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Epilepsy is one of the most frequent neurological diseases, with focal epilepsy accounting for the largest number of cases. The genetic alterations involved in focal epilepsy are far from being fully elucidated.Here, we show that defective lipid signalling caused by heterozygous ultra-rare variants in PIK3C2B, encoding for the class II phosphatidylinositol 3-kinase PI3K-C2β, underlie focal epilepsy in humans. We demonstrate that patients’ variants act as loss-of-function alleles, leading to impaired synthesis of the rare signalling lipid phosphatidylinositol 3,4-bisphosphate, resulting in mTORC1 hyperactivation. In vivo, mutant Pik3c2b alleles caused dose-dependent neuronal hyperexcitability and increased seizure susceptibility, indicating haploinsufficiency as a key driver of disease. Moreover, acute mTORC1 inhibition in mutant mice prevented experimentally induced seizures, providing a potential therapeutic option for a selective group of patients with focal epilepsy.Our findings reveal an unexpected role for class II PI3K-mediated lipid signalling in regulating mTORC1-dependent neuronal excitability in mice and humans.

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission, and the plastic modulation of their surface levels determines synaptic strength. AMPARs of different subunit compositions fulfill distinct roles in synaptic long-term potentiation (LTP) and depression (LTD) to enable learning. Largely unknown endocytic mechanisms mediate the subunit-selective regulation of the surface levels of GluA1-homomeric Ca 2+ -permeable (CP) versus heteromeric Ca 2+ -impermeable (CI) AMPARs. Here, we report that the Alzheimer’s disease risk factor CALM controls the surface levels of CP-AMPARs and thereby reciprocally regulates LTP and LTD in vivo to modulate learning. We show that CALM selectively facilitates the endocytosis of ubiquitinated CP-AMPARs via a mechanism that depends on ubiquitin recognition by its ANTH domain but is independent of clathrin. Our data identify CALM and related ANTH domain–containing proteins as the core endocytic machinery that determines the surface levels of CP-AMPARs to bidirectionally control synaptic plasticity and modulate learning in the mammalian brain.

Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3-phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2-mediated hyperactivation of Cdk5 downstream of receptor- and activity-dependent calcium influx. Our results unravel an unexpected function for PI(3)P-containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P-producing VPS34 kinase in neurological disease and neurodegeneration.

SUMMARYInformation processing in the brain is encoded as electrical impulses in neurons that are relayed from the presynaptic compartment to postsynaptic neurons by regulated neurotransmitter release. Neurons are known to rely on autophagy for the removal of defective proteins or organelles to maintain synaptic neurotransmission and to counteract neurodegeneration. In spite of its importance for neuronal health, the physiological substrates of neuronal autophagy in the absence of proteotoxic challenge have remained largely elusive. We use knockout mice conditionally lacking the essential autophagy protein ATG5 and quantitative proteomics to demonstrate that loss of neuronal autophagy causes the selective accumulation of tubular endoplasmic reticulum (ER) in axons, resulting in increased excitatory neurotransmission and compromised postnatal viability in vivo. The gain in excitatory neurotransmission is shown to be a consequence of elevated calcium release from ER stores via ryanodine receptors accumulated in axons and at presynaptic sites. We propose a model in which neuronal autophagy controls axonal ER calcium stores to regulate neurotransmission in healthy neurons and in the brain.

Aging is associated with functional alterations of synapses thought to contribute to age-dependent memory impairment (AMI). While therapeutic avenues to protect from AMI are largely elusive, supplementation of spermidine, a polyamine normally declining with age, has been shown to restore defective proteostasis and to protect from AMI in Drosophila. Here we demonstrate that dietary spermidine protects from age-related synaptic alterations at hippocampal mossy fiber (MF)-CA3 synapses and prevents the aging-induced loss of neuronal mitochondria. Dietary spermidine rescued age-dependent decreases in synaptic vesicle density and largely restored defective presynaptic MF-CA3 long-term potentiation (LTP) at MF-CA3 synapses (MF-CA3) in aged animals. In contrast, spermidine failed to protect CA3-CA1 hippocampal synapses characterized by postsynaptic LTP from age-related changes in function and morphology. Our data demonstrate that dietary spermidine attenuates age-associated deterioration of MF-CA3 synaptic transmission and plasticity. These findings provide a physiological and molecular basis for the future therapeutic usage of spermidine.

