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.

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.

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.