Developing Iron Nanochelating Agents: Preliminary Investigation of Effectiveness and Safety for Central Nervous System Applications.

Excessive iron levels are believed to contribute to the development of neurodegenerative disorders by promoting oxidative stress and harmful protein clustering. Novel chelation treatments that can effectively remove excess iron while minimizing negative effects on the nervous system are being explored. This study focuses on the creation and evaluation of innovative nanobubble (NB) formulations, shelled with various polymers such as glycol-chitosan (GC) and glycol-chitosan conjugated with deferoxamine (DFO), to enhance their ability to bind iron. Various methods were used to evaluate their physical and chemical properties, chelation capacity in diverse iron solutions and impact on reactive oxygen species (ROS). Notably, the GC-DFO NBs demonstrated the ability to decrease amyloid-ß protein misfolding caused by iron. To assess potential toxicity, in vitro cytotoxicity testing was conducted using organotypic brain cultures from the substantia nigra, revealing no adverse effects at appropriate concentrations. Additionally, the impact of NBs on spontaneous electrical signaling in hippocampal neurons was examined. Our findings suggest a novel nanochelation approach utilizing DFO-conjugated NBs for the removal of excess iron in cerebral regions, potentially preventing neurotoxic effects.

p140Cap regulates the composition and localization of the NMDAR complex in synaptic lipid rafts.

The N-Methyl-D-aspartate receptors (NMDAR) are key players in both physiological and pathological synaptic plasticity because of their involvement in many aspects of neuronal transmission as well as learning and memory. The contribution in these events of different types of GluN2A-interacting proteins is still unclear. The p140Cap scaffold protein acts as a hub for postsynaptic complexes relevant to psychiatric and neurological disorders and regulates synaptic functions like the stabilization of mature dendritic spine, memory consolidation, long-term potentiation, and depression. Here we demonstrate that p140Cap directly binds the GluN2A subunit of NMDAR and modulates GluN2A-associated molecular network. Indeed, in p140Cap knockout male mice, GluN2A is less associated with PSD95 both in ex vivo synaptosomes and in cultured hippocampal neurons and p140Cap expression in knockout neurons can rescue GluN2A and PSD95 colocalization. p140Cap is crucial in the recruitment of GluN2A-containing NMDARs and, consequently, in regulating NMDARs intrinsic properties. p140Cap is associated to synaptic lipid-raft (LR) and to soluble postsynaptic membranes and GluN2A and PSD95 are less recruited into synaptic LR of p140Cap knockout male mice. g-STED microscopy on hippocampal neurons confirmed that p140Cap is required for embedding GluN2A clusters in LR in an activity-dependent fashion. In the synaptic compartment p140Cap influences the association between GluN2A and PSD95 and modulates GluN2A enrichment into LR. Overall, such increase in these membrane domains rich in signalling molecules results in improved signal transduction efficiency.SIGNIFICANT STATEMENTHere we originally show that the adaptor protein p140Cap directly binds the GluN2A subunit of NMDAR and modulates the GluN2A-associated molecular network. Moreover, we show for the first time that p140Cap also associates to synaptic lipid rafts and controls the selective recruitment of GluN2A and PSD95 to this specific compartment. Finally, g-STED microscopy on hippocampal neurons confirmed that p140Cap is required for embedding GluN2A clusters in lipid rafts in an activity-dependent fashion. Overall, our findings provide the molecular and functional dissection of p140Cap as a new active member of a highly dynamic synaptic network involved in memory consolidation, LTP and LTD that are known to be altered in neurological and psychiatric disorders.

Two firing modes and well-resolved Na+, K+, and Ca2+ currents at the cell-microelectrode junction of spontaneously active rat chromaffin cell on MEAs.

