Human Liver Stem Cell Derived Extracellular Vesicles Alleviate Kidney Fibrosis by Interfering with the ß-Catenin Pathway through miR29b.

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.

p140Cap Regulates GABAergic Synaptogenesis and Development of Hippocampal Inhibitory Circuits.

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.

Pre- and postnatal exposure to glyphosate-based herbicide causes behavioral and cognitive impairments in adult mice: evidence of cortical ad hippocampal dysfunction.

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.

Impaired chromaffin cell excitability and exocytosis in autistic Timothy syndrome TS2-neo mouse rescued by L-type calcium channel blockers.

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.

Cholesterol loss during glutamate-mediated excitotoxicity.

The deregulation of brain cholesterol metabolism is typical in acute neuronal injury (such as stroke, brain trauma and epileptic seizures) and chronic neurodegenerative diseases (Alzheimer's disease). Since both conditions are characterized by excessive stimulation of glutamate receptors, we have here investigated to which extent excitatory neurotransmission plays a role in brain cholesterol homeostasis. We show that a short (30 min) stimulation of glutamatergic neurotransmission induces a small but significant loss of membrane cholesterol, which is paralleled by release to the extracellular milieu of the metabolite 24S-hydroxycholesterol. Consistent with a cause-effect relationship, knockdown of the enzyme cholesterol 24-hydroxylase (CYP46A1) prevented glutamate-mediated cholesterol loss. Functionally, the loss of cholesterol modulates the magnitude of the depolarization-evoked calcium response. Mechanistically, glutamate-induced cholesterol loss requires high levels of intracellular Ca(2+), a functional stromal interaction molecule 2 (STIM2) and mobilization of CYP46A1 towards the plasma membrane. This study underscores the key role of excitatory neurotransmission in the control of membrane lipid composition, and consequently in neuronal membrane organization and function.

ADF/Cofilin Controls Synaptic Actin Dynamics and Regulates Synaptic Vesicle Mobilization and Exocytosis.

Actin is a regulator of synaptic vesicle mobilization and exocytosis, but little is known about the mechanisms that regulate actin at presynaptic terminals. Genetic data on LIMK1, a negative regulator of actin-depolymerizing proteins of the ADF/cofilin family, suggest a role for ADF/cofilin in presynaptic function. However, synapse physiology is fully preserved upon genetic ablation of ADF in mice, and n-cofilin mutant mice display defects in postsynaptic plasticity, but not in presynaptic function. One explanation for this phenomenon is overlapping functions of ADF and n-cofilin in presynaptic physiology. Here, we tested this hypothesis and genetically removed ADF together with n-cofilin from synapses. In double mutants for ADF and n-cofilin, synaptic actin dynamics was impaired and more severely affected than in single mutants. The resulting cytoskeletal defects heavily affected the organization, mobilization, and exocytosis of synaptic vesicles in hippocampal CA3-CA1 synapses. Our data for the first time identify overlapping functions for ADF and n-cofilin in presynaptic physiology and vesicle trafficking. We conclude that n-cofilin is a limiting factor in postsynaptic plasticity, a function which cannot be substituted by ADF. On the presynaptic side, the presence of either ADF or n-cofilin is sufficient to control actin remodeling during vesicle release.

Learning, AMPA receptor mobility and synaptic plasticity depend on n-cofilin-mediated actin dynamics.

Neuronal plasticity is an important process for learning, memory and complex behaviour. Rapid remodelling of the actin cytoskeleton in the postsynaptic compartment is thought to have an important function for synaptic plasticity. However, the actin-binding proteins involved and the molecular mechanisms that in vivo link actin dynamics to postsynaptic physiology are not well understood. Here, we show that the actin filament depolymerizing protein n-cofilin is controlling dendritic spine morphology and postsynaptic parameters such as late long-term potentiation and long-term depression. Loss of n-cofilin-mediated synaptic actin dynamics in the forebrain specifically leads to impairment of all types of associative learning, whereas exploratory learning is not affected. We provide evidence for a novel function of n-cofilin function in synaptic plasticity and in the control of extrasynaptic excitatory AMPA receptors diffusion. These results suggest a critical function of actin dynamics in associative learning and postsynaptic receptor availability.

Neuroligin-4 is localized to glycinergic postsynapses and regulates inhibition in the retina.

Neuroligins (NL1-NL4) are postsynaptic adhesion proteins that control the maturation and function of synapses in the central nervous system (CNS). Loss-of-function mutations in NL4 are linked to rare forms of monogenic heritable autism, but its localization and function are unknown. Using the retina as a model system, we show that NL4 is preferentially localized to glycinergic postsynapses and that the loss of NL4 is accompanied by a reduced number of glycine receptors mediating fast glycinergic transmission. Accordingly, NL4-deficient ganglion cells exhibit slower glycinergic miniature postsynaptic currents and subtle alterations in their stimulus-coding efficacy, and inhibition within the NL4-deficient retinal network is altered as assessed by electroretinogram recordings. These data indicate that NL4 shapes network activity and information processing in the retina by modulating glycinergic inhibition. Importantly, NL4 is also targeted to inhibitory synapses in other areas of the CNS, such as the thalamus, colliculi, brainstem, and spinal cord, and forms complexes with the inhibitory postsynapse proteins gephyrin and collybistin in vivo, indicating that NL4 is an important component of glycinergic postsynapses.

Acute compartment syndrome and fasciotomy after a viper bite in Italy: a case report.

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.

Extracellular Vesicles Derived from Human Liver Stem Cells Counteract Chronic Kidney Disease Development and Cardiac Dysfunction in Remnant Kidney Murine Model: The Possible Involvement of Proteases.

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.

Early formation of GABAergic synapses governs the development of adult-born neurons in the olfactory bulb.

