Humanin, a mitochondrial-derived peptide released by astrocytes, prevents synapse loss in hippocampal neurons

Astroglial cells are crucial for central nervous system (CNS) homeostasis. They undergo complex morpho-functional changes during aging and in response to hormonal milieu. Ovarian hormones positively affect different astroglia parameters, including regulation of cell morphology and release of neurotrophic and neuroprotective factors. Thus, ovarian hormone loss during menopause has profound impact in astroglial pathophysilogy and has been widely associated to the process of brain aging. Humanin (HN) is a secreted mitochondrial-encoded peptide with neuroprotective effects. It is localized in several tissues with high metabolic rate and its expression decreases with age. In the brain, humanin has been found in glial cells in physiological conditions. We previously reported that surgical menopause induces hippocampal mitochondrial dysfunction that mimics an aging phenotype. However, the effect of ovarian hormone deprivation on humanin expression in this area has not been studied. Also, whether astrocytes express and release humanin and the regulation of such processes by ovarian hormones remain elusive. Although humanin has also proven to be beneficial in ameliorating cognitive impairment induced by different insults, its putative actions on structural synaptic plasticity have not been fully addressed. In a model of surgical menopause in rats, we studied hippocampal humanin expression and localization by real-time quantitative polymerase chain reaction (RT-qPCR) and double immunohistochemistry, respectively. Humanin production and release and ovarian hormone regulation of such processes were studied in cultured astrocytes by flow cytometry and ELISA, respectively. Humanin effects on glutamate-induced structural synaptic alterations were determined in primary cultures of hippocampal neurons by immunocytochemistry. Humanin expression was lower in the hippocampus of ovariectomized rats and its immunoreactivity colocalized with astroglial markers. Chronic ovariectomy also promoted the presence of less complex astrocytes in this area. Ovarian hormones increased humanin intracellular content and release by cultured astrocytes. Humanin prevented glutamate-induced dendritic atrophy and reduction in puncta number and total puncta area for pre-synaptic marker synaptophysin in cultured hippocampal neurons. In conclusion, astroglial functional and morphological alterations induced by chronic ovariectomy resemble an aging phenotype and could affect astroglial support to neuronal function by altering synaptic connectivity and functionality. Reduced astroglial-derived humanin may represent an underlying mechanism for synaptic dysfunction and cognitive decline after menopause.Fil: Zarate, Sandra Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires.Facultad de Medicina. Departamento de Histología, Embriología, Biología Celular y Genética; ArgentinaFil: Traetta, Marianela Evelyn. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Codagnone, Martín Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Seilicovich, Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentina. Universidad de Buenos Aires.Facultad de Medicina. Departamento de Histología, Embriología, Biología Celular y Genética; ArgentinaFil: Reines, Analia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Humanin, a Mitochondrial-Derived Peptide Released by Astrocytes, Prevents Synapse Loss in Hippocampal Neurons

Astroglial cells are crucial for central nervous system (CNS) homeostasis. They undergo complex morpho-functional changes during aging and in response to hormonal milieu. Ovarian hormones positively affect different astroglia parameters, including regulation of cell morphology and release of neurotrophic and neuroprotective factors. Thus, ovarian hormone loss during menopause has profound impact in astroglial pathophysilogy and has been widely associated to the process of brain aging. Humanin (HN) is a secreted mitochondrial-encoded peptide with neuroprotective effects. It is localized in several tissues with high metabolic rate and its expression decreases with age. In the brain, humanin has been found in glial cells in physiological conditions. We previously reported that surgical menopause induces hippocampal mitochondrial dysfunction that mimics an aging phenotype. However, the effect of ovarian hormone deprivation on humanin expression in this area has not been studied. Also, whether astrocytes express and release humanin and the regulation of such processes by ovarian hormones remain elusive. Although humanin has also proven to be beneficial in ameliorating cognitive impairment induced by different insults, its putative actions on structural synaptic plasticity have not been fully addressed. In a model of surgical menopause in rats, we studied hippocampal humanin expression and localization by real-time quantitative polymerase chain reaction (RT-qPCR) and double immunohistochemistry, respectively. Humanin production and release and ovarian hormone regulation of such processes were studied in cultured astrocytes by flow cytometry and ELISA, respectively. Humanin effects on glutamate-induced structural synaptic alterations were determined in primary cultures of hippocampal neurons by immunocytochemistry. Humanin expression was lower in the hippocampus of ovariectomized rats and its immunoreactivity colocalized with astroglial markers. Chronic ovariectomy also promoted the presence of less complex astrocytes in this area. Ovarian hormones increased humanin intracellular content and release by cultured astrocytes. Humanin prevented glutamate-induced dendritic atrophy and reduction in puncta number and total puncta area for pre-synaptic marker synaptophysin in cultured hippocampal neurons. In conclusion, astroglial functional and morphological alterations induced by chronic ovariectomy resemble an aging phenotype and could affect astroglial support to neuronal function by altering synaptic connectivity and functionality. Reduced astroglial-derived humanin may represent an underlying mechanism for synaptic dysfunction and cognitive decline after menopause

