Publicaciones por año
Publicaciones del Departamento de Histología y Embriología
A nuclear fluorescent dye identifies pericytes at the neurovascular unit
J Neurochem 2021 157(4):1377-1391
Sandra P Mai-Morente 1 , Virginia M Marset 1 , Fabiana Blanco 2 , Eugenia E Isasi 3 , Verónica Abudara 1
1 Departamento de Fisiología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 2 Departamento de Biofísica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 3 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
DOI: 10.1111/jnc.15193
PMID: 32974913
Pubmed: https://pubmed.ncbi.nlm.nih.gov/32974913
Texto completo: https://doi.org/10.1111/jnc.15193
Abstract:
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.
Glial Metabolic Reprogramming in Amyotrophic Lateral Sclerosis
Neuroimmunomodulation 2021 28(4):204-212
Patricia Cassina 1 2 , Ernesto Miquel 1 2 , Laura Martínez-Palma 1 2 , Adriana Cassina 2 3
1 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 2 Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 3 Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
DOI: 10.1159/000516926
PMID: 34175843
Pubmed: https://pubmed.ncbi.nlm.nih.gov/34175843
Texto completo: https://doi.org/10.1159/000516926
Abstract:
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.
Sex-dependent role of CD300f immune receptor in generalized anxiety disorder
Brain Behav Immun Health 2020 11:100191
Fernanda N Kaufmann 1 , Natalia Lago 2 , Daniela Alí-Ruiz 2 , Karen Jansen 3 , Luciano D M Souza 3 , Ricardo A Silva 3 , Diogo R Lara 4 , Gabriele Ghisleni 3 , Hugo Peluffo 2 5 , Manuella P Kaster 6
1 Department of Psychiatry and Neuroscience, Faculty of Medicine and CERVO Brain Research Center, Université Laval, Quebec City, Canada. 2 Neuroinflammation and Gene Therapy Laboratory, Institut Pasteur de Montevideo, Montevideo, Uruguay. 3 Department of Life and Health Sciences, Catholic University of Pelotas, Rio Grande do Sul, Brazil. 4 Department of Cellular and Molecular Biology, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil. 5 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 6 Department of Biochemistry, Federal University of Santa Catarina, Florianópolis, Santa Catarina, Brazil.
DOI: 10.1016/j.bbih.2020.100191
PMID: 34589728
Pubmed: https://pubmed.ncbi.nlm.nih.gov/34589728
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/S2666-3546(20)30156-3
Abstract:
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.
Mitochondrial bioenergetics, glial reactivity, and pain-related behavior can be restored by dichloroacetate treatment in rodent pain models
Pain 2020 161(12):2786-2797
Valentina Lagos-Rodríguez 1 , Laura Martínez-Palma 1 , Soledad Marton 1 , Ernesto Miquel 1 , Ricardo Escobar-Pintos 1 , Adriana Cassina 2 , Natalia Lago 3 , Patricia Cassina 1
1 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 2 Departamento de Bioquímica, Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 3 Neuroinflammation and Gene Therapy Laboratory, Institut Pasteur de Montevideo, Montevideo, Uruguay.
DOI: 10.1097/j.pain.0000000000001992
PMID: 32658145
Pubmed: https://pubmed.ncbi.nlm.nih.gov/32658145
Texto completo: https://doi.org/10.1097/j.pain.0000000000001992
Abstract:
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.
Reorganization of perineuronal nets in the medial Preoptic Area during the reproductive cycle in female rats
Sci Rep 2020 10(1):5479
Natalia Uriarte 1 , Marcela Ferreño 1 , Diego Méndez 2 , Javier Nogueira 3
1 Laboratorio de Neurociencias, Facultad de Ciencias, Universidad de la República, Montevideo, 11400, Uruguay. 2 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, 11800, Uruguay. 3 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, 11800, Uruguay. nogueira@fmed.edu.uy.
DOI: 10.1038/s41598-020-62163-z
PMID: 32214157
Pubmed: https://pubmed.ncbi.nlm.nih.gov/32214157
Texto completo: https://doi.org/10.1038/s41598-020-62163-z
Abstract:
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.
