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BACKGROUND: Choroid plexus (ChP) is the secretory epithelial structure located in brain ventricles. Choroid plexus tumors (CPTs) are rare neoplasms predominantly occurring in young patients with intensified malignancy in children. CPT treatment is hindered by insufficient knowledge of the tumor pathology and limited availability of valid models. METHODS: Genomic and transcriptomic data from CPT patients were analyzed to identify the putative pathological pathway. Cellular and molecular techniques were employed to validate bioinformatic results in CPT patient samples. Pharmacologic inhibition of Wnt/ß-catenin signaling was assessed in CPT cells. Cell-based assays of ChP cell lines were performed following CRISPR-Cas9-derived knockout and over-expression of Wnt/ß-catenin pathway genes. 3D CPT model was generated through CRISPR-Cas9-derived knockout of APC. RESULTS: We discovered that Wnt/ß-catenin signaling is activated in human CPTs, likely as a consequence of large-scale chromosomal instability events of the CPT genomes. We demonstrated that CPT-derived cells depend on autocrine Wnt/ß-catenin signaling for survival. Constitutive Wnt/ß-catenin pathway activation, either through knock-out of the negative regulator APC or overexpression of the ligand WNT3A, induced tumorigenic properties in ChP 2D in vitro models. Increased activation of Wnt/ß-catenin pathway in ChP organoids, through treatment with a potent GSK3ß inhibitor, reduced the differentiation of mature ChP epithelia cells. Remarkably, the depletion of APC was sufficient to induce the oncogenic transformation of ChP organoids. CONCLUSIONS: Our research identifies Wnt/ß-catenin signaling as a critical driver of CPT tumorigenesis and provides the first 3D in vitro model for future pathological and therapeutic studies of CPT.
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Fibrosis is a marker of chronic kidney disease (CKD) and consists of the accumulation of the extracellular matrix (ECM) components, causing the progressive deterioration of kidney function. Human liver stem cells (HLSCs) have anti-fibrotic activity, and HLSC-derived extracellular vesicles (EVs) mediate this effect. Herein, we evaluated the ability of HLSC-EVs to reverse renal and cardiac alterations in a murine model of partial nephrectomy (PNx) that mimics human CKD development. Furthermore, we investigated the contribution of extracellular matrix remodeling-related proteases to the anti-fibrotic effect of HLSC-EVs. PNx was performed by ligation of both poles of the left kidney, followed one week later by the removal of the right kidney. EV treatment started 4 weeks after the nephrectomy, when renal and cardiac alternations were already established, and mice were sacrificed at week eight. HLSC-EV treatment improved renal function and morphology, significantly decreasing interstitial fibrosis, glomerular sclerosis, and capillary rarefaction. This improvement was confirmed by the decreased expression of pro-fibrotic genes. Moreover, EV treatment improved cardiac function and reduced cardiac fibrosis. HLSC-EVs shuttled different proteases with ECM remodeling activity, and matrix metalloproteinase 1 (MMP-1) was involved in their anti-fibrotic effect on renal tissue. HLSC-EV treatment interferes with CKD development and ameliorates cardiomyopathy in PNx mice.
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BACKGROUND: Bites caused by European vipers are rare medical emergencies but can occasionally cause life-threatening complications. Viper venom causes local symptoms, which can be accompanied by systemic manifestations in severe cases. The local effects of snakebites include edema and, more rarely, necrosis and compartment syndrome. The consequences of envenomation are often more pronounced in children due to their smaller body size. CASE PRESENTATION: We present the case of a 6-year-old girl who experienced multiple viper bites in the lower limb in northwest Italy. The girl received supportive care but progressed to develop compartment syndrome that required emergency fasciotomy. The patient's condition improved promptly after surgical decompression and administration of antivenom, but full recovery required prolonged hospitalization and rehabilitation. CONCLUSIONS: This case highlights the importance of obtaining a timely assessment of the severity of viper envenomation without delaying the administration of antivenom in most serious cases. The presence of multiple bite marks on the patient is one factor that may help to predict the clinical severity of snakebites and anticipate symptom progression.