We recorded spontaneous extracellular action potentials (eAPs) from rat chromaffin cells (CCs) at 37 °C using microelectrode arrays (MEAs) and compared them with intracellularly recorded APs (iAPs) through conventional patch clamp recordings at 22 °C. We show the existence of two distinct firing modes on MEAs: a ~ 4 Hz irregular continuous firing and a frequent intermittent firing mode where periods of high-intraburst frequency (~ 8 Hz) of ~ 7 s duration are interrupted by silent periods of ~ 12 s. eAPs occurred either as negative- or positive-going signals depending on the contact between cell and microelectrode: either predominantly controlled by junction-membrane ion channels (negative-going) or capacitive/ohmic coupling (positive-going). Negative-going eAPs were found to represent the trajectory of the Na+, Ca2+, and K+ currents passing through the cell area in tight contact with the microelectrode during an AP (point-contact junction). The inward Nav component of eAPs was blocked by TTX in a dose-dependent manner (IC50 ~ 10 nM) while the outward component was strongly attenuated by the BK channel blocker paxilline (200 nM) or TEA (5 mM). The SK channel blocker apamin (200 nM) had no effect on eAPs. Inward Nav and Cav currents were well-resolved after block of Kv and BK channels or in cells showing no evident outward K+ currents. Unexpectedly, on the same type of cells, we could also resolve inward L-type currents after adding nifedipine (3 µM). In conclusion, MEAs provide a direct way to record different firing modes of rat CCs and to estimate the Na+, Ca2+, and K+ currents that sustain cell firing and spontaneous catecholamines secretion.

Missense variants in RPH3A cause defects in excitatory synaptic function and are associated with a clinically variable neurodevelopmental disorder.

PURPOSE: RPH3A encodes a protein involved in the stabilization of GluN2A subunit of N-methyl-D-aspartate (NMDA)-type glutamate receptors at the cell surface, forming a complex essential for synaptic plasticity and cognition. We investigated the effect of variants in RPH3A in patients with neurodevelopmental disorders. METHODS: By using trio-based exome sequencing, GeneMatcher, and screening of 100,000 Genomes Project data, we identified 6 heterozygous variants in RPH3A. In silico and in vitro models, including rat hippocampal neuronal cultures, have been used to characterize the effect of the variants. RESULTS: Four cases had a neurodevelopmental disorder with untreatable epileptic seizures [p.(Gln73His)dn; p.(Arg209Lys); p.(Thr450Ser)dn; p.(Gln508His)], and 2 cases [p.(Arg235Ser); p.(Asn618Ser)dn] showed high-functioning autism spectrum disorder. Using neuronal cultures, we demonstrated that p.(Thr450Ser) and p.(Asn618Ser) reduce the synaptic localization of GluN2A; p.(Thr450Ser) also increased the surface levels of GluN2A. Electrophysiological recordings showed increased GluN2A-dependent NMDA ionotropic glutamate receptor currents for both variants and alteration of postsynaptic calcium levels. Finally, expression of the Rph3AThr450Ser variant in neurons affected dendritic spine morphology. CONCLUSION: Overall, we provide evidence that missense gain-of-function variants in RPH3A increase GluN2A-containing NMDA ionotropic glutamate receptors at extrasynaptic sites, altering synaptic function and leading to a clinically variable neurodevelopmental presentation ranging from untreatable epilepsy to autism spectrum disorder.

The ryanodine receptor-calstabin interaction stabilizer S107 protects hippocampal neurons from GABAergic synaptic alterations induced by Abeta42 oligomers.

The oligomeric form of the peptide amyloid beta 42 (Abeta42) contributes to the development of synaptic abnormalities and cognitive impairments associated with Alzheimer's disease (AD). To date, there is a gap in knowledge regarding how Abeta42 alters the elementary parameters of GABAergic synaptic function. Here we found that Abeta42 increased the frequency and amplitude of miniature GABAergic currents as well as the amplitude of evoked inhibitory postsynaptic currents. When we focused on paired pulse depression (PPD) to establish whether GABA release probability was affected by Abeta42, we did not observe any significant change. On the other hand, a more detailed investigation of the presynaptic effects induced by Abeta42 by means of multiple probability fluctuation analysis and cumulative amplitude analysis showed an increase in both the size of the readily releasable pool responsible for synchronous release and the number of release sites. We further explored whether ryanodine receptors (RyRs) contributed to exacerbating these changes by stabilizing the interaction between RyRs and the accessory protein calstabin. We observed that the RyR-calstabin interaction stabilizer S107 restored the synaptic parameters to values comparable to those measured in control conditions. In conclusion, our results clarify the mechanisms of potentiation of GABAergic synapses induced by Abeta42. We further suggest that RyRs are involved in the control of synaptic activity during the early stage of AD onset and that their stabilization could represent a new therapeutical approach for AD treatment. KEY POINTS: Accumulation of the peptide amyloid beta 42 (Abeta42) is a key characteristic of Alzheimer's disease (AD) and causes synaptic dysfunctions. To date, the effects of Abeta42 accumulation on GABAergic synapses are poorly understood. The findings reported here suggest that, similarly to what is observed on glutamatergic synapses, Abeta42 modifies GABAergic synapses by targeting ryanodine receptors and causing calcium dysregulation. The GABAergic impairments can be restored by the ryanodine receptor-calstabin interaction stabilizer S107. Based on this research, RyRs stabilization may represent a novel pharmaceutical strategy for preventing or delaying AD.