Brain development and function depend on the directed and coordinated migration of neurons from proliferative zones to their final position. The secreted glycoprotein Reelin is an important factor directing neuronal migration. Loss of Reelin function results in the severe developmental disorder lissencephaly and is associated with neurological diseases in humans. Reelin signals via the lipoprotein receptors very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), but the exact mechanism by which these receptors control cellular function is poorly understood. We report that loss of the signaling scaffold intersectin 1 (ITSN1) in mice leads to defective neuronal migration and ablates Reelin stimulation of hippocampal long-term potentiation (LTP). Knockout (KO) mice lacking ITSN1 suffer from dispersion of pyramidal neurons and malformation of the radial glial scaffold, akin to the hippocampal lamination defects observed in VLDLR or ApoER2 mutants. ITSN1 genetically interacts with Reelin receptors, as evidenced by the prominent neuronal migration and radial glial defects in hippocampus and cortex seen in double-KO mice lacking ITSN1 and ApoER2. These defects were similar to, albeit less severe than, those observed in Reelin-deficient or VLDLR/ ApoER2 double-KO mice. Molecularly, ITSN1 associates with the VLDLR and its downstream signaling adaptor Dab1 to facilitate Reelin signaling. Collectively, these data identify ITSN1 as a component of Reelin signaling that acts predominantly by facilitating the VLDLR-Dab1 axis to direct neuronal migration in the cortex and hippocampus and to augment synaptic plasticity.

Summary Neurons are known to rely on autophagy for removal of defective proteins or organelles to maintain synaptic neurotransmission and counteract neurodegeneration. In spite of its importance for neuronal health, the physiological substrates of neuronal autophagy in the absence of proteotoxic challenge have remained largely elusive. We use knockout mice conditionally lacking the essential autophagy protein ATG5 and quantitative proteomics to demonstrate that loss of neuronal autophagy causes selective accumulation of tubular endoplasmic reticulum (ER) in axons, resulting in increased excitatory neurotransmission and compromised postnatal viability in vivo. The gain in excitatory neurotransmission is shown to be a consequence of elevated calcium release from ER stores via ryanodine receptors accumulated in axons and at presynaptic sites. We propose a model where neuronal autophagy controls axonal ER calcium stores to regulate neurotransmission in healthy neurons and in the brain., Graphical Abstract, Highlights • Neuronal autophagy controls the endoplasmic reticulum (ER) in axons • Loss of neuronal autophagy leads to increased excitatory neurotransmission • Increased neurotransmission is due to elevated calcium release from ER stores, Autophagy is crucial for nervous system function. However, its physiological substrates are largely unknown. Kuijpers et al. demonstrate, using knockout mice conditionally lacking the essential autophagy protein ATG5 and quantitative proteomics paired with electrophysiology and functional imaging experiments, that neuronal autophagy regulates presynaptic neurotransmission by controlling the axonal endoplasmic reticulum.

SummaryNeurotransmission depends on synaptic vesicle (SV) exocytosis driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation of vesicular synaptobrevin/VAMP2 (Syb2). Exocytic fusion is followed by endocytic SV membrane retrieval and the high-fidelity reformation of SVs. Syb2 is the most abundant SV protein with 70 copies per SV, yet, one to three Syb2 molecules appear to be sufficient for basal exocytosis. Here we demonstrate that loss of the Syb2-specific endocytic adaptor AP180 causes a moderate activity-dependent reduction of vesicular Syb2 levels, defects in SV reformation, and a corresponding impairment of neurotransmission that lead to excitatory/inhibitory imbalance, epileptic seizures, and premature death. Further reduction of Syb2 levels in AP180−/−/Syb2+/− mice results in perinatal lethality, whereas Syb2+/− mice partially phenocopy loss of AP180, indicating that reduced vesicular Syb2 levels underlie the observed defects in neurotransmission. Thus, a large vesicular Syb2 pool maintained by AP180 is crucial to sustain efficient neurotransmission and SV reformation.

Neurotransmission involves the calcium-regulated exocytic fusion of synaptic vesicles (SVs) and the subsequent retrieval of SV membranes followed by reformation of properly sized and shaped SVs. An unresolved question is whether each SV protein is sorted by its own dedicated adaptor or whether sorting is facilitated by association between different SV proteins. We demonstrate that endocytic sorting of the calcium sensor synaptotagmin 1 (Syt1) is mediated by the overlapping activities of the Syt1-associated SV glycoprotein SV2A/B and the endocytic Syt1-adaptor stonin 2 (Stn2). Deletion or knockdown of either SV2A/B or Stn2 results in partial Syt1 loss and missorting of Syt1 to the neuronal surface, whereas deletion of both SV2A/B and Stn2 dramatically exacerbates this phenotype. Selective missorting and degradation of Syt1 in the absence of SV2A/B and Stn2 impairs the efficacy of neurotransmission at hippocampal synapses. These results indicate that endocytic sorting of Syt1 to SVs is mediated by the overlapping activities of SV2A/B and Stn2 and favor a model according to which SV protein sorting is guarded by both cargo-specific mechanisms as well as association between SV proteins.