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Síndromes Compartimentales , Mordeduras de Serpientes , Niño , Femenino , Humanos , Antivenenos/uso terapéutico , Síndromes Compartimentales/diagnóstico , Síndromes Compartimentales/etiología , Síndromes Compartimentales/cirugía , Fasciotomía , Italia , Mordeduras de Serpientes/complicacionesRESUMEN
BACKGROUND: Neuroligin-3 is a postsynaptic adhesion molecule involved in synapse development and function. It is implicated in rare, monogenic forms of autism, and its shedding is critical to the tumor microenvironment of gliomas. While other members of the neuroligin family exhibit synapse-type specificity in localization and function through distinct interactions with postsynaptic scaffold proteins, the specificity of neuroligin-3 synaptic localization remains largely unknown. METHODS: We investigated the synaptic localization of neuroligin-3 across regions in mouse and human brain samples after validating antibody specificity in knockout animals. We raised a phospho-specific neuroligin antibody and used phosphoproteomics, cell-based assays, and in utero CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9) knockout and gene replacement to identify mechanisms that regulate neuroligin-3 localization to distinct synapse types. RESULTS: Neuroligin-3 exhibits region-dependent synapse specificity, largely localizing to excitatory synapses in cortical regions and inhibitory synapses in subcortical regions of the brain in both mice and humans. We identified specific phosphorylation of cortical neuroligin-3 at a key binding site for recruitment to inhibitory synapses, while subcortical neuroligin-3 remained unphosphorylated. In vitro, phosphomimetic mutation of that site disrupted neuroligin-3 association with the inhibitory postsynaptic scaffolding protein gephyrin. In vivo, phosphomimetic mutants of neuroligin-3 localized to excitatory postsynapses, while phospho-null mutants localized to inhibitory postsynapses. CONCLUSIONS: These data reveal an unexpected region-specific pattern of neuroligin-3 synapse specificity, as well as a phosphorylation-dependent mechanism that regulates its recruitment to either excitatory or inhibitory synapses. These findings add to our understanding of how neuroligin-3 is involved in conditions that may affect the balance of excitation and inhibition.
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Choroid plexuses (ChPs) produce cerebrospinal fluid and sense non-cell-autonomous stimuli to control the homeostasis of the central nervous system. They are mainly composed of epithelial multiciliated cells, whose development and function are still controversial. We have thus characterized the stepwise order of mammalian ChP epithelia cilia formation using a combination of super-resolution-microscopy approaches and mouse genetics. We show that ChP ciliated cells are built embryonically on a treadmill of spatiotemporally regulated events, starting with atypical centriole amplification and ending with the construction of nodal-like 9+0 cilia, characterized by both primary and motile features. ChP cilia undergo axoneme resorption at early postnatal stages through a microtubule destabilization process controlled by the microtubule-severing enzyme spastin and mitigated by polyglutamylation levels. Notably, this phenotype is preserved in humans, suggesting a conserved ciliary resorption mechanism in mammals.
