142 | DOI:10.1095/biolreprod.115.130989

  • J Neuroinflammation. 2015 Aug 12;12:145. doi: 10.1186/s12974-015-0364-y.


    BACKGROUND: It has recently become evident that activating/inhibitory cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (CNS). The immunoreceptor CD300f expressed on monocytes, neutrophils, and mast cells modulates inflammation, phagocytosis, and outcome in models of autoimmune demyelination, allergy, and systemic lupus erythematosus. On the other hand, a finely regulated inflammatory response is essential to induce regeneration after injury to peripheral nerves since hematogenous macrophages, together with resident macrophages and de-differentiated Schwann cells, phagocyte distal axonal and myelin debris in a well-orchestrated inflammatory response. The possible roles and expression of CD300f and its ligands have not been reported under these conditions.

    METHODS: By using quantitative PCR (QPCR) and CD300f-IgG2a fusion protein, we show the expression of CD300f and its ligands in the normal and crush injured sciatic nerve. The putative role of CD300f in peripheral nerve regeneration was analyzed by blocking receptor-ligand interaction with the same CD300f-IgG2a soluble receptor fusion protein in sciatic nerves of Thy1-YFP-H mice injected at the time of injury. Macrophage M1/M2 polarization phenotype was also analyzed by CD206 and iNOS expression.

    RESULTS: We found an upregulation of CD300f mRNA and protein expression after injury. Moreover, the ligands are present in restricted membrane patches of Schwann cells, which remain stable after the lesion. The lesioned sciatic nerves of Thy1-YFP-H mice injected with a single dose of CD300f-IgG2a show long lasting effects on nerve regeneration characterized by a lower number of YFP-positive fibres growing into the tibial nerve after 10 days post lesion (dpl) and a delayed functional recovery when compared to PBS- or IgG2a-administered control groups. Animals treated with CD300f-IgG2a show at 10 dpl higher numbers of macrophages and CD206-positive cells and lower levels of iNOS expression than both control groups. At later time points (28 dpl), increased numbers of macrophages and iNOS expression occur.

    CONCLUSIONS: Taken together, these results show that the pair CD300f ligand is implicated in Wallerian degeneration and nerve regeneration by modulating both the influx and phenotype of macrophages.

    PMID:26259611 | PMC:PMC4531482 | DOI:10.1186/s12974-015-0364-y

  • FEBS Lett. 2015 Nov 14;589(22):3479-91. doi: 10.1016/j.febslet.2015.07.031. Epub 2015 Jul 29.


    Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic disorder, characterized by both congenital and late onset defects. From the analysis of the mutational burden in patients to the functional characterization of the BBS proteins, this syndrome has become a model for both understanding oligogenic patterns of inheritance and the biology of a particular cellular organelle: the primary cilium. Here we briefly review the genetics of BBS to then focus on the function of the BBS proteins, not only in the context of the cilium but also highlighting potential extra-ciliary roles that could be relevant to the etiology of the disorder. Finally, we provide an overview of how the study of this rare syndrome has contributed to the understanding of cilia biology and how this knowledge has informed on the cellular basis of different clinical manifestations that characterize BBS and the ciliopathies.

    PMID:26231314 | DOI:10.1016/j.febslet.2015.07.031

  • Gen Comp Endocrinol. 2015 Oct 1;222:158-66. doi: 10.1016/j.ygcen.2015.06.015. Epub 2015 Jul 2.


    Secretoneurin (SN) in the preoptic area and pituitary of mammals and fish has a conserved close association with the vasopressin and oxytocin systems, members of a peptide family that are key in the modulation of sexual and social behaviors. Here we show the presence of SN-immunoreactive cells and projections in the brain of the electric fish, Brachyhypopomus gauderio. Secretoneurin colocalized with vasotocin (AVT) and isotocin in cells and fibers of the preoptic area. In the rostral pars distalis of the pituitary, many cells were both SN and prolactin-positive. In the hindbrain, at the level of the command nucleus of the electric behavior (pacemaker nucleus; PN), some of SN-positive fibers colocalized with AVT. We also explored the potential neuromodulatory role of SN on electric behavior, specifically on the rate of the electric organ discharge (EOD) that signals arousal, dominance and subordinate status. Each EOD is triggered by the command discharge of the PN, ultimately responsible for the basal EOD rate. SN modulated diurnal basal EOD rate in freely swimming fish in a context-dependent manner; determined by the initial value of EOD rate. In brainstem slices, SN partially mimicked the in vivo behavioral effects acting on PN firing rate. Taken together, our results suggest that SN may regulate electric behavior, and that its effect on EOD rate may be explained by direct action of SN at the PN level through either neuroendocrine and/or endocrine mechanisms.

    PMID:26141148 | DOI:10.1016/j.ygcen.2015.06.015

  • Cell Tissue Res. 2015 Dec;362(3):557-68. doi: 10.1007/s00441-015-2223-7. Epub 2015 Jun 18.


    Tissue injury triggers a complex network of cellular and molecular responses. Although cell migration and proliferation are the most conspicuous, several other responses, such as apoptosis and increased protease activity, are necessary for a proper restitution of the tissue. In this work, we study the leukocyte elastase inhibitor (LEI) expression during wound healing of bovine corneal endothelial monolayers in culture. LEI is a multifunctional protein with anti-protease and anti-apoptotic activity. When properly cleaved, it is transformed into L-DNase II, a pro-apoptotic enzyme and translocated to the nucleus. We found that early after injury LEI increases its protein and mRNA expressions, without nuclear translocation and returns to basal levels immediately after wound closure. This increase is blocked by N-acetylcysteine, suggesting that production of reactive oxygen species immediately after wounding is involved in the LEI increase. Another finding of this work is that there is an acidification of the cells at the wound border which, in contrast to other cell types, does not determine nuclear translocation of the protein. Taken together, the results of this work suggest that the function of LEI during wound healing is related to its activity as a protease inhibitor and/or to its anti-apoptotic activity.

    PMID:26085342 | DOI:10.1007/s00441-015-2223-7

  • Mol Ther Methods Clin Dev. 2014 Oct 15;1:14047. doi: 10.1038/mtm.2014.47. eCollection 2014.


    Traumatic brain injury (TBI) remains as one of the leading causes of mortality and morbidity worldwide and there are no effective treatments currently available. Gene therapy applications have emerged as important alternatives for the treatment of diverse nervous system injuries. New strategies are evolving with the notion that each particular pathological condition may require a specific vector. Moreover, the lack of detailed comparative studies between different vectors under similar conditions hampers the selection of an ideal vector for a given pathological condition. The potential use of lentiviral vectors versus several modular protein-based nanovectors was compared using a controlled cortical impact model of TBI under the same gene therapy conditions. We show that variables such as protein/DNA ratio, incubation volume, and presence of serum or chloroquine in the transfection medium impact on both nanovector formation and transfection efficiency in vitro. While lentiviral vectors showed GFP protein 1 day after TBI and increased expression at 14 days, nanovectors showed stable and lower GFP transgene expression from 1 to 14 days. No toxicity after TBI by any of the vectors was observed as determined by resulting levels of IL-1β or using neurological sticky tape test. In fact, both vector types induced functional improvement per se.

    PMID:26015985 | PMC:PMC4362363 | DOI:10.1038/mtm.2014.47

  • Reprod Fertil Dev. 2015 May 20. doi: 10.1071/RD14460. Online ahead of print.


    This study analysed the temporal association between ovarian cyst development induced by neonatal androgenisation and sympathetic innervation. Neonatal rats (postnatal Days 1 to 5) were treated with testosterone or dihydrotestosterone and the effects were evaluated at postnatal Days 20, 40, 90 or 180. Ovulation rate, number of cystic follicles and density of sympathetic fibres were analysed. The effects of surgical denervation or gonadotrophin stimulation were also assessed. Rats exposed to testosterone showed no oestrous cycle activity and did not ovulate, maintaining a polycystic ovarian morphology at all ages studied. Also, a significant increase in ovarian density of noradrenergic fibres was detected at postnatal Days 90 and 180. Sympathectomy was unable to re-establish ovarian activity; however, human chorionic gonadotrophin stimulation was enough to induce ovulation. The impact of dihydrotestosterone on ovarian function was less noticeable, showing the coexistence of corpora lutea and cystic structures without changes in sympathetic innervation. Our findings suggest that a remodelling of ovarian sympathetic innervation occurs as a response to modifications in the pattern of follicular growth induced by testosterone. A role of sympathetic innervation in the maintenance of the polycystic condition is suggested.

    PMID:25989716 | DOI:10.1071/RD14460

  • PLoS One. 2015 Apr 30;10(4):e0123928. doi: 10.1371/journal.pone.0123928. eCollection 2015.


    CMRF35-like molecule-1 (CLM-1) belongs to a receptor family mainly expressed in myeloid cells that include activating and inhibitory receptors. CLM-1 contains two ITIMs and a single immunoreceptor tyrosine-based switch motif (ITSM), although also displays a binding site for p85α regulatory subunit of PI3K. By using murine primary microglial cultures, we show the presence of all CLM members in microglial cells and characterize the expression of CLM-1 both in basal conditions and during microglial activation. The TLR4 agonist lipopolysaccharide (LPS) and the TLR3 agonist polyinosinic-polycytidylic acid (Poly I:C) induce an increase in microglial CLM-1 mRNA levels in vitro, whereas the TLR2/6 heterodimer agonist peptidoglycan (PGN) produces a marked decrease. In this study we also describe a new soluble isoform of CLM-1 that is detected at mRNA and protein levels in basal conditions in primary microglial cultures. Interestingly, CLM-1 engagement enhances the transcription of the pro-inflammatory mediators TNFα, COX-2 and NOS-2 in microglial cells challenged with LPS. These results reveal that CLM-1 can acts as a co-activating receptor and suggest that this receptor could play a key role in the regulation of microglial activation.

    PMID:25927603 | PMC:PMC4415817 | DOI:10.1371/journal.pone.0123928

  • Biotechnol Adv. 2015 Mar-Apr;33(2):277-87. doi: 10.1016/j.biotechadv.2015.02.004. Epub 2015 Feb 16.


    The increasing incidence of diseases affecting the central nervous system (CNS) demands the urgent development of efficient drugs. While many of these medicines are already available, the Blood Brain Barrier and to a lesser extent, the Blood Spinal Cord Barrier pose physical and biological limitations to their diffusion to reach target tissues. Therefore, efforts are needed not only to address drug development but specially to design suitable vehicles for delivery into the CNS through systemic administration. In the context of the functional and structural versatility of proteins, recent advances in their biological fabrication and a better comprehension of the physiology of the CNS offer a plethora of opportunities for the construction and tailoring of plain nanoconjugates and of more complex nanosized vehicles able to cross these barriers. We revise here how the engineering of functional proteins offers drug delivery tools for specific CNS diseases and more transversally, how proteins can be engineered into smart nanoparticles or 'artificial viruses' to afford therapeutic requirements through alternative administration routes.

    PMID:25698504 | DOI:10.1016/j.biotechadv.2015.02.004

  • Reprod Biol. 2014 Dec;14(4):289-92. doi: 10.1016/j.repbio.2014.07.002. Epub 2014 Aug 8.