CD300f immunoreceptor is associated with major depressive disorder and decreased microglial metabolic fitness
Proc Natl Acad Sci U S A 2020 117(12):6651-6662
Natalia Lago 1 , Fernanda N Kaufmann 2 , María Luciana Negro-Demontel 1 3 , Daniela Alí-Ruiz 1 , Gabriele Ghisleni 4 , Natalia Rego 5 , Andrea Arcas-García 6 , Nathalia Vitureira 1 7 , Karen Jansen 4 , Luciano M Souza 4 , Ricardo A Silva 4 , Diogo R Lara 8 , Bruno Pannunzio 1 3 , Juan Andrés Abin-Carriquiry 9 , Jesús Amo-Aparicio 10 , Celia Martin-Otal 6 , Hugo Naya 6 , Dorian B McGavern 11 , Joan Sayós 6 , Rubèn López-Vales 10 , Manuella P Kaster 2 , Hugo Peluffo 12 3
1 Neuroinflammation and Gene Therapy Laboratory, Institut Pasteur de Montevideo, 11400 Montevideo, Uruguay. 2 Department of Biochemistry, Federal University of Santa Catarina, Florianópolis, 88040-900 Santa Catarina, Brazil. 3 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, 11200 Montevideo, Uruguay. 4 Department of Life and Health Sciences, Catholic University of Pelotas, 96015-560 Rio Grande do Sul, Brazil. 5 Bioinformatics Unit, Institut Pasteur de Montevideo, 11400 Montevideo, Uruguay. 6 Immune Regulation and Immunotherapy Group, CIBBIM-Nanomedicine, Vall d'Hebrón Institut de Recerca, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain. 7 Department of Physiology, Facultad de Medicina, Universidad de la República, 11200 Montevideo, Uruguay. 8 Department of Cellular and Molecular Biology, Pontifical Catholic University of Rio Grande do Sul, 90619-900 Porto Alegre, Brazil. 9 Instituto de Investigaciones Biológicas Clemente Estable, 11600 Montevideo, Uruguay. 10 Departament de Biologia Cel·lular, Fisiologia i Immunologia, Institut de Neurociències, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. 11 Viral Immunology and Intravital Imaging Section, National Institute for Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892. 12 Neuroinflammation and Gene Therapy Laboratory, Institut Pasteur de Montevideo, 11400 Montevideo, Uruguay; hugo.peluffo@pasteur.edu.uy.
DOI: 10.1073/pnas.1911816117
PMID: 32152116
Pubmed: https://pubmed.ncbi.nlm.nih.gov/32152116
Texto completo: https://www.pnas.org/doi/full/10.1073/pnas.1911816117
Abstract:
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.
Signaling pathways in cytoskeletal responses to plasma membrane depolarization in corneal endothelial cells
J Cell Physiol 2020 235(3):2947-2962
Frances Evans 1 , Julio A Hernández 2 , Silvia Chifflet 3
1 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 2 Sección Biofísica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay. 3 Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
DOI: 10.1002/jcp.29200
PMID: 31535377
Pubmed: https://pubmed.ncbi.nlm.nih.gov/31535377
Texto completo: https://doi.org/10.1002/jcp.29200
Abstract:
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.
Signaling pathways in cytoskeletal responses to plasma membrane depolarization in corneal endothelial cells
J Cell Physiol 2020 235(3):2947-2962
Frances Evans 1 , Julio A Hernández 2 , Silvia Chifflet 3
1 Departamento de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. 2 Sección Biofísica, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay. 3 Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
DOI: 10.1002/jcp.29200
PMID: 31535377
Pubmed: https://pubmed.ncbi.nlm.nih.gov/31535377
Texto completo: https://doi.org/10.1002/jcp.29200
Abstract:
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.
Spotlight edition on South America
Gene Ther 2020 27(1-2):1
Ursula Matte 1 , Hugo Peluffo 2
1 Post-Graduation Program on Genetics and Molecular Biology, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, 2350, Porto Alegre, 90035-903, Brazil. umatte@hcpa.edu.br. 2 Department of Histology and Embryology, Facultad de Medicina, Universidad de la República, Mataojo 2020, Montevideo, 11400, Uruguay.
DOI: 10.1038/s41434-020-0129-9
PMID: 32099107
Pubmed: https://pubmed.ncbi.nlm.nih.gov/32099107
Texto completo: https://doi.org/10.1038/s41434-020-0129-9
Abstract:
Safe and neuroprotective vectors for long-term traumatic brain injury gene therapy
Gene Ther 2020 27(1-2):96-103
Daniela Blanco-Ocampo 1 2 , Fabio Andrés Cawen 3 , Luis Angel Álamo-Pindado 1 3 , María Luciana Negro-Demontel 1 3 , Hugo Peluffo 4 5
1 Department of Histology and Embryology, Faculty of Medicine, Universidad de la República, Montevideo, Uruguay. 2 Department of Physiopathology, Faculty of Medicine, Universidad de la República, Montevideo, Uruguay. 3 Neuroinflammation and Gene Therapy Laboratory, Institut Pasteur de Montevideo, Montevideo, Uruguay. 4 Department of Histology and Embryology, Faculty of Medicine, Universidad de la República, Montevideo, Uruguay. hugo.peluffo@pasteur.edu.uy. 5 Neuroinflammation and Gene Therapy Laboratory, Institut Pasteur de Montevideo, Montevideo, Uruguay. hugo.peluffo@pasteur.edu.uy.
DOI: 10.1038/s41434-019-0073-8
PMID: 30926962
Pubmed: https://pubmed.ncbi.nlm.nih.gov/30926962
Texto completo: https://doi.org/10.1038/s41434-019-0073-8
Abstract:
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.