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.

Harmful Iron-Calcium Relationship in Pantothenate kinase Associated Neurodegeneration.

Pantothenate Kinase-associated Neurodegeneration (PKAN) belongs to a wide spectrum of diseases characterized by brain iron accumulation and extrapyramidal motor signs. PKAN is caused by mutations in PANK2, encoding the mitochondrial pantothenate kinase 2, which is the first enzyme of the biosynthesis of Coenzyme A. We established and characterized glutamatergic neurons starting from previously developed PKAN Induced Pluripotent Stem Cells (iPSCs). Results obtained by inductively coupled plasma mass spectrometry indicated a higher amount of total cellular iron in PKAN glutamatergic neurons with respect to controls. PKAN glutamatergic neurons, analyzed by electron microscopy, exhibited electron dense aggregates in mitochondria that were identified as granules containing calcium phosphate. Calcium homeostasis resulted compromised in neurons, as verified by monitoring the activity of calcium-dependent enzyme calpain1, calcium imaging and voltage dependent calcium currents. Notably, the presence of calcification in the internal globus pallidus was confirmed in seven out of 15 genetically defined PKAN patients for whom brain CT scan was available. Moreover, we observed a higher prevalence of brain calcification in females. Our data prove that high amount of iron coexists with an impairment of cytosolic calcium in PKAN glutamatergic neurons, indicating both, iron and calcium dys-homeostasis, as actors in pathogenesis of the disease.

Synapsin I deletion reduces neuronal damage and ameliorates clinical progression of experimental autoimmune encephalomyelitis.

The classical view of multiple sclerosis (MS) pathogenesis states that inflammation-mediated demyelination is responsible for neuronal damage and loss. However, recent findings show that impairment of neuronal functions and demyelination can be independent events, suggesting the coexistence of other pathogenic mechanisms. Due to the inflammatory milieu, subtle alterations in synaptic function occur, which are probably at the basis of the early cognitive decline that often precedes the neurodegenerative phases in MS patients. In particular, it has been reported that inflammation enhances excitatory synaptic transmission while it decreases GABAergic transmission in vitro and ex vivo. This evidence points to the idea that an excitation/inhibition imbalance occurs in the inflamed MS brain, even though the exact molecular mechanisms leading to this synaptic dysfunction are as yet not completely clear. Along this line, we observed that acute treatment of primary hippocampal neurons in culture with pro-inflammatory cytokines leads to an increased phosphorylation of synapsin I (SynI) by ERK1/2 kinase and to an increase in the frequency of spontaneous synaptic vesicle release events, which is prevented by SynI deletion. In vivo, the ablation of SynI expression is protective in terms of disease progression and neuronal damage in the experimental autoimmune encephalomyelitis mouse model of MS. Our results point to a possible key role in MS pathogenesis of the neuronal protein SynI, a regulator of excitation/inhibition balance in neuronal networks.

AAV-PHP.B-Mediated Global-Scale Expression in the Mouse Nervous System Enables GBA1 Gene Therapy for Wide Protection from Synucleinopathy.

The lack of technology for direct global-scale targeting of the adult mouse nervous system has hindered research on brain processing and dysfunctions. Currently, gene transfer is normally achieved by intraparenchymal viral injections, but these injections target a restricted brain area. Herein, we demonstrated that intravenous delivery of adeno-associated virus (AAV)-PHP.B viral particles permeated and diffused throughout the neural parenchyma, targeting both the central and the peripheral nervous system in a global pattern. We then established multiple procedures of viral transduction to control gene expression or inactivate gene function exclusively in the adult nervous system and assessed the underlying behavioral effects. Building on these results, we established an effective gene therapy strategy to counteract the widespread accumulation of α-synuclein deposits throughout the forebrain in a mouse model of synucleinopathy. Transduction of A53T-SCNA transgenic mice with AAV-PHP.B-GBA1 restored physiological levels of the enzyme, reduced α-synuclein pathology, and produced significant behavioral recovery. Finally, we provided evidence that AAV-PHP.B brain penetration does not lead to evident dysfunctions in blood-brain barrier integrity or permeability. Altogether, the AAV-PHP.B viral platform enables non-invasive, widespread, and long-lasting global neural expression of therapeutic genes, such as GBA1, providing an invaluable approach to treat neurodegenerative diseases with diffuse brain pathology such as synucleinopathies.