    Electron microscopy analysis performed in five infertile human subjects after sperm selection by swim-up followed by magnetic activated cell sorting (MACS) demonstrated a decrease in the number of spermatozoa with characteristics compatible with cell death. However, no significant differences were found when the swim-up/MACS semen fraction was compared with swim-up fraction alone.

    PMID:25454495 | DOI:10.1016/j.repbio.2014.07.002

  • Free Radic Biol Med. 2014 May;70:204-13. doi: 10.1016/j.freeradbiomed.2014.02.019. Epub 2014 Feb 26.


    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motor neuron degeneration that ultimately results in progressive paralysis and death. Growing evidence indicates that mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in ALS. To further explore the hypothesis that mitochondrial dysfunction and nitroxidative stress contribute to disease pathogenesis at the in vivo level, we assessed whether the mitochondria-targeted antioxidant [10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl]triphenylphosphonium methane sulfonate (MitoQ) can modify disease progression in the SOD1(G93A) mouse model of ALS. To do this, we administered MitoQ (500 µM) in the drinking water of SOD1(G93A) mice from a time when early symptoms of neurodegeneration become evident at 90 days of age until death. This regime is a clinically plausible scenario and could be more easily translated to patients as this corresponds to initiating treatment of patients after they are first diagnosed with ALS. MitoQ was detected in all tested tissues by liquid chromatography/mass spectrometry after 20 days of administration. MitoQ treatment slowed the decline of mitochondrial function, in both the spinal cord and the quadriceps muscle, as measured by high-resolution respirometry. Importantly, nitroxidative markers and pathological signs in the spinal cord of MitoQ-treated animals were markedly reduced and neuromuscular junctions were recovered associated with a significant increase in hindlimb strength. Finally, MitoQ treatment significantly prolonged the life span of SOD1(G93A) mice. Our results support a role for mitochondrial nitroxidative damage and dysfunction in the pathogenesis of ALS and suggest that mitochondria-targeted antioxidants may be of pharmacological use for ALS treatment.

    PMID:24582549 | DOI:10.1016/j.freeradbiomed.2014.02.019

  • Neuroscience. 2014 Jan 31;258:347-54. doi: 10.1016/j.neuroscience.2013.11.021. Epub 2013 Nov 21.


    The intrinsic properties of spherical neurons play a fundamental role in the sensory processing of self-generated signals along a fast electrosensory pathway in electric fish. Previous results indicate that the spherical neuron's intrinsic properties depend mainly on the presence of two resonant currents that tend to clamp the voltage near the resting potential. Here we show that these are: a low-threshold potassium current blocked by 4-aminopyridine and a mixed cationic current blocked by cesium chloride. We also show that the low-threshold potassium current also causes the long refractory period, explaining the necessary properties that implement the dynamic filtering of the self-generated signals previously described. Comparative data from other fish and from the auditory system indicate that other single spiking onset neurons might differ in the channel repertoire observed in the spherical neurons of Gymnotus omarorum.

    PMID:24269939 | DOI:10.1016/j.neuroscience.2013.11.021

  • J Exp Biol. 2013 Jul 1;216(Pt 13):2380-92. doi: 10.1242/jeb.082651.


    This review deals with the question: what is the relationship between the properties of a neuron and the role that the neuron plays within a given neural circuit? Answering this kind of question requires collecting evidence from multiple neuron phenotypes and comparing the role of each type in circuits that perform well-defined computational tasks. The focus here is on the spherical neurons in the electrosensory lobe of the electric fish Gymnotus omarorum. They belong to the one-spike-onset phenotype expressed at the early stages of signal processing in various sensory modalities and diverse taxa. First, we refer to the one-spike neuron intrinsic properties, their foundation on a low-threshold K(+) conductance, and the potential roles of this phenotype in different circuits within a comparative framework. Second, we present a brief description of the active electric sense of weakly electric fish and the particularities of spherical one-spike-onset neurons in the electrosensory lobe of G. omarorum. Third, we introduce one of the specific tasks in which these neurons are involved: the trade-off between self- and allo-generated signals. Fourth, we discuss recent evidence indicating a still-undescribed role for the one-spike phenotype. This role deals with the blockage of the pathway after being activated by the self-generated electric organ discharge and how this blockage favors self-generated electrosensory information in the context of allo-generated interference. Based on comparative analysis we conclude that one-spike-onset neurons may play several functional roles in animal sensory behavior. There are specific adaptations of the neuron's 'response function' to the circuit and task. Conversely, the way in which a task is accomplished depends on the intrinsic properties of the neurons involved. In short, the role of a neuron within a circuit depends on the neuron and its functional context.

    PMID:23761463 | DOI:10.1242/jeb.082651

  • Neurol Res. 2013 Apr;35(3):308-19. doi: 10.1179/1743132812Y.0000000139. Epub 2012 Dec 26.


    BACKGROUND: The zinc finger protein A20 is an ubiquitinating/deubiquitinating enzyme essential for the termination of inflammatory reactions through the inhibition of nuclear factor kappaB (NF-kappaB) signaling. Moreover, it also shows anti-apoptotic activities in some cell types and proapoptotic/pronecrotic effects in others. Although it is known that the regulation of inflammatory and cell death processes are critical in proper brain functioning and that A20 mRNA is expressed in the CNS, its role in the brain under physiological and pathological conditions is still unknown.

    METHODS: In the present study, we have evaluated the effects of A20 overexpression in mixed cortical cultures in basal conditions: the in vivo pattern of endogenous A20 expression in the control and N-methyl-d-aspartate (NMDA) excitotoxically damaged postnatal day 9 immature rat brain, and the post-injury effects of A20 overexpression in the same lesion model.

    RESULTS: Our results show that overexpression of A20 in mixed cortical cultures induced significant neuronal death by decreasing neuronal cell counts by 45 ± 9%. in vivo analysis of endogenous A20 expression showed widespread expression in gray matter, mainly in neuronal cells. However, after NMDA-induced excitotoxicity, neuronal A20 was downregulated in the neurodegenerating cortex and striatum at 10-24 hours post-lesion, and it was re-expressed at longer survival times in reactive astrocytes located mainly in the lesion border. When A20 was overexpressed in vivo 2 hours after the excitotoxic damage, the lesion volume at 3 days post-lesion showed a significant increase (20.8 ± 7.0%). No A20-induced changes were observed in the astroglial response to injury.

    CONCLUSIONS: A20 is found in neuronal cells in normal conditions and is also expressed in astrocytes after brain damage, and its overexpression is neurotoxic for cortical neurons in basal mixed neuron-glia culture conditions and exacerbates postnatal brain excitotoxic damage.

    PMID:23336395 | DOI:10.1179/1743132812Y.0000000139

  • PLoS One. 2012;7(4):e34776. doi: 10.1371/journal.pone.0034776. Epub 2012 Apr 3.


    Mitochondrial dysfunction is one of the pathogenic mechanisms that lead to neurodegeneration in Amyotrophic Lateral Sclerosis (ALS). Astrocytes expressing the ALS-linked SOD1(G93A) mutation display a decreased mitochondrial respiratory capacity associated to phenotypic changes that cause them to induce motor neuron death. Astrocyte-mediated toxicity can be prevented by mitochondria-targeted antioxidants, indicating a critical role of mitochondria in the neurotoxic phenotype. However, it is presently unknown whether drugs currently used to stimulate mitochondrial metabolism can also modulate ALS progression. Here, we tested the disease-modifying effect of dichloroacetate (DCA), an orphan drug that improves the functional status of mitochondria through the stimulation of the pyruvate dehydrogenase complex activity (PDH). Applied to astrocyte cultures isolated from rats expressing the SOD1(G93A) mutation, DCA reduced phosphorylation of PDH and improved mitochondrial coupling as expressed by the respiratory control ratio (RCR). Notably, DCA completely prevented the toxicity of SOD1(G93A) astrocytes to motor neurons in coculture conditions. Chronic administration of DCA (500 mg/L) in the drinking water of mice expressing the SOD1(G93A) mutation increased survival by 2 weeks compared to untreated mice. Systemic DCA also normalized the reduced RCR value measured in lumbar spinal cord tissue of diseased SOD1(G93A) mice. A remarkable effect of DCA was the improvement of grip strength performance at the end stage of the disease, which correlated with a recovery of the neuromuscular junction area in extensor digitorum longus muscles. Systemic DCA also decreased astrocyte reactivity and prevented motor neuron loss in SOD1(G93A) mice. Taken together, our results indicate that improvement of the mitochondrial redox status by DCA leads to a disease-modifying effect, further supporting the therapeutic potential of mitochondria-targeted drugs in ALS.

    PMID:22509356 | PMC:PMC3318006 | DOI:10.1371/journal.pone.0034776

  • Horm Metab Res. 2012 Jul;44(8):569-76. doi: 10.1055/s-0032-1306343. Epub 2012 Mar 27.


    Steroid hormones modulate a wide array of physiological processes including development, metabolism, and reproduction in various species. It is generally believed that these biological effects are predominantly mediated by their binding to specific intracellular receptors resulting in conformational change, dimerization, and recruitment of coregulators for transcription-dependent genomic actions (classical mechanism). In addition, to their cognate ligands, intracellular steroid receptors can also be activated in a "ligand-independent" manner by other factors including neurotransmitters. Recent studies indicate that rapid, nonclassical steroid effects involve extranuclear steroid receptors located at the membrane, which interact with cytoplasmic kinase signaling molecules and G-proteins. The current review deals with various mechanisms that function together in an integrated manner to promote hormone-dependent actions on the central and sympathetic nervous systems.

    PMID:22454239 | PMC:PMC3646380 | DOI:10.1055/s-0032-1306343

  • BMC Res Notes. 2012 Jan 26;5:69. doi: 10.1186/1756-0500-5-69.


    BACKGROUND: Complex traits like cancer, diabetes, obesity or schizophrenia arise from an intricate interaction between genetic and environmental factors. Complex disorders often cluster in families without a clear-cut pattern of inheritance. Genomic wide association studies focus on the detection of tens or hundreds individual markers contributing to complex diseases. In order to test if a subset of single nucleotide polymorphisms (SNPs) from candidate genes are associated to a condition of interest in a particular individual or group of people, new techniques are needed. High-resolution melting (HRM) analysis is a new method in which polymerase chain reaction (PCR) and mutations scanning are carried out simultaneously in a closed tube, making the procedure fast, inexpensive and easy. Preterm birth (PTB) is considered a complex disease, where genetic and environmental factors interact to carry out the delivery of a newborn before 37 weeks of gestation. It is accepted that inflammation plays an important role in pregnancy and PTB.

    METHODS: Here, we used real time-PCR followed by HRM analysis to simultaneously identify several gene variations involved in inflammatory pathways on preterm labor. SNPs from TLR4, IL6, IL1 beta and IL12RB genes were analyzed in a case-control study. The results were confirmed either by sequencing or by PCR followed by restriction fragment length polymorphism.

    RESULTS: We were able to simultaneously recognize the variations of four genes with similar accuracy than other methods. In order to obtain non-overlapping melting temperatures, the key step in this strategy was primer design. Genotypic frequencies found for each SNP are in concordance with those previously described in similar populations. None of the studied SNPs were associated with PTB.