Susceptibility to obesity is linked to genes regulating neurotransmission, pancreatic beta-cell function and energy homeostasis. Genome-wide association studies have identified associations between body mass index and two loci near cell adhesion molecule 1 (CADM1) and cell adhesion molecule 2 (CADM2), which encode membrane proteins that mediate synaptic assembly. We found that these respective risk variants associate with increased CADM1 and CADM2 expression in the hypothalamus of human subjects. Expression of both genes was elevated in obese mice, and induction of Cadm1 in excitatory neurons facilitated weight gain while exacerbating energy expenditure. Loss of Cadm1 protected mice from obesity, and tract-tracing analysis revealed Cadm1-positive innervation of POMC neurons via afferent projections originating from beyond the arcuate nucleus. Reducing Cadm1 expression in the hypothalamus and hippocampus promoted a negative energy balance and weight loss. These data identify essential roles for Cadm1-mediated neuronal input in weight regulation and provide insight into the central pathways contributing to human obesity.

Neurotransmission is mediated by the exocytic release of neurotransmitters from readily releasable synaptic vesicles (SVs) at the active zone. To sustain neurotransmission during periods of elevated activity, release-ready vesicles need to be replenished from the reserve pool of SVs. The SV-associated synapsins are crucial for maintaining this reserve pool and regulate the mobilization of reserve pool SVs. How replenishment of release-ready SVs from the reserve pool is regulated and which other factors cooperate with synapsins in this process is unknown. Here we identify the endocytic multidomain scaffold protein intersectin as an important regulator of SV replenishment at hippocampal synapses. We found that intersectin directly associates with synapsin I through its Src-homology 3 A domain, and this association is regulated by an intramolecular switch within intersectin 1. Deletion of intersectin 1/2 in mice alters the presynaptic nanoscale distribution of synapsin I and causes defects in sustained neurotransmission due to defective SV replenishment. These phenotypes were rescued by wild-type intersectin 1 but not by a locked mutant of intersectin 1. Our data reveal intersectin as an autoinhibited scaffold that serves as a molecular linker between the synapsin-dependent reserve pool and the presynaptic endocytosis machinery.

Reggie-1 and -2 (flotillins) reside at recycling vesicles and promote jointly with Rab11a the targeted delivery of cargo. Recycling is essential for synapse formation suggesting that reggies and Rab11a may regulate the development of spine synapses. Recycling vesicles provide cargo for dendritic growth and recycle surface glutamate receptors (AMPAR, GluA) for long-term potentiation (LTP) induced surface exposure. Here, we show reduced number of spine synapses and impairment of an in vitro correlate of LTP in hippocampal neurons from reggie-1 k.o. (Flot2-/-) mice maturating in culture. These defects apparently result from reduced trafficking of PSD-95 revealed by live imaging of 10 div reggie-1 k.o. (Flot2-/-) neurons and likely impairs co-transport of cargo destined for spines: N-cadherin and the glutamate receptors GluA1 and GluN1. Impaired cargo trafficking and fewer synapses also emerged in reggie-1 siRNA, reggie-2 siRNA, and reggie-1 and -2 siRNA-treated neurons and was in siRNA and k.o. neurons rescued by reggie-1-EGFP and CA-Rab11a-EGFP. While correlative expressional changes of specific synapse proteins were observed in reggie-1 k.o. (Flot2-/-) brains in vivo, this did not occur in neurons maturating in vitro. Our work suggests that reggie-1 and reggie-2 function at Rab11a recycling containers in the transport of PSD-95, N-cadherin, GluA1 and GluN1, and promote (together with significant signaling molecules) spine-directed trafficking, spine synapse formation and the in vitro correlate of LTP. published

Neural cell adhesion molecule (NCAM) is the predominant carrier of the unusual glycan polysialic acid (PSA). Deficits in PSA and/or NCAM expression cause impairments in hippocampal long-term potentiation and depression (LTP and LTD) and are associated with schizophrenia and aging. In this study, we show that impaired LTP in adult NCAM-deficient (NCAM−/−) mice is restored by increasing the activity of the NMDA subtype of glutamate receptor (GluN) through either reducing the extracellular Mg2+concentration or applyingd-cycloserine (DCS), a partial agonist of the GluN glycine binding site. Pharmacological inhibition of the GluN2A subtype reduced LTP to the same level inNCAM−/−and wild-type (NCAM+/+) littermate mice and abolished the rescue by DCS inNCAM−/−mice, suggesting that the effects of DCS are mainly mediated by GluN2A. The insufficient contribution of GluN to LTD inNCAM−/−mice was also compensated for by DCS. Furthermore, impaired contextual and cued fear conditioning levels were restored inNCAM−/−mice by administration of DCS before conditioning. In 12-month-oldNCAM−/−, but notNCAM+/+mice, there was a decline in LTP compared with 3-month-old mice that could be rescued by DCS. In 24-month-old mice of both genotypes, there was a reduction in LTP that could be fully restored by DCS inNCAM+/+mice but only partially restored inNCAM−/−mice. Thus, several deficiencies ofNCAM−/−mice can be ameliorated by enhancing GluN2A-mediated neurotransmission with DCS.

