Browsing by Author "van Zundert, Brigitte"

Now showing 1 - 9 of 9
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    Astroglial gliotransmitters released via Cx43 hemichannels regulate NMDAR-dependent transmission and short-term fear memory in the basolateral amygdala
    (2022) Linsambarth, Sergio; Carvajal Cachaña, Francisco Javier; Moraga Amaro, Rodrigo; Mendez, Luis; Tamburini, Giovanni; Jimenez, Ivanka; Antonio Verdugo, Daniel; Gomez, Gonzalo, I; Jury, Nur; Martinez, Pablo; van Zundert, Brigitte; Varela Nallar, Lorena; Retamal, Mauricio A.; Martin, Claire; Altenberg, Guillermo A.; Fiori, Mariana C.; Cerpa Nebott, Waldo Francisco; Orellana Roca, Juan Andrés; Stehberg, Jimmy
    Astrocytes release gliotransmitters via connexin 43 (Cx43) hemichannels into neighboring synapses, which can modulate synaptic activity and are necessary for fear memory consolidation. However, the gliotransmitters released, and their mechanisms of action remain elusive. Here, we report that fear conditioning training elevated Cx43 hemichannel activity in astrocytes from the basolateral amygdala (BLA). The selective blockade of Cx43 hemichannels by microinfusion of TAT-Cx43L2 peptide into the BLA induced memory deficits 1 and 24 h after training, without affecting learning. The memory impairments were prevented by the co-injection of glutamate and D-serine, but not by the injection of either alone, suggesting a role for NMDA receptors (NMDAR). The incubation with TAT-Cx43L2 decreased NMDAR-mediated currents in BLA slices, effect that was also prevented by the addition of glutamate and D-serine. NMDARs in primary neuronal cultures were unaffected by TAT-Cx43L2, ruling out direct effects of the peptide on NMDARs. Finally, we show that D-serine permeates through purified Cx43 hemichannels reconstituted in liposomes. We propose that the release of glutamate and D-serine from astrocytes through Cx43 hemichannels is necessary for the activation of post-synaptic NMDARs during training, to allow for the formation of short-term and subsequent long-term memory, but not for learning per se.
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    c-Abl Phosphorylates MFN2 to Regulate Mitochondrial Morphology in Cells under Endoplasmic Reticulum and Oxidative Stress, Impacting Cell Survival and Neurodegeneration
    (De Gruyter, 2023) Martinez Saavedra, Alexis; Lamaizon Muñoz, Cristián Nicolás; Valls Jimenez, Cristián; Llambi, Fabien; Leal Reyes, Nancy Valeria; Fitzgerald, Patrick; Guy, Cliff; Kaminsk,i Marcin M.; Inestrosa Cantin, Nibaldo; van Zundert, Brigitte; Cancino, Gonzalo; Dulcey, Andrés E.; Zanlungo Matsuhiro, Silvana; Marugan, Juan J.; Hetz, Claudio; Green, Douglas R.; Alvarez Rojas, Alejandra
    The endoplasmic reticulum is a subcellular organelle key in the control of synthesis, folding, and sorting of proteins. Under endoplasmic reticulum stress, an adaptative unfolded protein response is activated; however, if this activation is prolonged, cells can undergo cell death, in part due to oxidative stress and mitochondrial fragmentation. Here, we report that endoplasmic reticulum stress activates c-Abl tyrosine kinase, inducing its translocation to mitochondria. We found that endoplasmic reticulum stress-activated c-Abl interacts with and phosphorylates the mitochondrial fusion protein MFN2, resulting in mitochondrial fragmentation and apoptosis. Moreover, the pharmacological or genetic inhibition of c-Abl prevents MFN2 phosphorylation, mitochondrial fragmentation, and apoptosis in cells under endoplasmic reticulum stress. Finally, in the amyotrophic lateral sclerosis mouse model, where endoplasmic reticulum and oxidative stress has been linked to neuronal cell death, we demonstrated that the administration of c-Abl inhibitor neurotinib delays the onset of symptoms. Our results uncovered a function of c-Abl in the crosstalk between endoplasmic reticulum stress and mitochondrial dynamics via MFN2 phosphorylation.