    CONCLUSIONS: Several gene variations related to the same inflammatory pathway were screened through a new flexible, fast and non expensive method with the purpose of analyzing their association to PTB. It can easily be used for simultaneously analyze any set of SNPs, either as the first choice for new association studies or as a complement to large-scale genotyping analysis. Given that inflammatory pathway is in the base of several diseases, it is potentially useful to analyze a broad range of disorders.

    PMID:22280494 | PMC:PMC3298535 | DOI:10.1186/1756-0500-5-69

  • Reproduction. 2012 Feb;143(2):183-93. doi: 10.1530/REP-11-0056. Epub 2011 Nov 11.


    Oestradiol (E(2)) is a key hormone in the regulation of reproductive processes. The aims of this work were a) to examine the distributions of oestrogen receptor α (ERα) and ERβ in the neurons of the superior mesenteric ganglion (SMG) in the oestrus stage by immunohistochemistry, b) to demonstrate whether E(2) in the SMG modifies progesterone (P(4)), androstenedione (A(2)) and nitrite release in the ovarian compartment on oestrus day and c) to demonstrate whether E(2) in the ganglion modifies the activity and gene expression in the ovary of the steroidogenic enzymes 3β-hydroxysteroid dehydrogenase (3β-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD). The ex vivo SMG-ovarian nervous plexus-ovary system was used. E(2), tamoxifen (Txf) and E(2) plus Txf were added in the ganglion to measure ovarian P(4) release, while E(2) alone was added to measure ovarian A(2) and nitrites release. Immunohistochemistry revealed cytoplasmic ERα immunoreactivity only in the neural somas in the SMG. E(2) increased ovarian P(4) and A(2) release at 15, 30 and 60 min but decreased nitrites. The activity and gene expression of 3β-HSD increased, while the activity and gene expression of 20α-HSD did not show changes with respect to the control. Txf in the ganglion diminished P(4) release only at 60 min. E(2) plus Txf in the ganglion reverted the effect of E(2) alone and the inhibitory effect of Txf. The results of this study demonstrate that ERα activation in the SMG has an impact on ovarian steroidogenesis in rats, thus providing evidence for the critical role of peripheral system neurons in the control of ovarian functions under normal and pathological conditions.

    PMID:22080140 | DOI:10.1530/REP-11-0056

  • Proc Natl Acad Sci U S A. 2011 Nov 1;108(44):18126-31. doi: 10.1073/pnas.1110689108. Epub 2011 Oct 18.


    Motoneuron loss and reactive astrocytosis are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a paralytic neurodegenerative disease that can be triggered by mutations in Cu-Zn superoxide dismutase (SOD1). Dysfunctional astrocytes contribute to ALS pathogenesis, inducing motoneuron damage and accelerating disease progression. However, it is unknown whether ALS progression is associated with the appearance of a specific astrocytic phenotype with neurotoxic potential. Here, we report the isolation of astrocytes with aberrant phenotype (referred as "AbA cells") from primary spinal cord cultures of symptomatic rats expressing the SOD1(G93A) mutation. Isolation was based on AbA cells' marked proliferative capacity and lack of replicative senescence, which allowed oligoclonal cell expansion for 1 y. AbA cells displayed astrocytic markers including glial fibrillary acidic protein, S100β protein, glutamine synthase, and connexin 43 but lacked glutamate transporter 1 and the glial progenitor marker NG2 glycoprotein. Notably, AbA cells secreted soluble factors that induced motoneuron death with a 10-fold higher potency than neonatal SOD1(G93A) astrocytes. AbA-like aberrant astrocytes expressing S100β and connexin 43 but lacking NG2 were identified in nearby motoneurons, and their number increased sharply after disease onset. Thus, AbA cells appear to be an as-yet unknown astrocyte population arising during ALS progression with unprecedented proliferative and neurotoxic capacity and may be potential cellular targets for slowing ALS progression.

    PMID:22010221 | PMC:PMC3207668 | DOI:10.1073/pnas.1110689108

  • Brain Pathol. 2012 May;22(3):318-28. doi: 10.1111/j.1750-3639.2011.00537.x. Epub 2011 Oct 31.


    It is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (CNS). We have analyzed the function of cluster of differentiation (CD)300f immunoreceptor in a model of excitotoxic rat brain damage. First, to explore the presence of endogenous ligand(s) for this receptor we used a human CD300f-Ig soluble protein and confocal microscopy, showing specific staining mainly in CNS white matter and on the surface of oligodendrocytes and certain astrocytes. Next, we demonstrated in a model of in vivo rat brain excitotoxic damage that the overexpression of human CD300f induced a significant reduction in the lesion volume. To validate these results, we cloned the rat ortholog of CD300f protein (rCD300f). The overexpression of rCD300f receptor had a comparable neuroprotective effect after the acute brain injury and a similar CNS staining pattern when stained with the rCD300f-Ig soluble protein. Interestingly, when we analyzed the expression pattern of rCD300f in brain cells by quantitative polymerase chain reaction and immunohistochemistry, we detected the expression of CD300f as expected in microglial cells, but also in oligodendrocytes and neurons. These data suggest that the neuroprotective role of CD300f would be the result of a complex network of cell interactions.

    PMID:21951326 | PMC:PMC8092960 | DOI:10.1111/j.1750-3639.2011.00537.x

  • Andrologia. 2012 Feb;44(1):59-65. doi: 10.1111/j.1439-0272.2010.01106.x. Epub 2011 May 19.


    Sperm morphology has consistently been the best indicator of male fertility. Transmission electron microscopy currently provides the most information on the subcellular details of sperm structure. Recently, assessment of sperm DNA damage has been employed to assess fertility potential. The purpose of this work was to link sperm DNA damage, evaluated by an intercalated fluorescent dye, with the structural characteristics of sperm. Conventional semen analysis was performed on samples from men undergoing fertility evaluation. Thirty men were evaluated and assigned to three subgroups based on strict criteria for sperm morphology: normal morphology (>14% normal forms), intermediate morphology (5-14% normal forms), and poor morphology (<5% normal forms). By quantifying acridine orange-positive cells and ultrastructural sperm defects, we found that the poor morphology pattern group showed a positive association between sperm carrying damaged DNA and the percentage of sperm nucleus with vacuoles (P = 0.01). No statistically significant correlations were established in other ultrastructural characteristics of sperm, including immature chromatin, lytic changes, or abnormal sperm tails. These results suggest that zones without chromatin in the sperm nucleus reflect underlying chromosomal or DNA defects in severe teratozoospermic men. This association should be considered in the evaluation of male fertility.

    PMID:21592172 | DOI:10.1111/j.1439-0272.2010.01106.x

  • Horm Behav. 2010 Nov;58(5):729-36. doi: 10.1016/j.yhbeh.2010.07.005. Epub 2010 Aug 3.


    Seasonal breeders are superb models for understanding natural relationships between reproductive behavior and its neural bases. We investigated the cellular bases of hormone effects in a weakly pulse-type electric fish with well-defined hormone-sensitive communication signals. Brachyhypopomus gauderio males emit social electric signals (SESs) consisting of rate modulations of the electric organ discharge during the breeding season. This discharge is commanded by a medullary pacemaker nucleus (PN), composed of pacemaker and relay neurons. We analyzed the contribution of androgen receptor (AR) expression to the seasonal generation of SESs, by examining the presence of ARs in the PN in different experimental groups: breeding, non-breeding, and testosterone (T)-implanted non-breeding males. AR presence and distribution in the CNS was assessed through western blotting and immunohistochemistry using the PG-21 antibody, which was raised against the human AR. We found AR immunoreactivity, for the first time in a pulse-type Gymnotiform, in several regions throughout the brain. In particular, this is the first report to reveal the presence of AR in both pacemaker and relay neurons within the Gymnotiform PN. The AR immunoreactivity was present in breeding males and could be induced in T-implanted non-breeding males. This seasonal and T-induced AR expression in the PN suggests that androgens may play an important role in the generation of SESs by modulating intrinsic electrophysiological properties of pacemaker and relay neurons.

    PMID:20688071 | DOI:10.1016/j.yhbeh.2010.07.005

  • Reprod Biol. 2010 Jul;10(2):125-39. doi: 10.1016/s1642-431x(12)60055-2.


    A specific cause of infertility cannot be identified in at least 25% of men referred to a specialized clinic. Diagnosis of infertile men is based mainly on standard semen analysis and the observation of sperm under light microscope. The aim of our study was to find the subcellular sperm characteristics that could explain infertility in a group of teratozoospermic infertile men. Morphological characteristics of sperm from non-teratozoospermic (control donors) and teratozoospermic infertile men were analyzed by transmission electron microscopy (TEM) and quantified. Our analysis showed that sperm cells from control donors presented a higher number of normal heads and tails than infertile men. Regarding subcellular characteristics of nucleus and tails, only the percentage of vacuolated nucleus, the absence of at least one pair of microtubules of the axoneme and the total distortion of the tail were statistically higher in infertile men than in control donors. There were no differences in the number of normal acrosomes between the groups. Although the ultrastructural sperm defects overlapped between control donors and infertile men, TEM permits the identification and differentiation of a larger amount of defects than light microscopy. Vacuolated nucleus and gross alterations of the tail are the major sperm defects that seem to have prognostic value in teratozoospermic men.

    PMID:20668504 | DOI:10.1016/s1642-431x(12)60055-2

  • J Neuroinflammation. 2010 Jun 9;7:33. doi: 10.1186/1742-2094-7-33.


    BACKGROUND: During pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X(7) receptors. In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X(7) receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1G93A astrocytes.

    METHODS: We evaluated motor neuron survival after co-culture with SOD1G93A or non-transgenic astrocytes pretreated with agents known to modulate ATP release or P2X(7) receptor. We also characterized astrocyte proliferation and extracellular ATP degradation.

    RESULTS: Repeated stimulation by ATP or the P2X(7)-selective agonist BzATP caused astrocytes to become neurotoxic, inducing death of motor neurons. Involvement of P2X(7) receptor was further confirmed by Brilliant blue G inhibition of ATP and BzATP effects. In SOD1G93A astrocyte cultures, pharmacological inhibition of P2X(7) receptor or increased extracellular ATP degradation with the enzyme apyrase was sufficient to completely abolish their toxicity towards motor neurons. SOD1G93A astrocytes also displayed increased ATP-dependent proliferation and a basal increase in extracellular ATP degradation.

    CONCLUSIONS: Here we found that P2X(7) receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death. Remarkably, the neurotoxic phenotype of SOD1G93A astrocytes depended upon basal activation the P2X(7) receptor. Thus, pharmacological inhibition of P2X(7) receptor might reduce neuroinflammation in ALS through astrocytes.

    PMID:20534165 | PMC:PMC2901222 | DOI:10.1186/1742-2094-7-33

  • Chem Res Toxicol. 2010 Apr 19;23(4):821-35. doi: 10.1021/tx900446r.