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Axonema , Cilios , Humanos , Ratones , Animales , Cilios/fisiología , Células Epiteliales/fisiología , Epitelio , Coroides , MamíferosRESUMEN
BACKGROUND: Choroid plexuses (ChPs) are intraventricular structures mainly composed by specialized epithelial cells interconnected by tight junctions that establish the blood-cerebrospinal fluid (CSF) barrier. ChPs are essential to produce CSF and transport solutes from and into the brain. Deterioration of ChP function and morphology has been correlated to worsening of neurodegenerative disorders. We here map morpho-functional changes in the ChP epithelial cells during healthy aging, starting from young adult to 2-years old mice. METHODS: We used a multi-tiered approach, including transmission electron microscopy (TEM), immunohistochemistry, RT-qPCR, Western Blot and 2-photon microscopy (2-PM) at multiple timepoints ranging from young adult to 2-years old mice. RESULTS: We identified distinct morpho-functional modifications in epithelial cells of ChP starting from 8 to 12 months of age, which mostly remained stable up to 2 years. These changes include flattening of the epithelium, reduction of microvilli length and an augmentation of interrupted tight junctions. We also found a decrease in mitochondria density together with elongation of mitochondria in older mice. Morphological mitochondrial rearrangements were accompanied by increased superoxide levels, decreased membrane potential and decreased mitochondrial motility in aged mice. Interestingly, most of the age-related changes were not accompanied by modification of protein and/or gene expression levels and aged mitochondria effectively responded to acute pharmacological stressful stimuli. CONCLUSIONS: Our study suggests a long-term progression of multiple morpho-functional features of the mouse choroid plexus epithelium during adulthood followed by structural remodeling during the aging process. These findings can lead to a better understanding on how functional and morphological rearrangements of ChP are correlated during aging.
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Plexo Coroideo , Envejecimiento Saludable , Ratones , Animales , Plexo Coroideo/metabolismo , Barrera Hematoencefálica/metabolismo , Células Epiteliales/metabolismo , MitocondriasRESUMEN
In this study, we investigated the use of wireless ultrasonography as an imaging system to study the reproductive ecology of the asp viper (Vipera aspis), a viviparous snake found in southwestern Europe. Female vipers were captured during the summer and immediately scanned to obtain an estimate of the number of embryos. Ultrasound imaging was performed with a pocket-sized wireless ultrasound probe interfaced with a tablet with a dedicated app. Vipers were then released at the exact capture site after collecting data on body size and weight. We validate wireless ultrasonography as a non-destructive, effective tool for ultrasonic investigations in the field. Wireless probes are light and compact, which facilitates carriage in rugged terrain. Moreover, the absence of cables simplifies the maneuvers to be made on a small, potentially dangerous snake. Importantly, ultrasound scans can be performed at the capture site, thus minimizing restraint time and handling of gravid females.
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Viperidae , Animales , Ecología , Europa (Continente) , Femenino , Reproducción/fisiología , Ultrasonografía , Viperidae/fisiologíaRESUMEN
Growing evidence demonstrates that serotonin (5-HT) depletion increases activity in the amygdala and medial prefrontal cortex (mPFC), ultimately leading to anxiety behavior. Previously, we showed that glyphosate-based herbicides (GBHs) increased anxiety levels and reduced the number of serotoninergic fibers within the mPFCs and amygdalas of exposed mice. However, the impact of this 5-HT depletion following GBH exposure on neuronal activity in these structures is still unknown. In this study, we investigated the effects of GBH on immediate early gene (IEG) activation within the mPFCs and amygdalas of treated mice from juvenile age to adulthood and its subsequent effects on anxiety levels. Mice were treated for subchronic (6 weeks) and chronic (12 weeks) periods with 250 or 500 mg/kg/day of GBH and subjected to behavioral testing using the open field and elevated plus maze paradigms. Then, we analyzed the expression levels of c-Fos and pCREB and established the molecular proxies of neuronal activation within the mPFC and the amygdala. Our data revealed that repeated exposure to GBH triggers anxiogenic behavior in exposed mice. Confocal microscopy investigations into the prelimbic/infralimbic regions of the mPFC and in basolateral/central nuclei of the amygdala disclosed that the behavioral alterations are paralleled by a robust increase in the density and labelling intensity of c-Fos- and pCREB-positive cells. Taken together, these data show that mice exposed to GBH display the hyperactivation of the mPFC-amygdala areas, suggesting that this is a potential mechanism underlying the anxiety-like phenotype.