COUP-TFI/Nr2f1 Orchestrates Intrinsic Neuronal Activity during Development of the Somatosensory Cortex.

The formation of functional cortical maps in the cerebral cortex results from a timely regulated interaction between intrinsic genetic mechanisms and electrical activity. To understand how transcriptional regulation influences network activity and neuronal excitability within the neocortex, we used mice deficient for Nr2f1 (also known as COUP-TFI), a key determinant of primary somatosensory (S1) area specification during development. We found that the cortical loss of Nr2f1 impacts on spontaneous network activity and synchronization of S1 cortex at perinatal stages. In addition, we observed alterations in the intrinsic excitability and morphological features of layer V pyramidal neurons. Accordingly, we identified distinct voltage-gated ion channels regulated by Nr2f1 that might directly influence intrinsic bioelectrical properties during critical time windows of S1 cortex specification. Altogether, our data suggest a tight link between Nr2f1 and neuronal excitability in the developmental sequence that ultimately sculpts the emergence of cortical network activity within the immature neocortex.

Amyloid Beta42 oligomers up-regulate the excitatory synapses by potentiating presynaptic release while impairing postsynaptic NMDA receptors.

KEY POINTS: NMDA receptors (NMDARs) are key molecules for controlling neuronal plasticity, learning and memory processes. Their function is impaired during Alzheimer's disease (AD) but the exact consequence on synaptic function is not yet fully identified. An important hallmark of AD onset is represented by the neuronal accumulation of Amyloid Beta42 oligomers (Abeta42) that we have recently shown to be responsible for the increased intracellular Ca2+ concentration through ryanodine receptors (RyRs). Here we characterized the effects of Abeta42 on NMDA synapses showing specific pre- and post-synaptic functional changes that lead to a potentiation of basal and synchronous NMDA synaptic transmission. These overall effects can be abolished by decreasing Ca2+ release from RyRs with specific inhibitors that we propose as new pharmacological tools for AD treatment. ABSTRACT: We have recently shown that Amyloid Beta42 oligomers (Abeta42) cause calcium dysregulation in hippocampal neurons by stimulating Ca2+ release from ryanodine receptors (RyRs) and inhibiting Ca2+ entry through NMDA receptors (NMDARs). Here, we found that Abeta42 decrease the average NMDA-activated inward current and that Ca2+ entry through NMDARs is accompanied by Ca2+ release from the stores. The overall amount of intraellular Ca2+ concentration([Ca2+ ]i ) increase during NMDA application is 50% associated with RyR opening and 50% with NMDARs activation. Addition of Abeta42 does not change this proportion. We estimated the number of NMDARs expressed in hippocampal neurons and their unitary current. We found that Abeta42 decrease the number of NMDARs without altering their unitary current. Paradoxically, the oligomer increases the size of electrically evoked eEPSCs induced by NMDARs activation. We found that this is the consequence of the increased release probability (p) of glutamate and the number of release sites (N) of NMDA synapses, while the quantal size (q) is significantly decreased as expected from the decreased number of NMDARs. An increased number of release sites induced by Abeta42 is also supported by the increased size of the ready releasable pool (RRPsyn) and by the enhanced percentage of paired pulse depression (PPD). Interestingly, the RyRs inhibitor dantrolene prevents the increase of PPD induced by Abeta42 oligomers. In conclusion, Abeta42 up-regulates NMDA synaptic responses with a mechanism involving RyRs that occurs during the early stages of Alzheimer's disease (AD) onset. This suggests that new selective modulators of RyRs may be useful for designing effective therapies to treat AD patients.

Cav1.2 channelopathies causing autism: new hallmarks on Timothy syndrome.