Direct generation of functional dopaminergic neurons from mouse and human fibroblasts.

Transplantation of dopaminergic neurons can potentially improve the clinical outcome of Parkinson's disease, a neurological disorder resulting from degeneration of mesencephalic dopaminergic neurons. In particular, transplantation of embryonic-stem-cell-derived dopaminergic neurons has been shown to be efficient in restoring motor symptoms in conditions of dopamine deficiency. However, the use of pluripotent-derived cells might lead to the development of tumours if not properly controlled. Here we identified a minimal set of three transcription factors--Mash1 (also known as Ascl1), Nurr1 (also known as Nr4a2) and Lmx1a--that are able to generate directly functional dopaminergic neurons from mouse and human fibroblasts without reverting to a progenitor cell stage. Induced dopaminergic (iDA) cells release dopamine and show spontaneous electrical activity organized in regular spikes consistent with the pacemaker activity featured by brain dopaminergic neurons. The three factors were able to elicit dopaminergic neuronal conversion in prenatal and adult fibroblasts from healthy donors and Parkinson's disease patients. Direct generation of iDA cells from somatic cells might have significant implications for understanding critical processes for neuronal development, in vitro disease modelling and cell replacement therapies.

Synchronous inhibitory potentials precede seizure-like events in acute models of focal limbic seizures.

Interictal spikes in models of focal seizures and epilepsies are sustained by the synchronous activation of glutamatergic and GABAergic networks. The nature of population spikes associated with seizure initiation (pre-ictal spikes; PSs) is still undetermined. We analyzed the networks involved in the generation of both interictal and PSs in acute models of limbic cortex ictogenesis induced by pharmacological manipulations. Simultaneous extracellular and intracellular recordings from both principal cells and interneurons were performed in the medial entorhinal cortex of the in vitro isolated guinea pig brain during focal interictal and ictal discharges induced in the limbic network by intracortical and brief arterial infusions of either bicuculline methiodide (BMI) or 4-aminopyridine (4AP). Local application of BMI in the entorhinal cortex did not induce seizure-like events (SLEs), but did generate periodic interictal spikes sensitive to the glutamatergic non-NMDA receptor antagonist DNQX. Unlike local applications, arterial perfusion of either BMI or 4AP induced focal limbic SLEs. PSs just ahead of SLE were associated with hyperpolarizing potentials coupled with a complete blockade of firing in principal cells and burst discharges in putative interneurons. Interictal population spikes recorded from principal neurons between two SLEs correlated with a depolarizing potential. We demonstrate in two models of acute limbic SLE that PS events are different from interictal spikes and are sustained by synchronous activation of inhibitory networks. Our findings support a prominent role of synchronous network inhibition in the initiation of a focal seizure.

Selective activation of parvalbumin- or somatostatin-expressing interneurons triggers epileptic seizurelike activity in mouse medial entorhinal cortex.