Hippocampal neurons isolated from rats subjected to the valproic acid model mimic in vivo synaptic pattern: evidence of neuronal priming during early development in autism spectrum disorders

Background: Autism spectrum disorders (ASD) are synaptopathies characterized by area-specific synaptic alterations and neuroinflammation. Structural and adhesive features of hippocampal synapses have been described in the valproic acid (VPA) model. However, neuronal and microglial contribution to hippocampal synaptic pattern and its time-course of appearance is still unknown. Methods: Male pups born from pregnant rats injected at embryonic day 10.5 with VPA (450 mg/kg, i.p.) or saline (control) were used. Maturation, exploratory activity and social interaction were assessed as autistic-like traits. Synaptic, cell adhesion and microglial markers were evaluated in the CA3 hippocampal region at postnatal day (PND) 3 and 35. Primary cultures of hippocampal neurons from control and VPA animals were used to study synaptic features and glutamate-induced structural remodeling. Basal and stimuli-mediated reactivity was assessed on microglia primary cultures isolated from control and VPA animals. Results: At PND3, before VPA behavioral deficits were evident, synaptophysin immunoreactivity and the balance between the neuronal cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) were preserved in the hippocampus of VPA animals along with the absence of microgliosis. At PND35, concomitantly with the establishment of behavioral deficits, the hippocampus of VPA rats showed fewer excitatory synapses and increased NCAM/PSA-NCAM balance without microgliosis. Hippocampal neurons from VPA animals in culture exhibited a preserved synaptic puncta number at the beginning of the synaptogenic period in vitro but showed fewer excitatory synapses as well as increased NCAM/PSA-NCAM balance and resistance to glutamate-induced structural synaptic remodeling after active synaptogenesis. Microglial cells isolated from VPA animals and cultured in the absence of neurons showed similar basal and stimuli-induced reactivity to the control group. Results indicate that in the absence of glia, hippocampal neurons from VPA animals mirrored the in vivo synaptic pattern and suggest that while neurons are primed during the prenatal period, hippocampal microglia are not intrinsically altered. Conclusions: Our study suggests microglial role is not determinant for developing neuronal alterations or counteracting neuronal outcome in the hippocampus and highlights the crucial role of hippocampal neurons and structural plasticity in the establishment of the synaptic alterations in the VPA rat model.Fil: Traetta, Marianela Evelyn. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Codagnone, Martín Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Uccelli, Nonthué Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Ramos, Alberto Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Zarate, Sandra Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Reines, Analia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Mild VPA behavioral phenotype in female rats: Evidence of structural synapse remodeling in the medial prefrontal cortex and the hippocampus

Autism spectrum disorders (ASD) are a group of severe neurodevelopmental disabilities of unknown etiology, characterized by social interaction deficits and increased stereotyped behaviors. Although ASD incidence is four times higher in boys than in girls, sex differences have not been clarified. The rat model of autism induced by prenatal exposure to valproic acid (VPA) has been characterized in males (MVPA), while female rat VPA (FVPA) phenotype is still controversial. The aim of this work was to further characterize the behavioral profile of FVPA and explore structural synapse markers, cell adhesion molecules and microglia morphology in the medial prefrontal cortex (mPFC) and the hippocampus. At early postnatal days (PND)7-15, and similar to MVPA, FVPA showed growth and maturation deficits: delayed eye opening, lower body weight, altered negative geotaxis, higher latencies to nest seeking response and a deficit in swimming performance. At PND30-35, like MVPA, FVPA showed a reduced number of interactions in the social play behavior test, but they exhibited distinctive pinning features. Contrary to MVPA that showed an exploratory deficit and increased stereotypical activities, FVPA matched control female rat behavior. Notably, at PND35, mPFC of FVPA and MVPA showed an increase in synaptophysin (SYN) and neural cell adhesion molecule (NCAM) and a similar ramified/unramified microglia (Iba+) ratio. However, the polysialylated form of NCAM (PSA-NCAM) was increased in FVPA but decreased in MVPA. In the hippocampus, both FVPA and MVPA showed reduced SYN labeling and increased NCAM but only FVPA displayed a higher proportion of unramified microglia. Also, PSA-NCAM levels were preserved in FVPA but reduced in MVPA. To sum up, FVPA exhibit a mild behavioral phenotype accompanied with a distinctive microglia profile and NCAM/PSA-NCAM ratio that may facilitate structural synapse remodeling and plasticity.Fil: Litvak, Einav Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Traetta, Marianela Evelyn. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Codagnone, Martín Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Malleville Corpa, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Uccelli, Nonthué Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Reines, Analia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaLXV Reunión Anual de la Sociedad Argentina de Investigación Clínica; LXVIII Reunión Anual de la Sociedad Argentina de Inmunología y Reunión Anual de la Sociedad Argentina de Fisiología.VirtualArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de InmunologíaSociedad Argentina de Fisiologí

Long-lasting changes in glial cells isolated from rats subjected to the valproic acid model of autism spectrum disorder.