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Despite Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) -induced Oxidative Stress (OxS) being well documented in different organs, the molecular pathways underlying placental OxS in late-pregnancy women with SARS-CoV-2 infection are poorly understood. Herein, we performed an observational study to determine whether placentae of women testing positive for SARS-CoV-2 during the third trimester of pregnancy showed redox-related alterations involving Catalase (CAT) and Superoxide Dismutase (SOD) antioxidant enzymes as well as placenta morphological anomalies relative to a cohort of healthy pregnant women. Next, we evaluated if placental redox-related alterations and mitochondria pathological changes were correlated with the presence of maternal symptoms. We observed ultrastructural alterations of placental mitochondria accompanied by increased levels of oxidative stress markers Thiobarbituric Acid Reactive Substances (TBARS) and Hypoxia Inducible Factor-1 α (HIF-1α) in SARS-CoV-2 women during the third trimester of pregnancy. Importantly, we found an increase in placental CAT and SOD antioxidant enzymes accompanied by physiological neonatal outcomes. Our findings strongly suggest a placenta-mediated OxS inhibition in response to SARS-CoV-2 infection, thus contrasting the cytotoxic profile caused by Coronavirus Disease 2019 (COVID-19).
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Human liver stem-cell-derived extracellular vesicles (HLSC-EVs) exhibit therapeutic properties in various pre-clinical models of kidney injury. We previously reported an overall improvement in kidney function following treatment with HLSC-EVs in a model of aristolochic acid nephropathy (AAN). Here, we provide evidence that HLSC-EVs exert anti-fibrotic effects by interfering with ß-catenin signalling. A mouse model of AAN and an in vitro pro-fibrotic model were used. The ß-catenin mRNA and protein expression, together with the pro-fibrotic markers α-SMA and collagen 1, were evaluated in vivo and in vitro following treatment with HLSC-EVs. Expression and functional analysis of miR29b was performed in vitro following HLSC-EV treatments through loss-of-function experiments. Results showed that expression of ß-catenin was amplified both in vivo and in vitro, and ß-catenin gene silencing in fibroblasts prevented AA-induced up-regulation of pro-fibrotic genes, revealing that ß-catenin is an important factor in fibroblast activation. Treatment with HLSC-EVs caused increased expression of miR29b, which was significantly inhibited in the presence of α-amanitin. The suppression of the miR29b function with a selective inhibitor abolished the anti-fibrotic effects of HLSC-EVs, resulting in the up-regulation of ß-catenin and pro-fibrotic α-Sma and collagen type 1 genes. Together, these data suggest a novel HLSC-EV-dependent regulatory mechanism in which ß-catenin is down regulated by HLSC-EVs-induced miR29b expression.
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Vesículas Extracelulares/fisiología , Fibrosis/prevención & control , Enfermedades Renales/prevención & control , Hígado/citología , Células Madre/citología , beta Catenina/metabolismo , Animales , Apoptosis , Biomarcadores/metabolismo , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Fibrosis/etiología , Fibrosis/metabolismo , Fibrosis/patología , Regulación de la Expresión Génica , Humanos , Enfermedades Renales/etiología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , MicroARNs/genética , Células Madre/metabolismo , beta Catenina/genéticaRESUMEN
In the rodent olfactory bulb the smooth dendrites of the principal glutamatergic mitral cells (MCs) form reciprocal dendrodendritic synapses with large spines on GABAergic granule cells (GC), where unitary release of glutamate can trigger postsynaptic local activation of voltage-gated Na+-channels (Navs), that is a spine spike. Can such single MC input evoke reciprocal release? We find that unitary-like activation via two-photon uncaging of glutamate causes GC spines to release GABA both synchronously and asynchronously onto MC dendrites. This release indeed requires activation of Navs and high-voltage-activated Ca2+-channels (HVACCs), but also of NMDA receptors (NMDAR). Simulations show temporally overlapping HVACC- and NMDAR-mediated Ca2+-currents during the spine spike, and ultrastructural data prove NMDAR presence within the GABAergic presynapse. This cooperative action of presynaptic NMDARs allows to implement synapse-specific, activity-dependent lateral inhibition, and thus could provide an efficient solution to combinatorial percept synthesis in a sensory system with many receptor channels.