In mammals, olfactory bulb granule cells (GCs) are generated throughout life in the subventricular zone. GABAergic inputs onto newborn neurons likely regulate their maturation, but the details of this process remain still elusive. Here, we investigated the differentiation, synaptic integration, and survival of adult-born GCs when their afferent GABAergic inputs are challenged by conditional gene targeting. Migrating GC precursors were targeted with Cre-eGFP-expressing lentiviral vectors in mice with a floxed gene encoding the GABA(A) receptor α2-subunit (i.e., Gabra2). Ablation of the α2-subunit did not affect GC survival but dramatically delayed their maturation. We found a reduction in postsynaptic α2-subunit and gephyrin clusters accompanied by a decrease in the frequency and amplitude of GABAergic postsynaptic currents beginning ∼14 d post-injection (dpi). In addition, mutant cells exhibited altered dendritic branching and spine density. Spine loss appeared with mislocation of glutamatergic synapses on dendritic shafts and a reduction of spontaneous glutamatergic postsynaptic currents, underscoring the relevance of afferent GABAergic transmission for a proper synaptic integration of newborn GCs. To test the role of GABAergic signaling during much early stages of GC maturation, we used a genetic strategy to selectively inactivate Gabra2 in precursor cells of the subventricular zone. In these mice, labeling of newborn GCs with eGFP lentiviruses revealed similar morphological alterations as seen on delayed Gabra2 inactivation in migrating neuroblasts, with reduced dendritic branching and spine density at 7 dpi. Collectively, these results emphasize the critical role of GABAergic synaptic signaling for structural maturation of adult-born GCs and formation of glutamatergic synapses.

Morphological and mitochondrial changes in murine choroid plexus epithelial cells during healthy aging.

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.

Choroid plexuses carry nodal-like cilia that undergo axoneme regression from early adult stage.

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.

Region-Specific Phosphorylation Determines Neuroligin-3 Localization to Excitatory Versus Inhibitory Synapses.

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.

ERK activation in axonal varicosities modulates presynaptic plasticity in the CA3 region of the hippocampus through synapsin I.

Activity-dependent changes in the strength of synaptic connections in the hippocampus are central for cognitive processes such as learning and memory storage. In this study, we reveal an activity-dependent presynaptic mechanism that is related to the modulation of synaptic plasticity. In acute mouse hippocampal slices, high-frequency stimulation (HFS) of the mossy fiber (MF)-CA3 pathway induced a strong and transient activation of extracellular-regulated kinase (ERK) in MF giant presynaptic terminals. Remarkably, pharmacological blockade of ERK disclosed a negative role of this kinase in the regulation of a presynaptic form of plasticity at MF-CA3 contacts. This ERK-mediated inhibition of post-tetanic enhancement (PTE) of MF-CA3 synapses was both frequency- and pathway-specific and was observed only with HFS at 50 Hz. Importantly, blockade of ERK was virtually ineffective on PTE of MF-CA3 synapses in mice lacking synapsin I, 1 of the major presynaptic ERK substrates, and triple knockout mice lacking all synapsin isoforms displayed PTE kinetics resembling that of wild-type mice under ERK inhibition. These findings reveal a form of short-term synaptic plasticity that depends on ERK and is finely tuned by the firing frequency of presynaptic neurons. Our results also demonstrate that presynaptic activation of the ERK signaling pathway plays part in the activity-dependent modulation of synaptic vesicle mobilization and transmitter release.

Use of a wireless ultrasound probe as a portable, noninvasive method for studying reproductive biology in the asp viper, Vipera aspis.

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.

Anxiety and Gene Expression Enhancement in Mice Exposed to Glyphosate-Based Herbicide.

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.

Increased Placental Anti-Oxidant Response in Asymptomatic and Symptomatic COVID-19 Third-Trimester Pregnancies.

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).

Complex role of collybistin and gephyrin in GABAA receptor clustering.

Gephyrin and collybistin are key components of GABA(A) receptor (GABA(A)R) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA(A)R subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA(A)R α2 and α3 subunit intracellular M3-M4 domain (but not α1, α4, α5, α6, ß1-3, or γ1-3) with gephyrin. Curiously, GABA(A)R α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA(A)R α2 also overlap at the start of the gephyrin E domain. This suggests that although GABA(A)R α3 interacts with gephyrin, GABA(A)R α2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA(A)R α2 and collybistin or GABA(A)R α2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA(A)R α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA(A)R α2 is capable of "activating " collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA(A)R α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA(A)R and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with α2 subunit containing GABA(A)Rs, but not GlyRs or other GABA(A)R subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABA(A)Rs containing the α2 subunit.

Synapse formation and clustering of neuroligin-2 in the absence of GABAA receptors.

GABAergic synapses are crucial for brain function, but the mechanisms underlying inhibitory synaptogenesis are unclear. Here, we show that postnatal Purkinje cells (PCs) of GABA(A)alpha1 knockout (KO) mice express transiently the alpha3 subunit, leading to the assembly of functional GABA(A) receptors and initial normal formation of inhibitory synapses, that are retained until adulthood. Subsequently, down-regulation of the alpha3 subunit causes a complete loss of GABAergic postsynaptic currents, resulting in a decreased rate of inhibitory synaptogenesis and formation of mismatched synapses between GABAergic axons and PC spines. Notably, the postsynaptic adhesion molecule neuroligin-2 (NL2) is correctly targeted to inhibitory synapses lacking GABA(A) receptors and the scaffold molecule gephyrin, but is absent from mismatched synapses, despite innervation by GABAergic axons. Our data indicate that GABA(A) receptors are dispensable for synapse formation and maintenance and for targeting NL2 to inhibitory synapses. However, GABAergic signaling appears to be crucial for activity-dependent regulation of synapse density during neuronal maturation.