SNAP-25 is a key component of the synaptic-vesicle fusion machinery, involved in several psychiatric diseases including schizophrenia and ADHD. SNAP-25 protein expression is lower in different brain areas of schizophrenic patients and in ADHD mouse models. How the reduced expression of SNAP-25 alters the properties of synaptic transmission, leading to a pathological phenotype, is unknown. We show that, unexpectedly, halved SNAP-25 levels at 13-14 DIV not only fail to impair synaptic transmission but instead enhance evoked glutamatergic neurotransmission. This effect is possibly dependent on presynaptic voltage-gated calcium channel activity and is not accompanied by changes in spontaneous quantal events or in the pool of readily releasable synaptic vesicles. Notably, synapses of 13-14 DIV neurons with reduced SNAP-25 expression show paired-pulse depression as opposed to paired-pulse facilitation occurring in their wild-type counterparts. This phenotype disappears with synapse maturation. As alterations in short-term plasticity represent a new mechanism contributing to cognitive impairments in intellectual disabilities, our data provide mechanistic clues for neuronal circuit alterations in psychiatric diseases characterized by reduced expression of SNAP-25.

The neural cell adhesion molecule, NCAM, is a pivotal regulator of neural development, with key roles in axonal and dendritic growth and synaptic plasticity. Alterations in NCAM expression or proteolytic cleavage have been linked to human neuropsychiatric disorders such as schizophrenia, bipolar disorder and Alzheimer's disease, and may contribute to cognitive dysfunction. We have generated mice overexpressing the NCAM extracellular (EC) proteolytic cleavage fragment which has been reported to be increased in schizophrenic versus normal brains. These mice show impaired GABAergic innervation and reduced number of apical dendritic spines on pyramidal neurons in the prefrontal cortex (PFC). Here, these NCAM-EC transgenic mice were subjected to behavioral tasks and electrophysiological measurements to determine the impact of structural abnormalities in the PFC on synaptic and cognitive functions. NCAM-EC mice exhibited impaired working memory in a delayed non-match-to-sample task, which requires PFC function, but showed no differences in anxiety, olfactory abilities, or sociability. Transgenic mice displayed impaired long- and short-term potentiation in the PFC but normal synaptic plasticity in the hippocampus, suggesting that the abnormal synaptic innervation in NCAM-EC mice impairs PFC plasticity and alters working memory. These findings may have implications for cognitive dysfunctions observed in neuropsychiatric disorders.

The neural cell adhesion molecule (NCAM) is the predominant carrier of α2,8 polysialic acid (PSA) in the mammalian brain. Abnormalities in PSA and NCAM expression are associated with schizophrenia in humans and cause deficits in hippocampal synaptic plasticity and contextual fear conditioning in mice. Here, we show that PSA inhibits opening of recombinant NMDA receptors composed of GluN1/2B (NR1/NR2B) or GluN1/2A/2B (NR1/NR2A/NR2B) but not of GluN1/2A (NR1/NR2A) subunits. Deficits in NCAM/PSA increase GluN2B-mediated transmission and Ca2+transients in the CA1 region of the hippocampus. In line with elevation of GluN2B-mediated transmission, defects in long-term potentiation in the CA1 region and contextual fear memory in NCAM/PSA-deficient mice are abrogated by application of a GluN2B-selective antagonist. Furthermore, treatment with the glutamate scavenger glutamic-pyruvic transaminase, ablation ofRas-GRF1(a mediator of GluN2B signaling to p38 MAPK), or direct inhibition of hyperactive p38 MAPK can restore impaired synaptic plasticity in brain slices lacking PSA/NCAM. Thus, PSA carried by NCAM regulates plasticity and learning by inhibition of the GluN2B-Ras-GRF1-p38 MAPK signaling pathway. These findings implicate carbohydrates carried by adhesion molecules in modulating NMDA receptor signaling in the brain and demonstrate reversibility of cognitive deficits associated with ablation of a schizophrenia-related adhesion molecule.