GABAergic interneurons are thought to play a critical role in eliciting interictal spikes (IICs) and triggering ictal discharges in temporal lobe epilepsy, yet the contribution of different interneuronal subtypes to seizure initiation is still largely unknown. Here we took advantage of optogenetic techniques combined with patch-clamp and field recordings to selectively stimulate parvalbumin (PV)- or somatostatin (SOM)-positive interneurons expressing channelrhodopsin-2 (CHR-2) in layers II-III of adult mouse medial entorhinal cortical slices during extracellular perfusion with the proconvulsive compound 4-aminopyridine (4-AP, 100-200 µM). In control conditions, blue laser photostimulation selectively activated action potential firing in either PV or SOM interneurons and, in both cases, caused a robust GABAA-receptor-mediated inhibition in pyramidal cells (PCs). During perfusion with 4-AP, brief photostimuli (300 ms) activating either PV or SOM interneurons induced patterns of epileptiform activity that closely replicated spontaneously occurring IICs and tonic-clonic ictal discharges. Laser-induced synchronous firing in both interneuronal types elicited large compound GABAergic inhibitory postsynaptic currents (IPSCs) correlating with IICs and preictal spikes. In addition, spontaneous and laser-induced epileptic events were similarly initiated in concurrence with a large increase in extracellular potassium concentration. Finally, interneuron activation was unable to stop or significantly shorten the progression of seizurelike episodes. These results suggest that entorhinal PV and SOM interneurons are nearly equally effective in triggering interictal and ictal discharges that closely resemble human temporal lobe epileptic activity.

Down-sizing of neuronal network activity and density of presynaptic terminals by pathological acidosis are efficiently prevented by Diminazene Aceturate.

Local acidosis is associated with neuro-inflammation and can have significant effects in several neurological disorders, including multiple sclerosis, brain ischemia, spinal cord injury and epilepsy. Despite local acidosis has been implicated in numerous pathological functions, very little is known about the modulatory effects of pathological acidosis on the activity of neuronal networks and on synaptic structural properties. Using non-invasive MRI spectroscopy we revealed protracted extracellular acidosis in the CNS of Experimental Autoimmune Encephalomyelitis (EAE) affected mice. By multi-unit recording in cortical neurons, we established that acidosis affects network activity, down-sizing firing and bursting behaviors as well as amplitudes. Furthermore, a protracted acidosis reduced the number of presynaptic terminals, while it did not affect the postsynaptic compartment. Application of the diarylamidine Diminazene Aceturate (DA) during acidosis significantly reverted both the loss of neuronal firing and bursting and the reduction of presynaptic terminals. Finally, in vivo DA delivery ameliorated the clinical disease course of EAE mice, reducing demyelination and axonal damage. DA is known to block acid-sensing ion channels (ASICs), which are proton-gated, voltage-insensitive, Na(+) permeable channels principally expressed by peripheral and central nervous system neurons. Our data suggest that ASICs activation during acidosis modulates network electrical activity and exacerbates neuro-degeneration in EAE mice. Therefore pharmacological modulation of ASICs in neuroinflammatory diseases could represent a new promising strategy for future therapies aimed at neuro-protection.

Glia-enriched stem-cell 3D model of the human brain mimics the glial-immune neurodegenerative phenotypes of multiple sclerosis.

The role of central nervous system (CNS) glia in sustaining self-autonomous inflammation and driving clinical progression in multiple sclerosis (MS) is gaining scientific interest. We applied a single transcription factor ( SOX10 )-based protocol to accelerate oligodendrocyte differentiation from hiPSC-derived neural precursor cells, generating self-organizing forebrain organoids. These organoids include neurons, astrocytes, oligodendroglia, and hiPSC-derived microglia to achieve immunocompetence. Over 8 weeks, organoids reproducibly generated mature CNS cell types, exhibiting single-cell transcriptional profiles similar to the adult human brain. Exposed to inflamed cerebrospinal fluid (CSF) from MS patients, organoids properly mimic macroglia-microglia neuro-degenerative phenotypes and intercellular communication seen in chronic active MS. Oligodendrocyte vulnerability emerged by day 6 post-MS-CSF exposure, with nearly 50% reduction. Temporally-resolved organoid data support and expand on the role of soluble CSF mediators in sustaining downstream events leading to oligodendrocyte death and inflammatory neurodegeneration. Such findings support implementing this organoid model for drug screening to halt inflammatory neurodegeneration.

A glia-enriched stem cell 3D model of the human brain mimics the glial-immune neurodegenerative phenotypes of multiple sclerosis.