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    c-Abl Phosphorylates MFN2 to Regulate Mitochondrial Morphology in Cells under Endoplasmic Reticulum and Oxidative Stress, Impacting Cell Survival and Neurodegeneration
    (MDPI, 2023) Martinez Saavedra, Alexis; Lamaizon Muñoz, Cristián Nicolás; Valls Jimenez, Cristián; Llambi, Fabien; Leal Reyes, Nancy Valeria; Fitzgerald, Patrick; Guy, Cliff; Kaminsk,i Marcin M.; Inestrosa Cantin, Nibaldo; van Zundert, Brigitte; Cancino, Gonzalo; Dulcey, Andrés E.; Zanlungo Matsuhiro, Silvana; Marugan, Juan J.; Hetz, Claudio; Green, Douglas R.; Alvarez Rojas, Alejandra
    The endoplasmic reticulum is a subcellular organelle key in the control of synthesis, folding, and sorting of proteins. Under endoplasmic reticulum stress, an adaptative unfolded protein response is activated; however, if this activation is prolonged, cells can undergo cell death, in part due to oxidative stress and mitochondrial fragmentation. Here, we report that endoplasmic reticulum stress activates c-Abl tyrosine kinase, inducing its translocation to mitochondria. We found that endoplasmic reticulum stress-activated c-Abl interacts with and phosphorylates the mitochondrial fusion protein MFN2, resulting in mitochondrial fragmentation and apoptosis. Moreover, the pharmacological or genetic inhibition of c-Abl prevents MFN2 phosphorylation, mitochondrial fragmentation, and apoptosis in cells under endoplasmic reticulum stress. Finally, in the amyotrophic lateral sclerosis mouse model, where endoplasmic reticulum and oxidative stress has been linked to neuronal cell death, we demonstrated that the administration of c-Abl inhibitor neurotinib delays the onset of symptoms. Our results uncovered a function of c-Abl in the crosstalk between endoplasmic reticulum stress and mitochondrial dynamics via MFN2 phosphorylation.
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    Epigenetic silencing of the osteoblast-lineage gene program during hippocampal maturation
    (2021) Aguilar, Rodrigo; Bustos, Fernando J.; Nardocci, Gino; van Zundert, Brigitte; Montecino, Martin
    Accumulating evidence indicates that epigenetic control of gene expression plays a significant role during cell lineage commitment and subsequent cell fate maintenance. Here, we assess epigenetic mechanisms operating in the rat brain that mediate silencing of genes that are expressed during early and late stages of osteogenesis. We report that repression of the osteoblast master regulator Sp7 in embryonic (E18) hippocampus is mainly mediated through the Polycomb complex PRC2 and its enzymatic product H3K27me3. During early postnatal (P10), juvenile (P30), and adult (P90) hippocampal stages, the repressive H3K27me3 mark is progressively replaced by nucleosome enrichment and increased CpG DNA methylation at the Sp7 gene promoter. In contrast, silencing of the late bone phenotypic Bglap gene in the hippocampus is PRC2-independent and accompanied by strong CpG methylation from E18 through postnatal and adult stages. Forced ectopic expression of the primary master regulator of osteogenesis Runx2 in embryonic hippocampal neurons activates the expression of its downstream target Sp7 gene. Moreover, transcriptomic analyses show that several genes associated with the mesenchymal-osteogenic lineages are transcriptionally activated in these hippocampal cells that express Runx2 and Sp7. This effect is accompanied by a loss in neuronal properties, including a significant reduction in secondary processes at the dendritic arbor and reduced expression of critical postsynaptic genes like PSD95. Together, our results reveal a developmental progression in epigenetic control mechanisms that repress the expression of the osteogenic program in hippocampal neurons at embryonic, postnatal, and adult stages.
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    Excessive release of inorganic polyphosphate by ALS/FTD astrocytes causes non-cell-autonomous toxicity to motoneurons
    (2022) Arredondo, Cristian; Cefaliello, Carolina; Dyrda, Agnieszka; Jury, Nur; Martinez, Pablo; Diaz, Ivan; Amaro, Armando; Tran, Helene; Morales, Danna; Pertusa, Maria; Stoica, Lorelei; Fritz, Elsa; Corvalan, Daniela; Abarzua, Sebastian; Mendez-Ruette, Maxs; Fernandez, Paola; Rojas, Fabiola; Kumar, Meenakshi Sundaram; Aguilar, Rodrigo; Almeida, Sandra; Weiss, Alexandra; Bustos, Fernando J.; Gonzalez-Nilo, Fernando; Otero, Carolina; Tevy, Maria Florencia; Bosco, Daryl A.; Saez, Juan C.; Kahne, Thilo; Gao, Fen-Biao; Berry, James D.; Nicholson, Katharine; Sena-Esteves, Miguel; Madrid, Rodolfo; Varela, Diego; Montecino, Martin; Brown, Robert H.; van Zundert, Brigitte
    Non-cell-autonomous mechanisms contribute to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), in which astrocytes release unidentified factors that are toxic to motoneurons (MNs). We report here that mouse and patient iPSC-derived astrocytes with diverse ALS/FTD-linked mutations (SOD1, TARDBP, and C9ORF72) display elevated levels of intracellular inorganic polyphosphate (polyP), a ubiquitous, negatively charged biopolymer. PolyP levels are also increased in astrocyte-conditioned media (ACM) from ALS/FTD astrocytes. ACM-mediated MN death is prevented by degrading or neutralizing polyP in ALS/FTD astrocytes or ACM. Studies further reveal that postmortem familial and sporadic ALS spinal cord sections display enriched polyP staining signals and that ALS cerebrospinal fluid (CSF) exhibits increased polyP concentrations. Our in vitro results establish excessive astrocyte-derived polyP as a critical factor in non-cell-autonomous MN degeneration and a potential therapeutic target for ALS/ FTD. The CSF data indicate that polyP might serve as a new biomarker for ALS/FTD.