Cav1.2 L-type calcium channels play key roles in long-term synaptic plasticity, sensory transduction, muscle contraction, and hormone release. De novo mutations in the gene encoding Cav1.2 (CACNA1C) causes two forms of Timothy syndrome (TS1, TS2), characterized by a multisystem disorder inclusive of cardiac arrhythmias, long QT, autism, and adrenal gland dysfunction. In both TS1 and TS2, the missense mutation G406R is on the alternatively spliced exon 8 and 8A coding for the IS6-helix of Cav1.2 and is responsible for the penetrant form of autism in most TS individuals. The mutation causes specific gain-of-function changes to Cav1.2 channel gating: a "leftward shift" of voltage-dependent activation, reduced voltage-dependent inactivation, and a "leftward shift" of steady-state inactivation. How this occurs and how Cav1.2 gating changes alter neuronal firing and synaptic plasticity is still largely unexplained. Trying to better understanding the molecular basis of Cav1.2 gating dysfunctions leading to autism, here, we will present and discuss the properties of recently reported typical and atypical TS phenotypes and the effective gating changes exhibited by missense mutations associated with long QTs without extracardiac symptoms, unrelated to TS. We will also discuss new emerging views achieved from using iPSCs-derived neurons and the newly available autistic TS2-neo mouse model, both appearing promising for understanding neuronal mistuning in autistic TS patients. We will also analyze and describe recent proposals of molecular pathways that might explain mistuned Ca2+-mediated and Ca2+-independent excitation-transcription signals to the nucleus. Briefly, we will also discuss possible pharmacological approaches to treat autism associated with L-type channelopathies.

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.

Early Alterations of Hippocampal Neuronal Firing Induced by Abeta42.

We studied the effect of Amyloid ß 1-42 oligomers (Abeta42) on Ca2+ dependent excitability profile of hippocampal neurons. Abeta42 is one of the Amyloid beta peptides produced by the proteolytic processing of the amyloid precursor protein and participates in the initiating event triggering the progressive dismantling of synapses and neuronal circuits. Our experiments on cultured hippocampal network reveal that Abeta42 increases intracellular Ca2+ concentration by 46% and inhibits firing discharge by 19%. More precisely, Abeta42 differently regulates ryanodine (RyRs), NMDA receptors (NMDARs), and voltage gated calcium channels (VGCCs) by increasing Ca2+ release through RyRs and inhibiting Ca2+ influx through NMDARs and VGCCs. The overall increased intracellular Ca2+ concentration causes stimulation of K+ current carried by big conductance Ca2+ activated potassium (BK) channels and hippocampal network firing inhibition. We conclude that Abeta42 alters neuronal function by means of at least 4 main targets: RyRs, NMDARs, VGCCs, and BK channels. The development of selective modulators of these channels may in turn be useful for developing effective therapies that could enhance the quality of life of AD patients during the early onset of the pathology.

Electrochemical measurement of quantal exocytosis using microchips.

Carbon-fiber electrodes (CFEs) are the gold standard for quantifying the release of oxidizable neurotransmitters from single vesicles and single cells. Over the last 15 years, microfabricated devices have emerged as alternatives to CFEs that offer the possibility of higher throughput, subcellular spatial resolution of exocytosis, and integration with other techniques for probing exocytosis including microfluidic cell handling and solution exchange, optical imaging and stimulation, and electrophysiological recording and stimulation. Here we review progress in developing electrochemical electrode devices capable of resolving quantal exocytosis that are fabricated using photolithography.

Microelectrode Arrays of Diamond-Insulated Graphitic Channels for Real-Time Detection of Exocytotic Events from Cultured Chromaffin Cells and Slices of Adrenal Glands.

A microstructured graphitic 4 × 4 multielectrode array was embedded in a single-crystal diamond substrate (4 × 4 µG-SCD MEA) for real-time monitoring of exocytotic events from cultured chromaffin cells and adrenal slices. The current approach relies on the development of a parallel ion beam lithographic technique, which assures the time-effective fabrication of extended arrays with reproducible electrode dimensions. The reported device is suitable for performing amperometric and voltammetric recordings with high sensitivity and temporal resolution, by simultaneously acquiring data from 16 rectangularly shaped microelectrodes (20 × 3.5 µm(2)) separated by 200 µm gaps. Taking advantage of the array geometry we addressed the following specific issues: (i) detect both the spontaneous and KCl-evoked secretion simultaneously from several chromaffin cells directly cultured on the device surface, (ii) resolve the waveform of different subsets of exocytotic events, and (iii) monitoring quantal secretory events from thin slices of the adrenal gland. The frequency of spontaneous release was low (0.12 and 0.3 Hz, respectively, for adrenal slices and cultured cells) and increased up to 0.9 Hz after stimulation with 30 mM KCl in cultured cells. The spike amplitude as well as rise and decay time were comparable with those measured by carbon fiber microelectrodes and allowed to identify three different subsets of secretory events associated with "full fusion" events, "kiss-and-run" and "kiss-and-stay" exocytosis, confirming that the device has adequate sensitivity and time resolution for real-time recordings. The device offers the significant advantage of shortening the time to collect data by allowing simultaneous recordings from cell populations either in primary cell cultures or in intact tissues.