The role of central nervous system (CNS) glia in sustaining self-autonomous inflammation and driving clinical progression in multiple sclerosis (MS) is gaining scientific interest. We applied a single transcription factor (SOX10)-based protocol to accelerate oligodendrocyte differentiation from human induced pluripotent stem cell (hiPSC)-derived neural precursor cells, generating self-organizing forebrain organoids. These organoids include neurons, astrocytes, oligodendroglia, and hiPSC-derived microglia to achieve immunocompetence. Over 8 weeks, organoids reproducibly generated mature CNS cell types, exhibiting single-cell transcriptional profiles similar to the adult human brain. Exposed to inflamed cerebrospinal fluid (CSF) from patients with MS, organoids properly mimic macroglia-microglia neurodegenerative phenotypes and intercellular communication seen in chronic active MS. Oligodendrocyte vulnerability emerged by day 6 post-MS-CSF exposure, with nearly 50% reduction. Temporally resolved organoid data support and expand on the role of soluble CSF mediators in sustaining downstream events leading to oligodendrocyte death and inflammatory neurodegeneration. Such findings support the implementation of this organoid model for drug screening to halt inflammatory neurodegeneration.

Neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50-60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS.

Massive iron accumulation in PKAN-derived neurons and astrocytes: light on the human pathological phenotype.

Neurodegeneration associated with defective pantothenate kinase-2 (PKAN) is an early-onset monogenic autosomal-recessive disorder. The hallmark of the disease is the massive accumulation of iron in the globus pallidus brain region of patients. PKAN is caused by mutations in the PANK2 gene encoding the mitochondrial enzyme pantothenate kinase-2, whose function is to catalyze the first reaction of the CoA biosynthetic pathway. To date, the way in which this alteration leads to brain iron accumulation has not been elucidated. Starting from previously obtained hiPS clones, we set up a differentiation protocol able to generate inhibitory neurons. We obtained striatal-like medium spiny neurons composed of approximately 70-80% GABAergic neurons and 10-20% glial cells. Within this mixed population, we detected iron deposition in both PKAN cell types, however, the viability of PKAN GABAergic neurons was strongly affected. CoA treatment was able to reduce cell death and, notably, iron overload. Further differentiation of hiPS clones in a pure population of astrocytes showed particularly evident iron accumulation, with approximately 50% of cells positive for Perls staining. The analysis of these PKAN astrocytes indicated alterations in iron metabolism, mitochondrial morphology, respiratory activity, and oxidative status. Moreover, PKAN astrocytes showed signs of ferroptosis and were prone to developing a stellate phenotype, thus gaining neurotoxic features. This characteristic was confirmed in iPS-derived astrocyte and glutamatergic neuron cocultures, in which PKAN glutamatergic neurons were less viable in the presence of PKAN astrocytes. This newly generated astrocyte model is the first in vitro disease model recapitulating the human phenotype and can be exploited to deeply clarify the pathogenetic mechanisms underlying the disease.

Neural precursor cells tune striatal connectivity through the release of IGFBPL1.

The adult brain retains over life endogenous neural stem/precursor cells (eNPCs) within the subventricular zone (SVZ). Whether or not these cells exert physiological functions is still unclear. In the present work, we provide evidence that SVZ-eNPCs tune structural, electrophysiological, and behavioural aspects of striatal function via secretion of insulin-like growth factor binding protein-like 1 (IGFBPL1). In mice, selective ablation of SVZ-eNPCs or selective abrogation of IGFBPL1 determined an impairment of striatal medium spiny neuron morphology, a higher failure rate in GABAergic transmission mediated by fast-spiking interneurons, and striatum-related behavioural dysfunctions. We also found IGFBPL1 expression in the human SVZ, foetal and induced-pluripotent stem cell-derived NPCs. Finally, we found a significant correlation between SVZ damage, reduction of striatum volume, and impairment of information processing speed in neurological patients. Our results highlight the physiological role of adult SVZ-eNPCs in supporting cognitive functions by regulating striatal neuronal activity.

Cell-attached and Whole-cell Patch-clamp Recordings of Dopamine Neurons in the Substantia Nigra Pars Compacta of Mouse Brain Slices.