Synaptic alterations concomitant with neuroinflammation have been described in patients and experimental models of autism spectrum disorder (ASD). However, the role of microglia and astroglia in relation to synaptic changes is poorly understood. Male Wistar rats prenatally exposed to valproic acid (VPA, 450 mg/kg, i.p.) or saline (control) at embryonic day 10.5 were used to study synapses, microglia, and astroglia in the prefrontal cortex (PFC) at postnatal days 3 and 35 (PND3 and PND35). Primary cultures of cortical neurons, microglia, and astroglia isolated from control and VPA animals were used to study each cell type individually, neuron-microglia and microglia-astroglia crosstalk. In the PFC of VPA rats, synaptic changes characterized by an increase in the number of excitatory synapses were evidenced at PND3 and persisted until PND35. At PND3, microglia and astroglia from VPA animals were morphologically similar to those of age-matched controls, whereas at PND35, reactive microgliosis and astrogliosis were observed in the PFC of VPA animals. Cortical neurons isolated from VPA rats mimicked in vitro the synaptic pattern seen in vivo. Cortical microglia and astroglia isolated from VPA animals exhibited reactive morphology, increased pro-inflammatory cytokines, and a compromised miRNA processing machinery. Microglia from VPA animals also showed resistance to a phagocytic challenge. In the presence of neurons from VPA animals, microglia isolated from VPA rats revealed a non-reactive morphology and promoted neurite outgrowth, while microglia from control animals displayed a reactive profile and promoted dendritic retraction. In microglia-astroglia co-cultures, microglia from VPA animals displayed a reactive profile and exacerbated astrocyte reactivity. Our study indicates that cortical microglia from VPA animals are insensitive or adapted to neuronal cues expressed by neurons from VPA animals. Further, long-term in vivo microgliosis could be the result of altered microglia-astroglia crosstalk in VPA animals. Thus, our study highlights cortical microglia-astroglia communication as a new mechanism implicated in neuroinflammation in ASD; consequently, we propose that this crosstalk is a potential target for interventions in this disorder.Fil: Traetta, Marianela Evelyn. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Uccelli, Nonthué Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Zarate, Sandra Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; ArgentinaFil: Gomez Cuautle, Jose Dante Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Ramos, Alberto Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Reines, Analia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Neurobiological substrates underlying corpus callosum hypoconnectivity and brain metabolic patterns in the valproic acid rat model of autism spectrum disorder

Atypical connectivity between brain regions and altered structure of the corpus callosum (CC) in imaging studies supports the long-distance hypoconnectivity hypothesis proposed for autism spectrum disorder (ASD). The aim of this study was to unveil the CC ultrastructural and cellular changes employing the valproic acid (VPA) rat model of ASD. Male Wistar rats were exposed to VPA (450 mg/kg i.p.) or saline (control) during gestation (embryonic day 10.5), and maturation, exploration, and social behavior were subsequently tested. Myelin content, ultrastructure, and oligodendroglial lineage were studied in the CC at post-natal days 15 (infant) and 36 (juvenile). As a functional outcome, brain metabolic activity was determined by positron emission tomography. Concomitantly with behavioral deficits in juvenile VPA rats, the CC showed reduced myelin basic protein, conserved total number of axons, reduced percentage of myelinated axons, and aberrant and less compact arrangements of myelin sheath ultrastructure. Mature oligodendrocytes decreased and oligodendrocyte precursors increased in the absence of astrogliosis or microgliosis. In medial prefrontal and somatosensory cortices of juvenile VPA rats, myelin ultrastructure and oligodendroglial lineage were preserved. VPA animals exhibited global brain hypometabolism and local hypermetabolism in brain regions relevant for ASD. In turn, the CC of infant VPA rats showed reduced myelin content but preserved oligodendroglial lineage. Our findings indicate that CC hypomyelination is established during infancy and prior to oligodendroglial pattern alterations, which suggests that axon–oligodendroglia communication could be compromised in VPA animals. Thus, CC hypomyelination may underlie white matter alterations and contribute to atypical patterns of connectivity and metabolism found in ASD.Fil: Uccelli, Nonthué Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Codagnone, Martín Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Traetta, Marianela Evelyn. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; ArgentinaFil: Levanovich, Nadia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Rosato Siri, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Urrutia, Leandro. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Falasco, Germán Alfredo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Vázquez, Silvia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Pasquini, Juana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Reines, Analia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología; Argentin