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Células Dendríticas/fisiología , Neuronas/fisiología , Bulbo Olfatorio/citología , Receptores de N-Metil-D-Aspartato/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Potenciales de Acción/fisiología , Animales , Animales Modificados Genéticamente , Canales de Calcio , Estimulación Eléctrica , Femenino , Regulación de la Expresión Génica , Activación del Canal Iónico , Masculino , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/genética , Canales de Sodio , Ácido gamma-Aminobutírico/genéticaRESUMEN
Multiple myeloma is a plasma cell neoplasm characterized by the production of unfolded immunoglobulins, which cause endoplasmic reticulum (ER) stress and sensitivity to proteasome inhibition. The genomic landscape of multiple myeloma is characterized by the loss of several genes rarely mutated in other cancers that may underline specific weaknesses of multiple myeloma cells. One of these is FAM46C that is lost in more than 10% of patients with multiple myeloma. We show here that FAM46C is part of a new complex containing the ER-associated protein FNDC3A, which regulates trafficking and secretion and, by impairing autophagy, exacerbates proteostatic stress. Reconstitution of FAM46C in multiple myeloma cells that had lost it induced apoptosis and ER stress. Apoptosis was preceded by an increase of intracellular aggregates, which was not linked to increased translation of IgG mRNA, but rather to impairment of autophagy. Biochemical analysis showed that FAM46C requires interaction with ER bound protein FNDC3A to reside in the cytoplasmic side of the ER. FNDC3A was lost in some multiple myeloma cell lines. Importantly, depletion of FNDC3A increased the fitness of FAM46C-expressing cells and expression of FNDC3A in cells that had lost it recapitulated the effects of FAM46C, inducing aggregates and apoptosis. FAM46C and FNDC3A formed a complex that modulates secretion routes, increasing lysosome exocytosis. The cellular landscape generated by FAM46C/FNDC3A expression predicted sensitivity to sphingosine kinase inhibition. These results suggest that multiple myeloma cells remodel their trafficking machinery to cope with ER stress. SIGNIFICANCE: This study identifies a new multiple myeloma-specific tumor suppressor complex that regulates autophagy and unconventional secretion, highlighting the sensitivity of multiple myeloma cells to the accumulation of protein aggregates.
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Fibronectinas/metabolismo , Mieloma Múltiple/patología , Nucleotidiltransferasas/metabolismo , Agregación Patológica de Proteínas/metabolismo , Animales , Autofagia/fisiología , Genes Supresores de Tumor , Xenoinjertos , Humanos , Ratones , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Nucleotidiltransferasas/genética , Agregado de Proteínas/fisiología , Transporte de Proteínas/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Muscle dystrophin-glycoprotein complex (DGC) links the intracellular cytoskeleton to the extracellular matrix. In neurons, dystroglycan and dystrophin, two major components of the DGC, localize in a subset of GABAergic synapses, where their function is unclear. Here we used mouse models to analyze the specific role of the DGC in the organization and function of inhibitory synapses. Loss of full-length dystrophin in mdx mice resulted in a selective depletion of the transmembrane ß-dystroglycan isoform from inhibitory post-synaptic sites in cerebellar Purkinje cells. Remarkably, there were no differences in the synaptic distribution of the extracellular α-dystroglycan subunit, of GABAA receptors and neuroligin 2. In contrast, conditional deletion of the dystroglycan gene from Purkinje cells caused a disruption of the DGC and severely impaired post-synaptic clustering of neuroligin 2, GABAA receptors and scaffolding proteins. Accordingly, whole-cell patch-clamp analysis revealed a significant reduction in the frequency and amplitude of spontaneous IPSCs recorded from Purkinje cells. In the long-term, deletion of dystroglycan resulted in a significant decrease of GABAergic innervation of Purkinje cells and caused an impairment of motor learning functions. These results show that dystroglycan is an essential synaptic organizer at GABAergic synapses in Purkinje cells.