    Protein tyrosine dimerization and nitration by biologically relevant oxidants usually depend on the intermediate formation of tyrosyl radical ((*)Tyr). In the case of tyrosine oxidation in proteins associated with hydrophobic biocompartments, the participation of unsaturated fatty acids in the process must be considered since they typically constitute preferential targets for the initial oxidative attack. Thus, we postulate that lipid-derived radicals mediate the one-electron oxidation of tyrosine to (*)Tyr, which can afterward react with another (*)Tyr or with nitrogen dioxide ((*)NO(2)) to yield 3,3'-dityrosine or 3-nitrotyrosine within the hydrophobic structure, respectively. To test this hypothesis, we have studied tyrosine oxidation in saturated and unsaturated fatty acid-containing phosphatidylcholine (PC) liposomes with an incorporated hydrophobic tyrosine analogue BTBE (N-t-BOC l-tyrosine tert-butyl ester) and its relationship with lipid peroxidation promoted by three oxidation systems, namely, peroxynitrite, hemin, and 2,2'-azobis (2-amidinopropane) hydrochloride. In all cases, significant tyrosine (BTBE) oxidation was seen in unsaturated PC liposomes, in a way that was largely decreased at low oxygen concentrations. Tyrosine oxidation levels paralleled those of lipid peroxidation (i.e., malondialdehyde and lipid hydroperoxides), lipid-derived radicals and BTBE phenoxyl radicals were simultaneously detected by electron spin resonance spin trapping, supporting an association between the two processes. Indeed, alpha-tocopherol, a known reactant with lipid peroxyl radicals (LOO(*)), inhibited both tyrosine oxidation and lipid peroxidation induced by all three oxidation systems. Moreover, oxidant-stimulated liposomal oxygen consumption was dose dependently inhibited by BTBE but not by its phenylalanine analogue, BPBE (N-t-BOC l-phenylalanine tert-butyl ester), providing direct evidence for the reaction between LOO(*) and the phenol moiety in BTBE, with an estimated second-order rate constant of 4.8 x 10(3) M(-1) s(-1). In summary, the data presented herein demonstrate that LOO(*) mediates tyrosine oxidation processes in hydrophobic biocompartments and provide a new mechanistic insight to understand protein oxidation and nitration in lipoproteins and biomembranes.

    PMID:20170094 | PMC:PMC3506709 | DOI:10.1021/tx900446r

  • Neurobiol Dis. 2010 Mar;37(3):574-80. doi: 10.1016/j.nbd.2009.11.007. Epub 2009 Nov 13.


    Exposure to environmental lead (Pb) is a mild risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive degeneration of motor neurons. However, recent evidence has paradoxically linked higher Pb levels in ALS patients with longer survival. We investigated the effects of low-level Pb exposure on survival of mice expressing the ALS-linked superoxide dismutase-1 G93A mutation (SOD1(G93A)). SOD1(G93A) mice exposed to Pb showed longer survival and increased expression of VEGF in the ventral horn associated with reduced astrocytosis. Pretreatment of cultured SOD1(G93A) astrocytes with low, non toxic Pb concentrations upregulated VEGF expression and significantly abrogated motor neuron loss in coculture, an effect prevented by neutralizing antibodies to VEGF. The actions of Pb on astrocytes might explain its paradoxical slowing of disease progression in SOD1(G93A) mice and the improved survival of ALS patients. Understanding how Pb stimulates astrocytic VEGF production and reduces neuroinflammation may yield a new therapeutic approach for treating ALS.

    PMID:19914377 | PMC:PMC2824063 | DOI:10.1016/j.nbd.2009.11.007

  • Cell Motil Cytoskeleton. 2009 Dec;66(12):1087-99. doi: 10.1002/cm.20416.


    In previous works we showed that the depolarization of the plasma membrane potential (PMP) determines a reorganization of the cytoskeleton of diverse epithelia in culture, consisting mainly of a reallocation of peripheral actin toward the cell center, ultimately provoking intercellular disruption. In view of this evidence, we explored in this study the possible effects of membrane potential hyperpolarization on the cytoskeletal organization and adherens junction (AJ) morphology and the stability of confluent bovine corneal endothelial cells in culture. For this purpose, hyperpolarization was achieved by substitution of extracellular sodium by nondiffusible cations or via the incorporation of valinomycin to the control solution. Actin compactness at the cell periphery was assessed by quantitative analysis of fluorescence microscopy images. The stability of the AJ was challenged by calcium deprivation or temperature decrease. Our results showed that plasma membrane hyperpolarization provokes a compaction of AJ-associated actin filaments toward the plasma membrane and an increase in the stability of the AJs. We also observed that the hyperpolarizing procedures determined similar modifications in the actin cytoskeleton of endothelial cells in whole bovine corneas. Together with our previous work, the results of this study contribute to the idea that modifications in the PMP of nonexcitable cells participate in cellular adaptive responses involving reorganization of cytoskeletal components.

    PMID:19753628 | DOI:10.1002/cm.20416

  • Antioxid Redox Signal. 2009 Jul;11(7):1535-45. doi: 10.1089/ars.2009.2614.


    We studied the subcellular distribution of mitochondria and superoxide dismutase-1 (SOD1) in whole mounts of microdissected motor axons of rats expressing the ALS-linked SOD1-G93A mutation. The rationale was to determine whether physical interactions between the enzyme and mitochondria were linked to the axonopathy of motor fibers occurring in amyotrophic lateral sclerosis (ALS). Mitochondria and SOD1 displayed a homogeneous distribution along motor axons both in nontransgenic rats and in those overexpressing wild-type SOD1. In contrast, axons from SOD1-G93A rats (older than 35 days) showed accumulation of mitochondria in discrete clusters located at regular intervals. Most of SOD1 immunoreactivity was enriched in these clusters and colocalized with mitochondria, suggesting a recruitment of SOD1-G93A to the organelle. The SOD1/mitochondrial clusters were abundant in motor axons but scarcely seen in sensory axons. Clusters also were stained for neuronal nitric oxide synthase, nitrotyrosine, and cytochrome c. The later also was detected surrounding clusters. Ubiquitin colocalized with clusters only at late stages of the disease. The cytoskeleton was not overtly altered in clusters. These results suggest that mutant SOD1 and defective mitochondria create localized dysfunctional domains in motor axons, which may lead to progressive axonopathy in ALS.

    PMID:19344250 | PMC:PMC2842590 | DOI:10.1089/ars.2009.2614

  • Biochem J. 2009 Mar 15;418(3):595-604. doi: 10.1042/BJ20081981.


    The epimastigote stage of Trypanosoma cruzi undergoes PCD (programmed cell death) when exposed to FHS (fresh human serum). Although it has been known for over 30 years that complement is responsible for FHS-induced death, the link between complement activation and triggering of PCD has not been established. We have previously shown that the mitochondrion participates in the orchestration of PCD in this model. Several changes in mitochondrial function were described, and in particular it was shown that mitochondrion-derived O(2)(*-) (superoxide radical) is necessary for PCD. In the present study, we establish mitochondrial Ca(2+) overload as the link between complement deposition and the observed changes in mitochondrial physiology and the triggering of PCD. We show that complement activation ends with the assembly of the MAC (membrane attack complex), which allows influx of Ca(2+) and release of respiratory substrates to the medium. Direct consequences of these events are accumulation of Ca(2+) in the mitochondrion and decrease in cell respiration. Mitochondrial Ca(2+) causes partial dissipation of the inner membrane potential and consequent mitochondrial uncoupling. Moreover, we provide evidence that mitochondrial Ca(2+) overload is responsible for the increased O(2)(*-) production, and that if cytosolic Ca(2+) rise is not accompanied by the accumulation of the cation in the mitochondrion and consequent production of O(2)(*-), epimastigotes die by necrosis instead of PCD. Thus our results suggest a model in which MAC assembly on the parasite surface allows Ca(2+) entry and its accumulation in the mitochondrion, leading to O(2)(*-) production, which in turn constitutes a PCD signal.

    PMID:19053945 | DOI:10.1042/BJ20081981

  • Auton Neurosci. 2009 Jan 28;145(1-2):35-43. doi: 10.1016/j.autneu.2008.10.021. Epub 2008 Nov 25.


    Estradiol is a key hormone in the regulation of reproductive processes acting both on peripheral organs and sympathetic neurons associated to reproductive function. However, many of its regulatory effects on the development and function on the sympathetic neurons have not been completely clarified. Sympathetic neurons located in the celiac ganglion projects to visceral, vascular and glandular targets, and contribute to ovarian innervation, being the main source of sympathetic fibers. In the present study, we analyze the effects of elevated levels of exogenous estrogen during the prepubertal period in post-ganglionic sympathetic neurons. Estrogen exposure induced a significant increase in sympathetic celiac neuronal size and modified the expression of neurotrophin receptor p75. This change affected mainly small and medium size neurons. The effect of estrogens on innervation of celiac target organs was heterogeneous, inducing a significant increase in catecholaminergic innervation of the ovary, but not of the pyloric muscular layers. These findings further support the role of estrogen as a modulator of neuronal plasticity and suggest that estrogen could modify some features involved in the relation between sympathetic immature peripheral neurons and their target organs throughout a neurotrophin-dependent mechanism.

    PMID:19036644 | DOI:10.1016/j.autneu.2008.10.021

  • Mol Hum Reprod. 2008 Sep;14(9):555-9. doi: 10.1093/molehr/gan049. Epub 2008 Aug 22.


    Preterm birth (PTB) is a worldwide health problem and remains the leading cause of perinatal morbidity and mortality. Systemic and local intrauterine infections have been implicated in the pathogenesis of preterm labor and delivery. Common pathways between PTB, premature rupture of ovular membranes (PROM) and altered molecular routes of inflammation have been proposed. There is evidence to support a genetic component in these conditions. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is thought to play a key role in eliciting an inflammatory response. LPS is recognized by proteins of the innate immune system, including Toll-like receptor 4 (TLR4). Individuals from some European countries carrying the variant alleles resulting in an amino acid substitution (Asp299Gly) are at increased risk of Gram-negative infections and premature birth. The objective of this study was to determine if preterm newborns have different allele frequency of the Asp299Gly TLR4 variant from healthy term neonates in Uruguay. The impact of PROM was also examined. There was an increase in the risk for fetuses carrying the Asp299Gly substitution in TLR4 of being severely premature (<33 weeks) and to present PROM at the same time.

    PMID:18723631 | PMC:PMC2547094 | DOI:10.1093/molehr/gan049

  • Free Radic Biol Med. 2008 Sep 15;45(6):733-42. doi: 10.1016/j.freeradbiomed.2008.05.028. Epub 2008 Jun 12.


    Trypanosoma cruzi is the etiologic agent of Chagas' disease, an infection that affects several million people in Latin America. With no immediate prospect of a vaccine and problems associated with current chemotherapies, the development of new treatments is an urgent priority. Several aspects of the redox metabolism of this parasite differ enough from those in the mammalian host to be considered targets for drug development. Here, we review the information about a trypanosomatid-specific molecule centrally involved in redox metabolism, the dithiol trypanothione, and the main effectors of cellular antioxidant defense. We focus mainly on data from T. cruzi, making comparisons with other trypanosomatids whenever possible. In these parasites trypanothione participates in crucial thiol-disulfide exchange reactions and serves as electron donor in different metabolic pathways, from synthesis of DNA precursors to oxidant detoxification. Interestingly, the levels of several enzymes involved in trypanothione metabolism and oxidant detoxification increase during the transformation of T. cruzi to its mammalian-infective form and the overexpression of some of them has been associated with increased resistance to macrophage-dependent oxidative killing. Together, the evidence suggests a central role of the trypanothione-dependent antioxidant systems in the infection process.

    PMID:18588970 | DOI:10.1016/j.freeradbiomed.2008.05.028

  • J Neurosci. 2008 Apr 16;28(16):4115-22. doi: 10.1523/JNEUROSCI.5308-07.2008.


    Mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Recent reports indicate that astrocytes expressing the mutations of superoxide dismutase-1 (SOD1) may contribute to motor neuron injury in ALS. Here, we provide evidence that mitochondrial dysfunction in SOD1(G93A) rat astrocytes causes astrocytes to induce apoptosis of motor neurons. Mitochondria from SOD1(G93A) rat astrocytes displayed a defective respiratory function, including decreased oxygen consumption, lack of ADP-dependent respiratory control, and decreased membrane potential. Protein 3-nitrotyrosine was detected immunochemically in mitochondrial proteins from SOD1(G93A) astrocytes, suggesting that mitochondrial defects were associated with nitroxidative damage. Furthermore, superoxide radical formation in mitochondria was increased in SOD1(G93A) astrocytes. Similar defects were found in mitochondria isolated from the spinal cord of SOD1(G93A) rats, and pretreatment of animals with the spin trap 5,5-dimethyl-1-pyrroline N-oxide restored mitochondrial function, forming adducts with mitochondrial proteins in vivo. As shown previously, SOD1(G93A) astrocytes induced death of motor neurons in cocultures, compared with nontransgenic ones. This behavior was recapitulated when nontransgenic astrocytes were treated with mitochondrial inhibitors. Remarkably, motor neuron loss was prevented by preincubation of SOD1(G93A) astrocytes with antioxidants and nitric oxide synthase inhibitors. In particular, low concentrations (approximately 10 nm) of two mitochondrial-targeted antioxidants, ubiquinone and carboxy-proxyl nitroxide, each covalently coupled to a triphenylphosphonium cation (Mito-Q and Mito-CP, respectively), prevented mitochondrial dysfunction, reduced superoxide production in SOD1(G93A) astrocytes, and restored motor neuron survival. Together, our results indicate that mitochondrial dysfunction in astrocytes critically influences motor neuron survival and support the potential pharmacological utility of mitochondrial-targeted antioxidants in ALS treatment.

    PMID:18417691 | PMC:PMC3844766 | DOI:10.1523/JNEUROSCI.5308-07.2008

  • Reprod Fertil Dev. 2007;19(8):954-60. doi: 10.1071/rd07075.


    The present study analyses the participation of ovarian innervation during reproductive senescence. We use the model of acute peripheral pharmacological sympathetic denervation with guanethidine in young (3 months old), middle-aged (12 months old) or old (18 months old) rats with spontaneous or induced ovulation. Ovarian levels of norepinephrine (NE) were measured by HPLC and the oestrous cycle, the number of ovulating animals and the percentage of atretic follicles were also assessed. Aged animals showed a progressive reduction in ovulatory capacity and an increase in ovarian NE content. Acute denervation increased the percentage of healthy follicles in 12- and 18-month-old rats compared with control adult animals. Combined treatment of denervation plus stimulation with gonadotrophins doubled the number of ova shed in young adult rats and restablished a partial ovulation in 12-month-old rats. The results suggest that ovarian noradrenergic innervation plays a modulator role in ovarian physiology during the ageing ovary process. The action of ovarian noradrenergic innervation seems to be associated with folliculogenesis and the ovarian response to gonadotrophins.

    PMID:18076827 | DOI:10.1071/rd07075

  • Exp Parasitol. 2008 Feb;118(2):188-96. doi: 10.1016/j.exppara.2007.07.014. Epub 2007 Aug 23.


    In this work we studied the evolution of early inflammation, complement activation and parasite survival/death along the establishment phase of Echinococcus granulosus metacestode. Using a chamber model of infection in mice, we examined cell infiltration and C3 deposition on individual parasites during their development from protoscoleces to cystic forms. We found that the intensity of the initial inflammation decreased around undamaged but not around damaged parasites: at 43dpi undamaged parasites were mostly associated with poor/mild inflammation while damaged parasites with a strong inflammation. In addition, whereas complement activation participated in the induction of early inflammation, the deposition of C3 on undamaged parasites progressively diminished. Interestingly, we observed some parasites in pre-cystic stage with no/poor C3 deposition at 3dpi. Overall, these results indicated that the establishment and survival of the hydatid cyst is associated with the control of complement and, consequently, of local inflammation at the initial phases of infection.

    PMID:17905232 | DOI:10.1016/j.exppara.2007.07.014

  • Comp Biochem Physiol A Mol Integr Physiol. 2008 Nov;151(3):370-380. doi: 10.1016/j.cbpa.2007.04.012. Epub 2007 Apr 22.


    Pulse gymnotids extract information about the environment using the pulsed discharge of an electric organ. Cutaneous electroreceptor organs transduce and encode the changes that objects imprint on the self-generated transcutaneous electric field. This review deals with the role of a neural circuit, the fast electrosensory path of pulse gymnotids, in the streaming of self generated electrosensory signals. The activation of this path triggers a low-responsiveness window slightly shorter than the interval between electric organ discharges. This phenomenon occurs at the electrosensory lateral line lobe where primary afferent terminals project on the somata of spherical neurons. The main subservient mechanism of the low-responsiveness window rely on the intrinsic properties of spherical neurons (dominated by a voltage dependent, low-threshold, non-inactivating and slowly-deactivating K(+) conductance) determining the cell to respond with a single spike followed by a long refractory period. Externally generated signals that randomly occur within the interval between self-generated discharges are likely blocked by the low responsiveness window. Repetitive signals, as those emitted by conspecifics with a slightly lower rate, occur progressively at longer delays beyond the duration of the low responsiveness window. Transient increases of the discharge rate relocate the interference within the low-responsiveness window. We propose that this combination of sensory filtering and electromotor control favors the self-generated signals in detriment of other, securing the continuity of the electrolocation stream.

    PMID:17513149 | DOI:10.1016/j.cbpa.2007.04.012

  • Reprod Fertil Dev. 2006;18(5):525-31. doi: 10.1071/rd05053.


    In mammals, pregnancy induces a transient and extensive degeneration of uterine sympathetic innervation. We used the models of unilateral oviduct ligation and in oculo myometrium transplant in pregnant rats to address the role of stretching forces and/or hormone milieu in the loss of sympathetic innervation. The sympathetic fibres of the uterine horn and in oculo myometrial transplants were quantified on tissue sections processed by the glyoxylic acid technique. In normal pregnant rats, the density of uterine horn innervation was significantly reduced at late pregnancy and recovery took place during post partum. The empty horn of pregnant rats showed no significant changes in density of myometrial innervation during pregnancy or post partum. In oculo myometrial transplants were organotypically reinnervated in virgin animals. When the transplants were exposed to gestational hormonal milieu, few or no fibres were observed to the end of pregnancy; however, a significant increase at post partum was observed. Results showed that both the effects of stretching and the hormone milieu derived from the fetus-placenta complex play a role as inductors of changes on sympathetic myometrial innervation during pregnancy and support the idea that immature muscular uterine fibres are more susceptible to the effects of pregnancy than those originating from adult animals.

    PMID:16836959 | DOI:10.1071/rd05053

  • J Biol Chem. 2005 Jul 8;280(27):25571-9. doi: 10.1074/jbc.M501920200. Epub 2005 May 3.


    Fibroblast growth factor-1 (FGF-1) is highly expressed in motor neurons and can be released in response to sublethal cell injury. Because FGF-1 potently activates astroglia and exerts a direct neuroprotection after spinal cord injury or axotomy, we examined whether it regulated the expression of inducible and cytoprotective heme oxygenase-1 (HO-1) enzyme in astrocytes. FGF-1 induced the expression of HO-1 in cultured rat spinal cord astrocytes, which was dependent on FGF receptor activation and prevented by cycloheximide. FGF-1 also induced Nrf2 mRNA and protein levels and prompted its nuclear translocation. HO-1 induction was abolished by transfection of astrocytes with a dominant-negative mutant Nrf2, indicating that FGF-1 regulates HO-1 expression through Nrf2. FGF-1 also modified the expression of other antioxidant genes regulated by Nrf2. Both Nrf2 and HO-1 levels were increased and co-localized with reactive astrocytes in the degenerating lumbar spinal cord of rats expressing the amyotrophic lateral sclerosis-linked SOD1 G93A mutation. Overexpression of Nrf2 in astrocytes increased survival of co-cultured embryonic motor neurons and prevented motor neuron apoptosis mediated by nerve growth factor through p75 neurotrophin receptor. Taken together, these results emphasize the key role of astrocytes in determining motor neuron fate in amyotrophic lateral sclerosis.

    PMID:15870071 | DOI:10.1074/jbc.M501920200

  • J Neurochem. 2005 Apr;93(1):38-46. doi: 10.1111/j.1471-4159.2004.02984.x.


    Fibroblast growth factor-1 (FGF1 or acidic FGF) is highly expressed in motor neurons. FGF-1 is released from cells by oxidative stress, which might occur from SOD-1 aberrant function in amyotrophic lateral sclerosis (ALS). Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that FGF-1 could activate spinal cord astrocytes in a manner that decreased motor neuron survival in co-cultures. FGF-1 induced accumulation of the FGF receptor 1 (FGFR1) in astrocyte nuclei and potently stimulated nerve growth factor (NGF) expression and secretion. The FGFR1 tyrosine kinase inhibitor PD166866 prevented these effects. Previously, we have shown that NGF secretion by reactive astrocytes induces motor neuron apoptosis through a p75(NTR)-dependent mechanism. Embryonic motor neurons co-cultured on the top of astrocytes exhibiting activated FGFR1 underwent apoptosis, which was prevented by PD166866 or by adding either anti-NGF or anti-p75(NTR) neutralizing antibodies. In the degenerating spinal cord of mice carrying the ALS mutation G93A of Cu, Zn superoxide dismutase, FGF-1 was no longer localized only in the cytosol of motor neurons, while FGFR1 accumulated in the nuclei of reactive astrocytes. These results suggest that FGF-1 released by oxidative stress from motor neurons might have a role in activating astrocytes, which could in turn initiate motor neuron apoptosis in ALS through a p75(NTR)-dependent mechanism.

    PMID:15773903 | DOI:10.1111/j.1471-4159.2004.02984.x

  • Brain Res Brain Res Rev. 2004 Dec;47(1-3):263-74. doi: 10.1016/j.brainresrev.2004.05.003.


    A strong glial reaction typically surrounds the affected upper and lower motor neurons and degenerating descending tracts of ALS patients. Reactive astrocytes in ALS contain protein inclusions, express inflammatory makers such as the inducible forms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2), display nitrotyrosine immunoreactivity and downregulate the glutamate transporter EAAT2. In this review, we discuss the evidence sustaining an active role for astrocytes in the induction and propagation of motor neuron loss in ALS. Available evidence supports the view that glial activation could be initiated by proinflammatory mediators secreted by motor neurons in response to injury, axotomy or muscular pathology. In turn, reactive astrocytes produce nitric oxide and peroxynitrite, which cause mitochondrial damage in cultured neurons and trigger apoptosis in motor neurons. Astrocytes may also contribute to the excitotoxic damage of motor neurons by decreasing glutamate transport or actively releasing the excitotoxic amino acid. In addition, reactive astrocytes secrete pro-apoptotic mediators, such as nerve growth factor (NGF) or Fas-ligand, a mechanism that may serve to eliminate vulnerable motor neurons. The comprehensive understanding of the interactions between motor neurons and glia in ALS may lead to a more accurate theory of the pathogenesis of the disease.