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    H3K9 Methyltransferases Suv39h1 and Suv39h2 Control the Differentiation of Neural Progenitor Cells in the Adult Hippocampus
    (2022) Guerra, Miguel V.; Caceres, Matias I.; Herrera-Soto, Andrea; Arredondo, Sebastian B.; Varas-Godoy, Manuel; van Zundert, Brigitte; Varela-Nallar, Lorena
    In the dentate gyrus of the adult hippocampus new neurons are generated from neural precursor cells through different stages including proliferation and differentiation of neural progenitor cells and maturation of newborn neurons. These stages are controlled by the expression of specific transcription factors and epigenetic mechanisms, which together orchestrate the progression of the neurogenic process. However, little is known about the involvement of histone posttranslational modifications, a crucial epigenetic mechanism in embryonic neurogenesis that regulates fate commitment and neuronal differentiation. During embryonic development, the repressive modification trimethylation of histone H3 on lysine 9 (H3K9me3) contributes to the cellular identity of different cell-types. However, the role of this modification and its H3K9 methyltransferases has not been elucidated in adult hippocampal neurogenesis. We determined that during the stages of neurogenesis in the adult mouse dentate gyrus and in cultured adult hippocampal progenitors (AHPs), there was a dynamic change in the expression and distribution of H3K9me3, being enriched at early stages of the neurogenic process. A similar pattern was observed in the hippocampus for the dimethylation of histone H3 on lysine 9 (H3K9me2), another repressive modification. Among H3K9 methyltransferases, the enzymes Suv39h1 and Suv39h2 exhibited high levels of expression at early stages of neurogenesis and their expression decreased upon differentiation. Pharmacological inhibition of these enzymes by chaetocin in AHPs reduced H3K9me3 and concomitantly decreased neuronal differentiation while increasing proliferation. Moreover, Suv39h1 and Suv39h2 knockdown in newborn cells of the adult mouse dentate gyrus by retrovirus-mediated RNA interference impaired neuronal differentiation of progenitor cells. Our results indicate that H3K9me3 and H3K9 methyltransferases Suv39h1 and Suv39h2 are critically involved in the regulation of adult hippocampal neurogenesis by controlling the differentiation of neural progenitor cells.
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    Protein kinase B (AKT) upregulation and Thy‑1‑αvβ3 integrin‑induced phosphorylation of Connexin43 by activated AKT in astrogliosis
    (2023) Pérez-Núñez, Ramón; Chamorro, Alejandro; González, María F.; Contreras, Pamela; Artigas Barrenechea, Rocío; Corvalán R., Alejandro; van Zundert, Brigitte; Reyes, Christopher; Moya, Pablo R.; Avalos, Ana M.; Schneider, Pascal; Quest, Andrew F. G.; Leyton, Lisette
    Background: In response to brain injury or inflammation, astrocytes undergo hypertrophy, proliferate, and migrate to the damaged zone. These changes, collectively known as "astrogliosis", initially protect the brain; however, astrogliosis can also cause neuronal dysfunction. Additionally, these astrocytes undergo intracellular changes involving alterations in the expression and localization of many proteins, including αvβ3 integrin. Our previous reports indicate that Thy-1, a neuronal glycoprotein, binds to this integrin inducing Connexin43 (Cx43) hemichannel (HC) opening, ATP release, and astrocyte migration. Despite such insight, important links and molecular events leading to astrogliosis remain to be defined. Methods: Using bioinformatics approaches, we analyzed different Gene Expression Omnibus datasets to identify changes occurring in reactive astrocytes as compared to astrocytes from the normal mouse brain. In silico analysis was validated by both qRT-PCR and immunoblotting using reactive astrocyte cultures from the normal rat brain treated with TNF and from the brain of a hSOD1G93A transgenic mouse model. We evaluated the phosphorylation of Cx43 serine residue 373 (S373) by AKT and ATP release as a functional assay for HC opening. In vivo experiments were also performed with an AKT inhibitor (AKTi). Results: The bioinformatics analysis revealed that genes of the PI3K/AKT signaling pathway were among the most significantly altered in reactive astrocytes. mRNA and protein levels of PI3K, AKT, as well as Cx43, were elevated in reactive astrocytes from normal rats and from hSOD1G93A transgenic mice, as compared to controls. In vitro, reactive astrocytes stimulated with Thy-1 responded by activating AKT, which phosphorylated S373Cx43. Increased pS373Cx43 augmented the release of ATP to the extracellular medium and AKTi inhibited these Thy-1-induced responses. Furthermore, in an in vivo model of inflammation (brain damage), AKTi decreased the levels of astrocyte reactivity markers and S373Cx43 phosphorylation. Conclusions: Here, we identify changes in the PI3K/AKT molecular signaling network and show how they participate in astrogliosis by regulating the HC protein Cx43. Moreover, because HC opening and ATP release are important in astrocyte reactivity, the phosphorylation of Cx43 by AKT and the associated increase in ATP release identify a potential therapeutic window of opportunity to limit the adverse effects of astrogliosis.