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Glyphosate-based herbicides (GBH) are the most widely used pesticides worldwide. Despite considerable progress in describing the neurotoxic potential of GBH, the harmful effects on brain cytoarchitecture and behavior are still unclear. Here, we addressed the developmental impact of GBH by exposing female mice to 250 or 500 mg/kg doses of GBH during both pregnancy and lactation and then examined the downstream effects at the behavioral, neurochemical and molecular levels. We show that pre- and neonatal exposure to GBH impairs fertility and reproduction parameters as well as maternal behavior of exposed mothers. In offspring, GBH was responsible for a global delay in innate reflexes and a deficit in motor development. At the adult age, exposed animals showed a decrease of locomotor activity, sociability, learning and short- and long-term memory associated with alterations of cholinergic and dopaminergic systems. Furthermore, GBH-activated microglia and astrocytes, sign of neuroinflammation event in the medial prefrontal cortex and hippocampus. At the molecular level, a down-regulation of brain-derived neurotrophic factor (BDNF) expression and an up-regulation of tyrosine-related kinase receptor (TrkB), NR1 subunit of NMDA receptor as well as tumor necrosis factor α (TNFα) were found in the brain of GBH-exposed mice. The present work demonstrates that GBH induces numerous behavioral and cognitive abnormalities closely associated with significant histological, neurochemical and molecular impairments. It also raises fundamental concerns about the ability of current safety testing to assess risks of pesticide exposure during developmental periods of central nervous system.
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Glicina/análogos & derivados , Herbicidas/toxicidad , Efectos Tardíos de la Exposición Prenatal , Animales , Astrocitos , Disfunción Cognitiva , Femenino , Glicina/toxicidad , Hipocampo , Lactancia , Aprendizaje , Ratones , Síndromes de Neurotoxicidad , Embarazo , Receptores de N-Metil-D-Aspartato , Reproducción , Transducción de Señal , GlifosatoRESUMEN
KEY POINTS: Tymothy syndrome (TS) is a multisystem disorder featuring cardiac arrhythmias, autism and adrenal gland dysfunction that originates from a de novo point mutation in the gene encoding the Cav1.2 (CACNA1C) L-type channel. To study the role of Cav1.2 channel signals in autism, the autistic TS2-neo mouse has been generated bearing the G406R point-mutation associated with TS type-2. Using heterozygous TS2-neo mice, we report that the G406R mutation reduces the rate of inactivation and shifts leftward the activation and inactivation of L-type channels, causing marked increase of resting Ca2+ influx ('window' Ca2+ current). The increased 'window current' causes marked reduction of NaV channel density, switches normal tonic firing to abnormal burst firing, reduces mitochondrial metabolism, induces cell swelling and decreases catecholamine release. Overnight incubations with nifedipine rescue NaV channel density, normal firing and the quantity of catecholamine released. We provide evidence that chromaffin cell malfunction derives from altered Cav1.2 channel gating. ABSTRACT: L-type voltage-gated calcium (Cav1) channels have a key role in long-term synaptic plasticity, sensory transduction, muscle contraction and hormone release. A point mutation in the gene encoding Cav1.2 (CACNA1C) causes Tymothy syndrome (TS), a multisystem disorder featuring cardiac arrhythmias, autism spectrum disorder (ASD) and adrenal gland dysfunction. In the more severe type-2 form (TS2), the missense mutation G406R is on exon 8 coding for the IS6-helix of the Cav1.2 channel. The mutation causes reduced inactivation and induces autism. How this occurs and how Cav1.2 gating-changes alter cell excitability, neuronal firing and hormone release on a molecular basis is still largely unknown. Here, using the TS2-neo mouse model of TS we show that the G406R mutation altered excitability and reduced secretory activity in adrenal chromaffin cells (CCs). Specifically, the TS2 mutation reduced the rate of voltage-dependent inactivation and shifted leftward the activation and steady-state inactivation of L-type channels. This markedly increased the resting 'window' Ca2+ current that caused an increased percentage of CCs undergoing abnormal action potential (AP) burst firing, cell swelling, reduced mitochondrial metabolism and decreased catecholamine release. The increased 'window' Ca2+ current caused also decreased NaV channel density and increased steady-state inactivation, which contributed to the increased abnormal burst firing. Overnight incubation with the L-type channel blocker nifedipine rescued the normal AP firing of CCs, the density of functioning NaV channels and their steady-state inactivation. We provide evidence that CC malfunction derives from the altered Cav1.2 channel gating and that dihydropyridines are potential therapeutics for ASD.