    PMID:15572176 | DOI:10.1016/j.brainresrev.2004.05.003

  • J Biochem Biophys Methods. 2004 May 31;59(2):139-43. doi: 10.1016/j.jbbm.2003.12.004.


    We have developed a simple method for plasma fibronectin purification based on the well-known gelatin binding property of fibronectin. In this procedure we immobilize the melted gelatin to nitrocellulose membranes; these are then used to affinity-purify the fibronectin from the plasma sample. The fibronectin is eluted from the membrane by treatment with 8 M urea. The procedure described here gives a yield of up to 60% (from presumed fibronectin concentration) and the fibronectin obtained is homogeneous in SDS-PAGE and biologically active, as assessed by a cell migration assay. The method is rapid, simple, inexpensive, does not require the use of chromatographic equipment and is suitable for tissue culture applications.

    PMID:15163525 | DOI:10.1016/j.jbbm.2003.12.004

  • J Neurochem. 2004 May;89(3):602-12. doi: 10.1046/j.1471-4159.2004.02363.x.


    Peroxynitrite-dependent tyrosine nitration has been postulated to be involved in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Evidence supporting this supposition includes the appearance of both free and protein-linked 3-nitro-l-tyrosine (nitrotyrosine) in both sporadic and familial ALS, as well as of increased free nitrotyrosine levels in the spinal cord of transgenic mice expressing ALS-linked superoxide dismutase mutants at symptom onset. Here we demonstrate that incubation with clinically relevant concentrations of nitrotyrosine induced apoptosis in motor neurons cultured with trophic factors. Nitrotyrosine was bound to proteins, but it was not incorporated into alpha-tubulin, as previously demonstrated for other cell types. Neither inhibition of nitric oxide production nor scavenging of superoxide and peroxynitrite prevented increases in cell nitrotyrosine immunoreactivity or motor neuron death, suggesting that these effects are not due to the endogenous formation of reactive nitrogen species. In contrast, some populations of astrocytes incorporated nitrotyrosine into alpha-tubulin, but free nitrotyrosine had no effect on the viability and phenotype of astrocytes in culture, as evaluated by glial fibrillary acidic protein immunoreactivity, cell growth and morphology. Co-culture of motor neurons on astrocyte monolayers delayed, but did not prevent, nitrotyrosine-induced motor neuron death. These results suggest that free nitrotyrosine may play a role in the induction of motor neuron apoptosis in ALS.

    PMID:15086517 | DOI:10.1046/j.1471-4159.2004.02363.x

  • Neurotox Res. 2003;5(6):399-406. doi: 10.1007/BF03033168.


    The vulnerability of oligodendrocytes to excitatory amino acids may account for the pathology of white matter occurring following hypoxia/ischemia or autoimmune attack. Here, we examined the vulnerability of immature oligodendrocytes (positively labeled by galactocerobroside-C and not expressing myelin basic protein) from neonatal rat spinal cord to kainate, an agonist of excitatory amino acid receptors that induces long-lasting inward currents in immature oligodendrocytes. In particular, we studied whether kainate toxicity was linked to the endogenous production of nitric oxide. We found cultured oligodendrocytes to be highly sensitive to 24-48 h exposure to 0.5-1 mM kainate. The toxin induced striking morphological changes in oligodendrocytes, characterized by the disruption of the process network around the cell body and the growth of one or two long, thick and non-branched processes. A longer exposure to kainate resulted in massive death of oligodendrocytes, which was prevented by 6,7, dinitroquinoxaline-2,3-dione (DNQX) (30 micro M), the antagonist of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic/kainate receptors. Remarkably, we found that those oligodendrocytes displaying bipolar morphology following kainate exposure, also expressed the inducible form of nitric oxide synthase (iNOS) and nitrotyrosine immunoreactivity, suggesting that peroxynitrite could be formed by the reaction of nitric oxide with superoxide. Moreover, kainate toxicity was significantly prevented by addition of the NOS inhibitor nitro-L-arginine methyl ester (L-NAME), further suggesting that nitric oxide-derived oxidants contribute to excitotoxic mechanisms in immature oligodendrocytes.

    PMID:14715442 | DOI:10.1007/BF03033168

  • Cell Tissue Res. 2003 Nov;314(2):191-205. doi: 10.1007/s00441-003-0799-9. Epub 2003 Sep 13.


    In the present study we investigated the effects of infantile/prepubertal chronic oestrogen treatment, chemical sympathectomy with guanethidine and combined sympathectomy and chronic oestrogen treatment on developing sensory nerves of the rat uterus. Changes in sensory innervation were assessed quantitatively on uterine cryostat tissue sections stained for calcitonin gene-related peptide (CGRP). Uterine levels of NGF protein, using immunohistochemistry and ELISA, and mRNA, using Northern blots and in situ hybridization, were also measured. Finally, levels of TrkA NGF receptor in sensory neurons of T13 and L1 dorsal root ganglia (DRG), which supply the uterus, were assessed using densitometric immunohistochemistry. These studies showed that: (1) chronic oestrogen treatment led to an 83% reduction in the intercept density of CGRP-immunoreactive nerves; (2) sympathectomy had no effect on the density of uterine sensory nerves or on the pattern of oestrogen-induced changes; (3) NGF mRNA and protein increased following sympathectomy or chronic oestrogen treatment; and (4) oestrogen produced increased intensity of labelling (28%) for TrkA receptors in small-diameter sensory neurons, but decreased labelling (13%) in medium-sized neurons, which represent the large majority of the DRG neurons supplying the upper part of the uterine horn. Contrary to expectations, increased levels of NGF after sympathectomy and oestrogen treatment did not lead to increased sensory innervation of the uterus. The possibility that alterations in neuronal levels of TrkA contribute to the lack of response of uterine sensory nerves to the oestrogen-induced increase in NGF levels is discussed.

    PMID:13680358 | DOI:10.1007/s00441-003-0799-9

  • J Exp Biol. 2003 Sep;206(Pt 17):3085-93. doi: 10.1242/jeb.00515.


    The spinal cords and brains--comprising dorsal cortex (DC), medial cortex (MC) and diencephalon (Dien)--of juvenile turtles acclimated to warm temperature [27-30 degrees C; warm-acclimated turtles (WATs)] revealed higher density values of bromodeoxyuridine-labeled cells (BrdU-LCs) than those acclimated to a cooler environment [5-14 degrees C; cold-acclimated turtles (CATs)]. Both populations were under the influence of the seasonal daily light-dark rhythms. Pronounced differences between WATs and CATs (independent t-test; confidence level, P<0.01) were found in the central area of the spinal gray matter and in the ependymal epithelium lining the brain ventricles. Forebrain regions (DC, MC and Dien) also revealed significant differences between WATs and CATs (independent t-test; confidence level, P<0.01-0.05). Unexplored biological clocks that may be affecting cell proliferation were equalized by performing paired experiments involving one WAT and one CAT. Both animals were injected on the same day at the same time and both were sacrificed 24 h later. These experiments confirmed that a warm environment increased cell proliferation in the CNS of turtles. Double- and triple-labeling experiments involving anti-BrdU antibody together with anti-glial protein antibodies revealed that temperature modulates not only cell populations expressing glial markers but also other cells that do not express them. As expected, in the case of short post-injection (BrdU) surviving time points, no cells were found colabeling for BrdU and NeuN (neuronal marker). The probable direct effect of temperature on the cell division rate should be analyzed together with potential indirect effects involving increased motor activity and increased food intake. The fate of the increased BrdU-LCs (death, permanence as progenitor cells or differentiation following neuronal or glial lines) remains a matter for further investigation. Results are discussed in the light of current opinions concerned with post-natal neurogenesis in vertebrates.

    PMID:12878675 | DOI:10.1242/jeb.00515

  • Neurotox Res. 2002 Mar;4(2):87-93. doi: 10.1080/10298420290015818.


    We have established a cell culture model of spinal cord astrocytes to study the cytotoxicity of peroxynitrite. Nitric oxide (NO) has been implicated as a key contributor to neurotoxicity. NO reacts with superoxide to generate peroxynitrite, a strong oxidant and nitrating agent with deleterious cytotoxic and pro-apoptotic effects. Peroxynitrite and nitrotyrosine are formed in damaged motor neurons in amyotrophic lateral sclerosis (ALS), which are surrounded by reactive astrocytes. To determine the effects of extracellular addition of peroxynitrite, purified astrocyte monolayers prepared from neonatal rat spinal cords were exposed to peroxynitrite (0.25-0.75 mM) for 5 min and further incubated in culture medium for 24-72h. Peroxynitrite exposure did not result in apparent cell loss or damage of the monolayer. However, a substantial number of cells adopted reactive features, with long processes displaying intense immunoreactivity to glial fibrillary acidic protein (GFAP). Western blot analysis performed 24h after peroxynitrite treatment showed that GFAP levels were not modified by the oxidant. There were no changes in cell viability parameters in astrocyte cultures after peroxyintrite, indicating that astrocytes are more resistant to the oxidant than other cell types. Peroxynitrite reacts with protein-bound tyrosine residues to form nitrotyrosine. We observed a modest to strong nitrotyrosine immunoreactivity in astrocytes 24h following peroxynitrite exposure. There was a remarkable association between nitrotyrosine and high-intensity GFAP immunoreactivity in astrocytes bearing long processes. These results suggest that peroxynitrite induces a characteristic long-lasting reactive astrocytic phenotype and provide new insight into understanding the origin of reactive astrocytes occurring in ALS.

    PMID:12829407 | DOI:10.1080/10298420290015818

  • J Neurobiol. 2003 Feb 15;54(3):525-36. doi: 10.1002/neu.10179.


    The Drosophila Rel transcription factor Dorsal and its inhibitor Cactus participate in a signal transduction pathway involved in several biologic processes, including embryonic pattern formation, immunity, and muscle development. In contrast with embryonic muscle, where Dorsal is reportedly absent, this protein and Cactus accumulates in the neuromuscular junctions in the muscle of both larvae and adults. The phenotype of homozygous dorsal mutant larvae suggested that Dorsal and Cactus maybe necessary for normal function and maintenance of the neuromuscular system. Here we investigate if these proteins can respond to synaptic activity. Using larval body wall preparations and antibodies specific for Dorsal or Cactus we show that the amount of these proteins at the neuromuscular junction is substantially decreased after electrical stimulation of the nerves or incubation in glutamate, the principal transmitter in this type of synapse. The specificity of the response was tested with a glutamate receptor antagonist (argiotoxin 636). Because the effect can be reproduced using a calcium ionophore (ionomycin treatment) as well as blocked by the inhibition of the muscle ryanodine receptor (tetracaine treatment), the involvement of calcium in this process seems likely. We also observed that the inhibition of the calcium dependent protein phosphatase calcineurin prevents the effect of glutamate on the fluorescence for Dorsal and Cactus, suggesting its participation in a signal transduction cascade that may activate Dorsal in the muscle independently of Toll. Our results are consistent with a novel function of the Rel factor Dorsal in a molecular pathway turned on by neural activity and/or contractile activity.

    PMID:12532402 | DOI:10.1002/neu.10179

  • Microsc Res Tech. 2002 Dec 15;59(6):484-9. doi: 10.1002/jemt.10231.