Firing properties of entorhinal cortex neurons and early alterations in an Alzheimer's disease transgenic model.

The entorhinal cortex (EC) is divided into medial (MEC) and lateral (LEC) anatomical areas, and layer II neurons of these two regions project to granule cells of the dentate gyrus through the medial and lateral perforant pathways (MPP and LPP), respectively. Stellate cells (SCs) represent the main neurons constituting the MPP inputs, while fan cells (FCs) represent the main LPP inputs. Here, we first characterized the excitability properties of SCs and FCs in adult wild-type (WT) mouse brain. Our data indicate that, during sustained depolarization, action potentials (APs) generated by SCs exhibit increased fast afterhyperpolarization and overshoot, making them able to fire at higher frequencies and to exhibit higher spike frequency adaptation (SFA) than FCs. Since the EC is one of the earliest brain regions affected during Alzheimer's disease (AD) progression, we compared SCs and FCs firing in 4-month-old WT and transgenic Tg2576 mice, a well-established AD mouse model. Tg2576-SCs displayed a slight increase in firing frequency during mild depolarization but otherwise normal excitability properties during higher stimulations. On the contrary, Tg2576-FCs exhibited a decreased firing frequency during mild and higher depolarizations, as well as an increased SFA. Our data identify the FCs as a neuronal population particularly sensitive to early pathological effects of chronic accumulation of APP-derived peptides, as it occurs in Tg2576 mice. As FCs represent the major input of sensory information to the hippocampus during memory acquisition, early alterations in their excitability profile could significantly contribute to the onset of cognitive decline in AD.

The Role of Ryanodine Receptors in Regulating Neuronal Activity and Its Connection to the Development of Alzheimer's Disease.

Research into the early impacts of Alzheimer's disease (AD) on synapse function is one of the most promising approaches to finding a treatment. In this context, we have recently demonstrated that the Abeta42 peptide, which builds up in the brain during the processing of the amyloid precursor protein (APP), targets the ryanodine receptors (RyRs) of mouse hippocampal neurons and potentiates calcium (Ca2+) release from the endoplasmic reticulum (ER). The uncontrolled increase in intracellular calcium concentration ([Ca2+]i), leading to the development of Ca2+ dysregulation events and related excitable and synaptic dysfunctions, is a consolidated hallmark of AD onset and possibly other neurodegenerative diseases. Since RyRs contribute to increasing [Ca2+]i and are thought to be a promising target for AD treatment, the goal of this review is to summarize the current level of knowledge regarding the involvement of RyRs in governing neuronal function both in physiological conditions and during the onset of AD.

Methodologies for Detecting Quantal Exocytosis in Adrenal Chromaffin Cells Through Diamond-Based MEAs.

Diamond-based multiarray sensors are suitable to detect in real-time exocytosis and action potentials from cultured, spontaneously firing chromaffin cells, primary hippocampal neurons, and midbrain dopaminergic neurons. Here, we focus on how amperometric measurements of catecholamine release are performed on micrographitic diamond multiarrays (µG-D-MEAs) with high temporal and spatial resolution by 16 electrodes simultaneously.

Alpha-synuclein oligomers alter the spontaneous firing discharge of cultured midbrain neurons.