The Substantia Nigra pars compacta (SNc) is a midbrain dopaminergic nucleus that plays a key role in modulating motor and cognitive functions. It is crucially involved in several disorders, particularly Parkinson's disease, which is characterized by a progressive loss of SNc dopaminergic cells. Electrophysiological studies on SNc neurons are of paramount importance to understand the role of dopaminergic transmission in health and disease. Here, we provide an extensive protocol to prepare SNc-containing mouse brain slices and record the electrical activity of dopaminergic cells. We describe all the necessary steps, including mouse transcardiac perfusion, brain extraction, slice cutting, and patch-clamp recordings.

TBL1XR1 Ensures Balanced Neural Development Through NCOR Complex-Mediated Regulation of the MAPK Pathway.

TBL1XR1 gene is associated with multiple developmental disorders presenting several neurological aspects. The relative protein is involved in the modulation of important cellular pathways and master regulators of transcriptional output, including nuclear receptor repressors, Wnt signaling, and MECP2 protein. However, TBL1XR1 mutations (including complete loss of its functions) have not been experimentally studied in a neurological context, leaving a knowledge gap in the mechanisms at the basis of the diseases. Here, we show that Tbl1xr1 knock-out mice exhibit behavioral and neuronal abnormalities. Either the absence of TBL1XR1 or its point mutations interfering with stability/regulation of NCOR complex induced decreased proliferation and increased differentiation in neural progenitors. We suggest that this developmental unbalance is due to a failure in the regulation of the MAPK cascade. Taken together, our results broaden the molecular and functional aftermath of TBL1XR1 deficiency associated with human disorders.

Inhibiting glycolysis rescues memory impairment in an intellectual disability Gdi1-null mouse.

OBJECTIVES: GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear. METHODS: We carried out proteomic analysis and monitored [18F]-fluoro-2-deoxy-d-glucose uptake into brain slices of Gdi1 knockout and wild type control mice. d-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP, d-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible Gdi1 knockout mouse carrying the Gdi1 deletion only in adult astrocytes and conducted behavioural tests. RESULTS: Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [18F]-fluoro-2-deoxy-d-glucose revealed an increased d-glucose uptake in Gdi1 knockout tissue versus wild type control mice, consistent with the facilitated d-glucose uptake determined by FRET measurements. In mice with Gdi1 deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-d-glucose injection. CONCLUSIONS: These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice.

Pharmacological antagonism of kainate receptor rescues dysfunction and loss of dopamine neurons in a mouse model of human parkin-induced toxicity.

Mutations in the PARK2 gene encoding the protein parkin cause autosomal recessive juvenile Parkinsonism (ARJP), a neurodegenerative disease characterized by dysfunction and death of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Since a neuroprotective therapy for ARJP does not exist, research efforts aimed at discovering targets for neuroprotection are critically needed. A previous study demonstrated that loss of parkin function or expression of parkin mutants associated with ARJP causes an accumulation of glutamate kainate receptors (KARs) in human brain tissues and an increase of KAR-mediated currents in neurons in vitro. Based on the hypothesis that such KAR hyperactivation may contribute to the death of nigral DA neurons, we investigated the effect of KAR antagonism on the DA neuron dysfunction and death that occur in the parkinQ311X mouse, a model of human parkin-induced toxicity. We found that early accumulation of KARs occurs in the DA neurons of the parkinQ311X mouse, and that chronic administration of the KAR antagonist UBP310 prevents DA neuron loss. This neuroprotective effect is associated with the rescue of the abnormal firing rate of nigral DA neurons and downregulation of GluK2, the key KAR subunit. This study provides novel evidence of a causal role of glutamate KARs in the DA neuron dysfunction and loss occurring in a mouse model of human parkin-induced toxicity. Our results support KAR as a potential target in the development of neuroprotective therapy for ARJP.

Recurrent collateral connections of striatal medium spiny neurons are disrupted in models of Parkinson's disease.