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Potenciales de Acción , Trastorno Autístico/genética , Canales de Calcio Tipo L/genética , Células Cromafines/metabolismo , Exocitosis , Síndrome de QT Prolongado/genética , Sindactilia/genética , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Catecolaminas/metabolismo , Células Cultivadas , Células Cromafines/efectos de los fármacos , Células Cromafines/fisiología , Activación del Canal Iónico , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Nifedipino/farmacología , Mutación Puntual , Canales de Sodio/metabolismoRESUMEN
The neuronal scaffold protein p140Cap was investigated during hippocampal network formation. p140Cap is present in presynaptic GABAergic terminals and its genetic depletion results in a marked alteration of inhibitory synaptic activity. p140Cap-/- cultured neurons display higher frequency of miniature inhibitory postsynaptic currents (mIPSCs) with no changes of their mean amplitude. Consistent with a potential presynaptic alteration of basal GABA release, p140Cap-/- neurons exhibit a larger synaptic vesicle readily releasable pool, without any variation of single GABAA receptor unitary currents and number of postsynaptic channels. Furthermore, p140Cap-/- neurons show a premature and enhanced network synchronization and appear more susceptible to 4-aminopyridine-induced seizures in vitro and to kainate-induced seizures in vivo. The hippocampus of p140Cap-/- mice showed a significant increase in the number of both inhibitory synapses and of parvalbumin- and somatostatin-expressing interneurons. Specific deletion of p140Cap in forebrain interneurons resulted in increased susceptibility to in vitro epileptic events and increased inhibitory synaptogenesis, comparable to those observed in p140Cap-/- mice. Altogether, our data demonstrate that p140Cap finely tunes inhibitory synaptogenesis and GABAergic neurotransmission, thus regulating the establishment and maintenance of the proper hippocampal excitatory/inhibitory balance.
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Proteínas Portadoras/fisiología , Neuronas GABAérgicas/fisiología , Hipocampo/fisiología , Red Nerviosa/fisiología , Inhibición Neural/fisiología , Sinapsis/fisiología , Animales , Células Cultivadas , Potenciales Postsinápticos Inhibidores/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
Sab (SH3 binding protein 5 or SH3BP5) is a mitochondrial scaffold protein involved in signaling associated with mitochondrial dysfunction and apoptosis; furthermore, Sab is a crucial signaling platform for neurodegenerative disease. To determine how this signaling nexus could have a significant effect on disease, we examined the regional abundance of Sab in the brain and sub-neuronal distribution, and we monitored the effect of Sab-mediated signaling on neuronal activity. We found that Sab is widely expressed in the adult mouse brain with increased abundance in hippocampus, ventral midbrain, and cerebellum. Sab was found in purified synaptosomes and in cultures of hippocampal neurons and astrocytes. Confocal and electron microscopy of mouse hippocampal sections confirmed the mitochondrial localization of Sab in the soma, dendrites, and axons. Given the localization and sub-neuronal distribution of Sab, we postulated that Sab-mediated signaling could affect neuronal function, so we measured the impact of inhibiting Sab-mediated events on the spontaneous activity in cultured hippocampal neurons. Treatment with a Sab-inhibitory peptide (Tat-SabKIM1), but not a scrambled control peptide, decreased the firing frequency and spike amplitudes. Our results demonstrate that brain-specific Sab-mediated signaling plays a role in neuronal activity through the manipulation of mitochondrial physiology by interacting kinases.