    Mammalian ovarian function is under endocrine and neural control. Although the extrinsic innervation of the ovary has been implicated in the control of both ovarian development and mature function, it is now clear that, from rats to humans, the ovary is endowed with a network of intrinsic neurons displaying diverse chemical phenotypes. This article describes the presence of these intrinsic neurons in the ovary of different mammalian species, and discusses the possible functions that they may have in the regulation of ovarian physiology.

    PMID:12467023 | DOI:10.1002/jemt.10231

  • Auton Neurosci. 2002 Oct 31;101(1-2):13-22. doi: 10.1016/s1566-0702(02)00173-x.


    Chronic administration of oestrogen to rats during the infantile/prepubertal period provokes, at 28 days of age, complete loss of noradrenaline-labelled intrauterine sympathetic nerves. It is not known whether oestrogen inhibits the growth or causes the degeneration of developing uterine sympathetic nerves, or whether the uterus recovers its innervation following cessation of infantile/prepubertal oestrogen treatment. In the present study, we analysed the time-course of the effects of oestrogen on the development of uterine sympathetic nerves in the rat, using histochemical methods. In addition, the pattern of sympathetic reinnervation of the uterus of intact and ovariectomised females was assessed 3 and 6 months after cessation of chronic oestrogen treatment. The ability of sympathetic nerves to reinnervate the oestrogenized uterine tissue was assessed in intraocular transplants of uterine myometrium into ovariectomised host rats. Early exposure to oestrogen did not inhibit the approach of sympathetic nerves to the uterus, but prevented the normal growth and maturation of intrauterine sympathetic fibres and abolished the innervation that reached the organ before initiation of treatment. Three or six months following cessation of oestrogen treatment, most of the sympathetic nerves were restricted to the mesometrium and mesometrial entrance, whereas intrauterine innervation remained persistently depressed as a consequence of a sustained oestrous-like state provoked by ovarian dysfunction (polycystic ovary). An organotypic regrowth of uterine sympathetic nerves was observed in ovariectomised infantile/prepubertal oestrogen-treated animals. After 5 weeks in oculo, the innervation of oestrogenized myometrial transplants was reduced by 50%, and substantial changes in the pattern of reinnervation were observed. In control transplants, 86% of the nerves were terminal varicose myometrial and perivascular nerve fibres, whereas 14% were preterminal nerve bundles. In oestrogenized myometrial transplants, 83% of the noradrenaline-labelled intercepting nerves were enlarged preterminal bundles and only 17% were terminal fibres. These results indicate that the oestrogenized myometrium is unattractive for sympathetic nerves and inhibits organotypic sympathetic reinnervation.

    PMID:12462355 | DOI:10.1016/s1566-0702(02)00173-x

  • J Histochem Cytochem. 2002 Jun;50(6):839-50. doi: 10.1177/002215540205000610.


    The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.

    PMID:12019300 | DOI:10.1177/002215540205000610

  • Cell Tissue Res. 2002 Apr;308(1):61-73. doi: 10.1007/s00441-002-0521-3. Epub 2002 Mar 8.


    Oestrogen is a key factor in the remodelling of uterine sympathetic nerves during puberty and the oestrous cycle; these nerves are influenced by changes in their target uterine tissue. The magnitude of oestrogen-induced responses might however be influenced by the maturation stage of sympathetic nerve fibres, the age of the neurons and/or the developmental state of the uterus. We have therefore compared the sympathetic innervation of the uterus following chronic oestrogen treatment of infantile/prepubertal and young adult intact and ovariectomised rats. Treatment of infantile/prepubertal rats resulted in the complete loss of intrauterine noradrenaline (NA)-labelled sympathetic nerves and a marked reduction in the total NA content in the uterine horn. Chronic treatment of young adult rats had little effect. To examine whether the age of the neurons or the degree of development of the uterus determined responsiveness of nerves to oestrogen, we assessed the effects of oestrogen on the sympathetic reinnervation of intraocular transplants of young adult uterine myometrium into ovariectomised adult host rats. Early treatment (10 days post-transplantation) resulted in less sympathetic innervation than late treatment (30 days post-transplantation). Measurements of nerve growth factor (NGF) levels in the uterine horn of control rats before and after puberty and following infantile/prepubertal chronic oestrogen treatment and acute oestrogen treatment of young adult rats revealed a coordinated increase between the growth of the uterus and NGF protein levels. Thus, developing and recently regrown sympathetic nerves are more susceptible to oestrogen-induced changes in the uterus than mature nerves, differential susceptibility is not related to the age of the neurons or the developmental state of the uterus and changes in NGF protein do not account for the differential susceptibility of developing and mature uterine sympathetic nerve fibres to oestrogen. Growing sympathetic fibres are more vulnerable to oestrogen than mature fibres and nerve fibres that have been in contact for longer periods with their target become less susceptible to oestrogen.

    PMID:12012206 | DOI:10.1007/s00441-002-0521-3

  • J Neurosci Res. 2002 Jan 1;67(1):21-9. doi: 10.1002/jnr.10107.


    Oxidative stress mediated by nitric oxide (NO) and its toxic metabolite peroxynitrite has previously been associated with motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Degenerating spinal motor neurons in familial and sporadic ALS are typically surrounded by reactive astrocytes expressing the inducible form of NO synthase (iNOS), suggesting that astroglia may have a pathogenic role in ALS. We report here that a brief exposure of spinal cord astrocyte monolayers to peroxynitrite (0.25-1 mM) provoked long-lasting reactive morphological changes characterized by process-bearing cells displaying intense glial fibrillary acidic protein and iNOS immunoreactivity. Furthermore, peroxynitrite caused astrocytes to promote apoptosis of embryonic motor neurons subsequently plated on the monolayers. Neuronal death occurred within 24 hr after plating, as evidenced by the presence of degenerating motor neurons positively stained for activated caspase-3 and nitrotyrosine. Motor neuron death was largely prevented by NOS inhibitors and peroxynitrite scavengers but not by trophic factors that otherwise will support motor neuron survival in the absence of astrocytes. The bacterial lipopolysaccharide, a well-known inflammatory stimulus that induces iNOS expression in astrocytes, provoked the same effects on astrocytes as peroxynitrite. Thus, spinal cord astrocytes respond to extracellular peroxynitrite by adopting a phenotype that is cytotoxic to motor neurons through peroxynitrite-dependent mechanisms.

    PMID:11754077 | DOI:10.1002/jnr.10107

  • Cell Tissue Res. 2001 Nov;306(2):231-7. doi: 10.1007/s004410100451.


    Mammalian ovarian function is regulated by both hormonal inputs and direct neural influences. Recent studies have shown that, in addition to the extrinsic innervation, the ovaries of nonhuman primates and a strain of rats contain a discrete population of intrinsic neurons. In the present study, we used histological and immunohistochemical approaches to identify the presence of neuronal cell bodies in the fetal and neonatal human ovary. Neurons containing neurofilament immunoreactivity were detected in the hilum and medulla of the ovary at all ages studied, ranging from 24 weeks of gestation to 10 months of postnatal age. Most of them coexpressed the low affinity neurotrophin receptor (p75NTR), and some were catecholaminergic, as determined by their content of immunoreactive tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. The presence of intrinsic neurons in the human ovary, similar to those previously found in other species, indicates that they may be engaged in regulating common, phylogenetically conserved, ovarian functions. It also raises the possibility that their dysfunction may contribute to the manifestation of particular ovarian pathologies.

    PMID:11702234 | DOI:10.1007/s004410100451

  • Prog Brain Res. 2001;132:413-25. doi: 10.1016/S0079-6123(01)32092-7.


    PMID:11545008 | DOI:10.1016/S0079-6123(01)32092-7

  • Cell Tissue Res. 2001 Jun;304(3):323-31. doi: 10.1007/s004410100355.


    The neural retina is a highly organized organ whose final histoarchitecture depends on the presence of diverse growth factors and on their interactions with extracellular matrix components. However, the role of growth factors on retinal development is not fully understood. Suramin has been shown to produce diverse cellular effects via the simultaneous block of the action of several growth factors. We have therefore studied the effects of suramin on organotypic culture of chick embryo neural retina in order to gain further insights into the participation of growth factors in neural retinal development. Neural retina was incubated for 24 h with suramin at 50-200 microM and then processed to determine cell proliferation, nuclear morphology, and actin distribution. Suramin provoked extensive morphological changes revealed by a decrease in BrdU incorporation, alterations in cellular organization, and disruption of the outer limiting membrane, with the emergence of cellular elements through it. All of these effects were dose-dependent and markedly attenuated by the simultaneous presence of suramin and fibroblast growth factor 2 (FGF-2) in the culture medium. These findings indicate that suramin induces pleiotropic effects on the histoarchitecture of the chicken neural retina in organ culture and suggest that FGF-2 is one of the biological modulators involved in the maintenance of the structural organization of the chicken neural retina.

    PMID:11456408 | DOI:10.1007/s004410100355

  • J Anat. 2000 Apr;196 ( Pt 3)(Pt 3):347-55. doi: 10.1046/j.1469-7580.2000.19630347.x.


    Previous studies have shown that chronic administration of oestrogen during postnatal rat development dramatically reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. In the present study we analysed if this response is due to a direct and selective effect of oestrogen on the uterine noradrenaline-containing sympathetic nerves, using the in oculo transplantation method. Small pieces of myometrium from prepubertal rats were transplanted into the anterior eye chamber of adult ovariectomised host rats. The effect of systemic chronic oestrogen treatment on the reinnervation of the transplants by noradrenaline-containing sympathetic fibres from the superior cervical ganglion was analysed on cryostat tissue sections processed by the glyoxylic acid technique. In addition, the innervation of the host iris was assessed histochemically and biochemically. The histology of the transplants and irises was examined in toluidine blue-stained semithin sections. These studies showed that after 5 wk in oculo, the overall size of the oestrogen-treated transplants was substantially larger than controls, and histology showed that this change was related to an increase in the size and number of smooth muscle cells within the transplant. Chronic oestrogen treatment did not provoke trophic changes in the irideal muscle. Histochemistry showed that control transplants had a rich noradrenergic innervation, associated with both myometrium and blood vessels. Conversely, in oestrogen-treated transplants only occasional fibres were recognised, showing a reduced NA fluorescence intensity. No changes in the pattern and density of innervation or in the total content of noradrenaline of the host irises were detected after chronic exposure to oestrogen. We interpreted these results to indicate that the effects of oestrogen on uterine noradrenaline-containing sympathetic nerves are neither selective or direct, but result from an interaction between sympathetic nerve fibres with the oestradiol-primed uterine tissue. A potential effect of oestrogen on the neurotrophic capacity of the uterus is discussed.

    PMID:10853957 | PMC:PMC1468071 | DOI:10.1046/j.1469-7580.2000.19630347.x

  • Cell Tissue Res. 2000 Apr;300(1):47-56. doi: 10.1007/s004419900130.