The aim of this work was to monitor the effects of extracellular α-synuclein on the firing activity of midbrain neurons dissociated from substantia nigra TH-GFP mice embryos and cultured on microelectrode arrays (MEA). We monitored the spontaneous firing discharge of the network for 21 days after plating and the role of glutamatergic and GABAergic inputs in regulating burst generation and network synchronism. Addition of GABA A , AMPA and NMDA antagonists did not suppress the spontaneous activity but allowed to identify three types of neurons that exhibited different modalities of firing and response to applied L-DOPA: high-rate (HR) neurons, low-rate pacemaking (LR-p), and low-rate non-pacemaking (LR-np) neurons. Most HR neurons were insensitive to L-DOPA, while the majority of LR-p neurons responded with a decrease of the firing discharge; less defined was the response of LR-np neurons. The effect of exogenous α-synuclein (α-syn) on the firing discharge of midbrain neurons was then studied by varying the exposure time (0-48 h) and the α-syn concentration (0.3-70 µM), while the formation of α-syn oligomers was monitored by means of AFM. Independently of the applied concentration, acute exposure to α-syn monomers did not exert any effect on the spontaneous firing rate of HR, LR-p, and LR-np neurons. On the contrary, after 48 h exposure, the firing activity was drastically altered at late developmental stages (14 days in vitro, DIV, neurons): α-syn oligomers progressively reduced the spontaneous firing discharge (IC50 = 1.03 µM), impaired burst generation and network synchronism, proportionally to the increased oligomer/monomer ratio. Different effects were found on early-stage developed neurons (9 DIV), whose firing discharge remained unaltered, regardless of the applied α-syn concentration and the exposure time. Our findings unravel, for the first time, the variable effects of exogenous α-syn at different stages of midbrain network development and provide new evidence for the early detection of neuronal function impairment associated to aggregated forms of α-syn.

Corrigendum: Alpha-synuclein oligomers alter the spontaneous firing discharge of cultured midbrain neurons.

[This corrects the article DOI: 10.3389/fncel.2023.1078550.].

mGluR5 PAMs rescue cortical and behavioural defects in a mouse model of CDKL5 deficiency disorder.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a devastating rare neurodevelopmental disease without a cure, caused by mutations of the serine/threonine kinase CDKL5 highly expressed in the forebrain. CDD is characterized by early-onset seizures, severe intellectual disabilities, autistic-like traits, sensorimotor and cortical visual impairments (CVI). The lack of an effective therapeutic strategy for CDD urgently demands the identification of novel druggable targets potentially relevant for CDD pathophysiology. To this aim, we studied Class I metabotropic glutamate receptors 5 (mGluR5) because of their important role in the neuropathological signs produced by the lack of CDKL5 in-vivo, such as defective synaptogenesis, dendritic spines formation/maturation, synaptic transmission and plasticity. Importantly, mGluR5 function strictly depends on the correct expression of the postsynaptic protein Homer1bc that we previously found atypical in the cerebral cortex of Cdkl5-/y mice. In this study, we reveal that CDKL5 loss tampers with (i) the binding strength of Homer1bc-mGluR5 complexes, (ii) the synaptic localization of mGluR5 and (iii) the mGluR5-mediated enhancement of NMDA-induced neuronal responses. Importantly, we showed that the stimulation of mGluR5 activity by administering in mice specific positive-allosteric-modulators (PAMs), i.e., 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) or RO6807794, corrected the synaptic, functional and behavioral defects shown by Cdkl5-/y mice. Notably, in the visual cortex of 2 CDD patients we found changes in synaptic organization that recapitulate those of mutant CDKL5 mice, including the reduced expression of mGluR5, suggesting that these receptors represent a promising therapeutic target for CDD.

The human channel gating-modifying A749G CACNA1D (Cav1.3) variant induces a neurodevelopmental syndrome-like phenotype in mice.

Germline de novo missense variants of the CACNA1D gene, encoding the pore-forming α1 subunit of Cav1.3 L-type Ca2+ channels (LTCCs), have been found in patients with neurodevelopmental and endocrine dysfunction, but their disease-causing potential is unproven. These variants alter channel gating, enabling enhanced Cav1.3 activity, suggesting Cav1.3 inhibition as a potential therapeutic option. Here we provide proof of the disease-causing nature of such gating-modifying CACNA1D variants using mice (Cav1.3AG) containing the A749G variant reported de novo in a patient with autism spectrum disorder (ASD) and intellectual impairment. In heterozygous mutants, native LTCC currents in adrenal chromaffin cells exhibited gating changes as predicted from heterologous expression. The A749G mutation induced aberrant excitability of dorsomedial striatum-projecting substantia nigra dopamine neurons and medium spiny neurons in the dorsal striatum. The phenotype observed in heterozygous mutants reproduced many of the abnormalities described within the human disease spectrum, including developmental delay, social deficit, and pronounced hyperactivity without major changes in gross neuroanatomy. Despite an approximately 7-fold higher sensitivity of A749G-containing channels to the LTCC inhibitor isradipine, oral pretreatment over 2 days did not rescue the hyperlocomotion. Cav1.3AG mice confirm the pathogenicity of the A749G variant and point toward a pathogenetic role of altered signaling in the dopamine midbrain system.