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Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/fisiología , Neuronas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Astrocitos/metabolismo , Encéfalo/metabolismo , Cerebelo/metabolismo , Dendritas/metabolismo , Regulación de la Expresión Génica/genética , Hipocampo/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Since the groundbreaking work of Ramon y Cajal, the cerebellar Purkinje cell has always represented an ideal model for studying the organization, development and function of synaptic circuits. Purkinje cells receive distinct types of glutamatergic and GABAergic synapses, each characterized by exquisite sub-cellular and molecular specificity. The formation and refinement of these connections results from a temporally-regulated sequence of events that involves molecular interactions between distinct sets of secreted and surface proteins, as well as activity-dependent competition between converging inputs. Insights into the mechanisms controlling synaptic specificity in Purkinje cells may help understand synapse development also in other brain regions and disclose circuit abnormalities that underlie neurodevelopmental disorders.
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Células de Purkinje/fisiología , Sinapsis/fisiología , Animales , Humanos , Modelos NeurológicosRESUMEN
Cyclin-dependent kinase-like 5 (CDKL5) mutations are found in severe neurodevelopmental disorders, including the Hanefeld variant of Rett syndrome (RTT; CDKL5 disorder). CDKL5 loss-of-function murine models recapitulate pathological signs of the human disease, such as visual attention deficits and reduced visual acuity. Here we investigated the cellular and synaptic substrates of visual defects by studying the organization of the primary visual cortex (V1) of Cdkl5-/y mice. We found a severe reduction of c-Fos expression in V1 of Cdkl5-/y mutants, suggesting circuit hypoactivity. Glutamatergic presynaptic structures were increased, but postsynaptic PSD-95 and Homer were significantly downregulated in CDKL5 mutants. Interneurons expressing parvalbumin, but not other types of interneuron, had a higher density in mutant V1, and were hyperconnected with pyramidal neurons. Finally, the developmental trajectory of pavalbumin-containing cells was also affected in Cdkl5-/y mice, as revealed by fainter appearance perineuronal nets at the closure of the critical period (CP). The present data reveal an overall disruption of V1 cellular and synaptic organization that may cause a shift in the excitation/inhibition balance likely to underlie the visual deficits characteristic of CDKL5 disorder. Moreover, ablation of CDKL5 is likely to tamper with the mechanisms underlying experience-dependent refinement of cortical circuits during the CP of development.
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BACKGROUND: Actin depolymerizing proteins of the actin depolymerizing factor (ADF)/cofilin family are essential for actin dynamics, which is critical for synaptic function. Two ADF/cofilin family members, ADF and n-cofilin, are highly abundant in the brain, where they are present in excitatory synapses. Previous studies demonstrated the relevance of n-cofilin for postsynaptic plasticity, associative learning, and anxiety. These studies also suggested overlapping functions for ADF and n-cofilin. METHODS: We performed pharmacobehavioral, electrophysiologic, and electron microscopic studies on ADF and n-cofilin single mutants and double mutants (named ACC mice) to characterize the importance of ADF/cofilin activity for synapse physiology and mouse behavior. RESULTS: The ACC mice, but not single mutants, exhibited hyperlocomotion, impulsivity, and impaired working memory. Hyperlocomotion and impulsive behavior were reversed by methylphenidate, a psychostimulant commonly used for the treatment of attention-deficit/hyperactivity disorder (ADHD). Also, ACC mice displayed a disturbed morphology of striatal excitatory synapses, accompanied by strongly increased glutamate release. Blockade of dopamine or glutamate transmission resulted in normal locomotion. CONCLUSIONS: Our study reveals that ADHD can result from a disturbed balance between excitation and inhibition in striatal circuits, providing novel insights into the mechanisms underlying this neurobehavioral disorder. Our results link actin dynamics to ADHD, suggesting that mutations in actin regulatory proteins may contribute to the etiology of ADHD in humans.