The principal neurons of the striatum, GABAergic medium spiny neurons (MSNs), are interconnected by local recurrent axon collateral synapses. Although critical to many striatal models, it is not clear whether these connections are random or whether they preferentially link functionally related groups of MSNs. To address this issue, dual whole patch-clamp recordings were made from striatal MSNs in brain slices taken from transgenic mice in which D(1) or D(2) dopamine receptor expression was reported with EGFP (enhanced green fluorescent protein). These studies revealed that unidirectional connections were common between both D(1) receptor-expressing MSN (D(1) MSN) pairs (26%) and D(2) receptor-expressing MSN (D(2) MSN) pairs (36%). D(2) MSNs also commonly formed synapses on D(1) MSNs (27% of pairs). Conversely, only 6% of the D(1) MSNs formed detectable connections with D(2) MSNs. Furthermore, synaptic connections formed by D(1) MSNs were weaker than those formed by D(2) MSNs, a difference that was attributable to fewer GABA(A) receptors at D(1) MSN synapses. The strength of detectable recurrent connections was dramatically reduced in Parkinson's disease models. The studies demonstrate that recurrent collateral connections between MSNs are not random but rather differentially couple D(1) and D(2) MSNs. Moreover, this recurrent collateral network appears to be disrupted in Parkinson's disease models, potentially contributing to pathological alterations in MSN activity patterns and psychomotor symptoms.

Membrane potential changes occurring upon acidification influence the binding of small-molecule inhibitors to ASIC1a.

Acid-sensing ion channels (ASICs) are proton-activated, sodium-permeable channels, highly expressed in both central and peripheral nervous systems. ASIC1a is the most abundant isoform in the central nervous system and is credited to be involved in several neurological disorders including stroke, multiple sclerosis, and epilepsy. Interestingly, the affinity of ASIC1a for two antagonists, diminazene and amiloride, has recently been proposed to be voltage sensitive. Based on this evidence, it is expected that the pharmacology of ASIC1cannot be properly characterized by single-cell voltage-clamp, an experimental condition in which membrane potential is maintained close to resting values. In particular, these measurements do not take into account the influence of the membrane potential depolarization induced by ASIC1a activation during acidosis or neuronal activity. We show here the voltage-dependence of some small molecules antagonists (diminazene, amiloride and a new patented drug from Merck), but not of Psalmotoxin 1, a peptide binding to regions other than the pore. We also demonstrate that the opening of ASIC1a induced by moderate acidosis determines a depolarization sufficient to change the affinity of small molecule antagonists. The characterization of this mechanism was performed on CHO-K1 expressing ASIC1a and further confirmed in hippocampal neurons in culture. Finally, perforated-patch experiments indicate that intracellular modulations do not play a role in the voltage-dependent binding of small molecules. Since ASIC1a activation promotes a membrane depolarization that may influence the binding of small molecules, we propose to adopt experimental methods that do not interfere with the membrane potential for the drug screening of ASIC1a modulators.

SETD5 Regulates Chromatin Methylation State and Preserves Global Transcriptional Fidelity during Brain Development and Neuronal Wiring.

Mutations in one SETD5 allele are genetic causes of intellectual disability and autistic spectrum disorders. However, the mechanisms by which SETD5 regulates brain development and function remain largely elusive. Herein, we found that Setd5 haploinsufficiency impairs the proliferative dynamics of neural progenitors and synaptic wiring of neurons, ultimately resulting in behavioral deficits in mice. Mechanistically, Setd5 inactivation in neural stem cells, zebrafish, and mice equally affects genome-wide levels of H3K36me3 on active gene bodies. Notably, we demonstrated that SETD5 directly deposits H3K36me3, which is essential to allow on-time RNA elongation dynamics. Hence, Setd5 gene loss leads to abnormal transcription, with impaired RNA maturation causing detrimental effects on gene integrity and splicing. These findings identify SETD5 as a fundamental epigenetic enzyme controlling the transcriptional landscape in neural progenitors and their derivatives and illuminate the molecular events that connect epigenetic defects with neuronal dysfunctions at the basis of related human diseases.