    Previous studies have shown the presence of neuronal perikarya in the primate ovary, but not in the ovary from Sprague-Dawley rats. We report here that while such intrinsic neurons are indeed absent in this strain of rats, they can be visualized in the ovary from Wistar rats. The neurons, identified by their morphology and by the expression of NeuN (a neuron-specific nuclear protein), were detected at all postnatal intervals examined, from 14 h after birth to 50 days of age. While they were present in the ovarian hilum and medulla at all ages studied, neurons first appeared in the ovarian cortex during the juvenile period (postnatal days 10-20). In all cases, the size of the neuronal soma increased significantly during prepubertal development, reaching maximal values before puberty. Some neurons were catecholaminergic, as indicated by their content of immunoreactive tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis. Some showed neuropeptide Y (NPY) immunoreactivity. TH-positive neurons were seen either in isolation or clustered in ganglion-like structures in both the ovarian cortex and medulla. These results indicate that ovarian neurons are not present in all strains of rats, but when present, the chemical phenotype of some of them is of a sympathetic nature, similar to that described in primates.

    PMID:10805074 | DOI:10.1007/s004419900130

  • Neurosci Lett. 1999 Nov 5;275(1):5-8. doi: 10.1016/s0304-3940(99)00686-2.


    The present study investigates the presence of nitric oxide synthase (NOS) by the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemical reaction and immunohistochemistry with neuronal NOS (nNOS) antibody during postnatal development of hypoglossal nucleus (XII) in the rat. Our results showed that the enzyme expression was limited to a subset of cells in the dorsal division of the nucleus. Retrogradely labeling with diamidino yellow (DY) from the tongue indicates that these cells were motoneurons. NOS expression was transient, being detectable during the first 3 postnatal weeks, suggesting a role for nitric oxide in the development and maturation of this subset of cranial motoneurons.

    PMID:10554971 | DOI:10.1016/s0304-3940(99)00686-2

  • Neurosci Lett. 1997 Jun 13;228(3):207-11. doi: 10.1016/s0304-3940(97)00384-4.


    In the present study we have assessed whether riluzole stimulates the production of trophic activities for motoneurons by spinal astrocyte cultures. Astrocyte monolayers prepared from new-born rats were exposed to vehicle or riluzole (1-10 microM) for 30-36 h, then washed and further incubated without riluzole for 24 h in L15 medium to obtain the astrocyte conditioned media (ACM). Motoneuron-enriched cultures were used to test the ability of the ACM to support motoneuron viability. Astrocyte monolayers exposed to 1 microM riluzole did not show changes in morphology or in DNA or protein synthesis. However, the conditioned medium obtained from astrocyte monolayers after this treatment increased motoneuron survival compared to that from vehicle-treated cultures. A similar effect was found when astrocytes were exposed to a higher riluzole concentration (10 microM) but with greater dilutions of the conditioned medium. This trophic activity was abolished by boiling or after treatment with trypsin. These findings strongly suggest the existence of a new trophic mechanism, through which riluzole may exert motoneuron protection.

    PMID:9218644 | DOI:10.1016/s0304-3940(97)00384-4

  • Neurosci Lett. 1996 Feb 16;205(1):65-7. doi: 10.1016/0304-3940(96)12361-2.


    The present study describes ganglia and isolated neurones in the ovary of the Wistar rat, employing histological and histochemical techniques. Four kinds of ganglia in the postpubertal and young adult rat were identified: the mesovarial, hilar, medullary and cortical ganglia. Isolated neurones were also found, being dispersed along blood vessels in the ovary medulla and near the follicles. The soma diameters of these neuronal cells ranged from 25 to 50 microns. In the prepubertal rat, only the mesovarial and hilar ganglia were observed. They contained small neurones with soma diameters ranging from 10 to 15 microns. NADPH-diaphorase activity was detected in some isolated neurones and in the cortical and hilar ganglia in all rats examined.

    PMID:8867022 | DOI:10.1016/0304-3940(96)12361-2

  • Braz J Med Biol Res. 1996 Feb;29(2):249-58.


    Although N-acetylaspartylglutamate (NAAG) is one of the neuropeptides found in highest concentrations in the mammalian central nervous system, its functional role in neuronal signaling has not been definitively established. In some neuronal populations, NAAG is concentrated in nerve terminals and thus, it may play a role in the cytoplasmic events underlying neurotransmitter exocytosis. In the present study we have validated the use of the synthetic derivative NAAG-acetoxymethyl triester (NAAG.AM) as a tool to increase the intracellular levels of the peptide and assessed the ability of NAAG to regulate [3H]-dopamine ([3H]-DA) secretion in PC12 cells. Enzymatic degradation of NAAG.AM by nonspecific brain esterases resulted in the progressive formation of NAAG and succinimidyl-NAAG (Asu-NAAG). However, only 8% of NAAG.AM was converted to NAAG. Significant amounts of NAAG (1 nmol/mg protein) were demonstrable in cultures of the neuroblastoma cell line N2A following incubation with NAAG.AM for 2 h, with the concentration of (Asu)-NAAG being at least 100-fold higher. The pheochromocytoma cell line PC12 was used to assess the influence of loaded NAAG derivatives on [3H]-DA exocytosis. Incubation with 0.1-1 mM NAAG.AM did not affect the basal efflux or total content of [3H]-DA. However, it induced a dose-dependent decrease of [3H]-DA secretion in response to 56 mM KCl depolarization reaching an inhibition of 49% with 1 mM NAAG.AM. In contrast, NAAG.AM did not affect secretion induced by the calcium ionophore A23187 (100 microM). The present study validates the use of NAAG.AM as a tool to load NAAG derivatives into intact cells and provides preliminary evidence for an intracellular role of the peptide.


  • Toxicon. 1995 Jul;33(7):909-16. doi: 10.1016/0041-0101(95)00025-h.


    Fasciculin 2 (FAS), a potent acetylcholinesterase (AChE, EC inhibitory peptide with affinity for the enzyme in the nanomolar range was utilized together with two other AChE inhibitors (Paroxon and BW284c51) to study the role of AChE in central nervous system development. When drugs were intracisternally injected at postnatal days 3 and 5, only FAS showed a significant inhibition of hippocampus and striatum AChE (39% and 77% inhibition, respectively). After FAS treatment, animals showed convulsive behaviour which was blocked by subcutaneous pretreatment with atropine sulfate (10 mg/kg). An assessment of developmental indices showed no alteration in neurological reflex maturation, motor behaviour or cell morphology. Body weight gain was significantly lower only in FAS-treated animals compared to controls during the preweaning period. To investigate the specificity of this effect a synthetic loop of FAS (showing no activity in vitro or in vivo) and oxidized FAS (showing a weak inhibition in vitro and no activity in vivo) were also intracisternally injected. Animals injected with the loop showed normal body weight development while those treated with oxidized FAS showed impairment in body weight. In conclusion, FAS was the most potent drug at inhibiting neonatal AChE in vivo without nonspecific brain damage. Impairment in body weight seems to be dependent on AChE involvement, although the possibility of a direct FAS effect is discussed. These results point to FAS intracisternal treatment as a useful in vivo model to study the role of AChE in the critical period of early postnatal central nervous system development.

    PMID:8588215 | DOI:10.1016/0041-0101(95)00025-h

  • Electrophoresis. 1991 Jul-Aug;12(7-8):588-91. doi: 10.1002/elps.1150120720.


    Polypeptides having different net electric charges and very similar molecular weights, visualized as one single band in sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE), can be readily analyzed by an improved method combining two electrophoretic procedures. The methodology consists of the identification and isolation of selected protein bands from SDS-PAGE, their equilibration in an isoelectric focusing (IEF) sample buffer, and their casting and separation in an IEF flat-bed gel. This method requires no extra equipment, is highly reproducible, is suitable for quantitative and comparative studies, and is especially useful in the case of small samples. As a particular example, we analyze here the subunit composition of alpha-crystallins of young and embryonic quail lenses.

    PMID:1915250 | DOI:10.1002/elps.1150120720

  • Differentiation. 1990 Dec;45(3):161-7. doi: 10.1111/j.1432-0436.1990.tb00469.x.


    We have investigated the localization of basic fibroblast growth factor (bFGF) binding sites during the development of the neural retina in the chick embryo. The specificity of the affinity of bFGF for its receptors was assessed by competition experiments with unlabelled growth factor or with heparin, as well as by heparitinase treatment of the samples. Two different types of binding sites were observed in the neural retina by light-microscopic autoradiography. The first type, localized mainly to basement membranes, was highly sensitive to heparitinase digestion and to competition with heparin. It was not developmentally regulated. The second type of binding site, resistant to heparin competition, appeared to be associated with retinal cells from the earliest stages studied (3-day-old embryo, stages 21-22 of Hamburger and Hamilton). Its distribution was found to vary during embryonic development, paralleling layering of the neural retina. Binding of bFGF to the latter sites was observed throughout the retinal neuroepithelium at early stages but displayed a distinct pattern at the time when the inner and outer plexiform layers were formed. During the development of the inner plexiform layer, a banded pattern of bFGF binding was observed. These bands, lying parallel to the vitreal surface, seemed to codistribute with the synaptic bands existing in the inner plexiform layer. The presence of intra-retinal bFGF binding sites whose distribution varies with embryonic development suggests a regulatory mechanism involving differential actions of bFGF on neural retinal cells.

    PMID:2090518 | DOI:10.1111/j.1432-0436.1990.tb00469.x

  • J Neural Transm Gen Sect. 1990;80(3):167-79. doi: 10.1007/BF01245118.


    The presynaptic actions of the potassium channel blocker Dendrotoxin (DTX) on the Ca+2-dependent release of endogenous glutamate (GLU) and aspartate (ASP) have been tested in synaptosome-enriched preparations from rat striatum. 24 hours after the intrastriatal administration of DTX the K(+)-evoked release of GLU and ASP from the striatal synaptosomes was decreased by 40-45%. No changes in the total synaptosomal content of the amino acids were observed. Superfusion of immobilized synaptosomes with DTX or 4-amino-pyridine resulted in a dose-dependent increase in the basal outflow of GLU and ASP. The release of GLU stimulated by DTX was Ca+2-dependent and was not abolished by superfusing the synaptosomes with 50 microM D-ASP. Moreover, continuous superfusion of DTX (7 microM) to synaptosomes almost completely dumped the subsequent release of GLU and ASP stimulated by 20 mM K+. It is concluded that blockade of presynaptic K+ channels by DTX leads to a massive release of the transmitter pool of GLU (and possible also ASP) from isolated nerve terminals and to a depletion of the amino acid releasable pool.

    PMID:1970482 | DOI:10.1007/BF01245118

  • Toxicon. 1988;26(11):1009-15. doi: 10.1016/0041-0101(88)90199-7.


    Unilateral striatal injection of dendrotoxin (DTX), a polypeptide isolated from the venom of the snake Dendroaspis angusticeps, in rats provoked a complex behavioral syndrome characterized by spontaneous circling towards the contralateral side, stereotypic like chewing movements and gnawing, abnormal postures and convulsions. All these symptoms achieved their maximum on the first day, disappearing during the first week after injection. Neurochemical analyses of striatal monoamines and monoamine metabolites showed a significant increase of dopamine and serotonin metabolites 20 hr after DTX injection. A group of animals sacrificed 15 days after toxin administration showed normal levels of monoamines and their metabolites, except for homovanillic acid levels which were still significantly increased. These data indicate that monoamines are involved in the behavioral syndrome elicited by DTX and are possibly related to its excitatory effect upon brain structures in vivo.

    PMID:3245048 | DOI:10.1016/0041-0101(88)90199-7