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    PSD95 regulates morphological development of adult-born granule neurons in the mouse hippocampus
    (2019) Mardones, Muriel D.; Jorquera, Patricia, V; Herrera-Soto, Andrea; Ampuero, Estibaliz; Bustos, Fernando J.; van Zundert, Brigitte; Varela-Nallar, Lorena
    In the adult hippocampus new neurons are generated in the dentate gyrus from neural progenitor cells. Adult-born neurons integrate into the hippocampal circuitry and contribute to hippocampal function. PSD95 is a major postsynaptic scaffold protein that is crucial for morphological maturation and synaptic development of hippocampal neurons. Here we study the function of PSD95 in adult hippocampal neurogenesis by downregulating PSD95 expression in newborn cells using retroviral-mediated RNA interference. Retroviruses coding for a control shRNA or an shRNA targeting PSD95 (shPSD95) were stereotaxically injected into the dorsal dentate gyms of 2-month-old C57BL/6 mice. PSD95 knockdown did not affect neuronal differentiation of newborn cells into neurons, or migration of newborn neurons into the granule cell layer. Morphological analysis revealed that newborn neurons expressing shPSD95 showed increased dendritic length and increased number of high-order dendrites. Concomitantly, dendrites from shPSD95-expressing newborn granule neurons showed a reduction in the density of dendritic spines. These results suggest that PSD95 is required for proper dendritic and spine maturation of adult-born neurons, but not for early stages of neurogenesis in the hippocampus.
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    Reduced repressive epigenetic marks, increased DNA damage and Alzheimer's disease hallmarks in the brain of humans and mice exposed to particulate urban air pollution
    (2020) Calderon-Garciduenas, Lilian; Herrera-Soto, Andrea; Jury, Nur; Maher, Barbara A.; Gonzalez-Maciel, Angelica; Reynoso-Robles, Rafael; Ruiz-Rudolph, Pablo; van Zundert, Brigitte; Varela-Nallar, Lorena
    Exposure to air pollutants is associated with an increased risk of developing Alzheimer's disease (AD). AD pathological hallmarks and cognitive deficits are documented in children and young adults in polluted cities (e.g. Metropolitan Mexico City, MMC). Iron-rich combustion- and friction-derived nanoparticles (CFDNPs) that are abundantly present in airborne particulate matter pollution have been detected in abundance in the brains of young urbanites. Epigenetic gene regulation has emerged as a candidate mechanism linking exposure to air pollution and brain diseases. A global decrease of the repressive histone post-translational modifications (HPTMs) H3K9me2 and H3K9me3 (H3K9me2/me3) has been described both in AD patients and animal models. Here, we evaluated nuclear levels of H3K9me2/me3 and the DNA double-strand-break marker gamma-H2AX by immunostaining in post-mortem prefrontal white matter samples from 23 young adults (age 29 +/- 6 years) who resided in MMC (n = 13) versus low-pollution areas (n = 10). Lower H3K9me2/me3 and higher gamma-H2A.X staining were present in MMC urbanites, who also displayed the presence of hyperphosphorylated tau and amyloid-beta (A beta) plaques. Transmission electron microscopy revealed abundant CFDNPs in neuronal, glial and endothelial nuclei in MMC residents' frontal samples. In addition, mice exposed to particulate air pollution (for 7 months) in urban Santiago (Chile) displayed similar brain impacts; reduced H3K9me2/me3 and increased gamma-H2A.X staining, together with increased levels of AD-related tau phosphorylation. Together, these findings suggest that particulate air pollution, including metal-rich CFDNPs, impairs brain chromatin silencing and reduces DNA integrity, increasing the risk of developing AD in young individuals exposed to high levels of particulate air pollution.