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Summary: Phosphatidylinositol 4-kinase IIα (PI4KIIα) generates essential phospholipids and is a cargo for endosomal adaptor proteins. Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle endocytosis mode during high neuronal activity and is sustained by glycogen synthase kinase 3β (GSK3β) activity. We reveal the GSK3β substrate PI4KIIα is essential for ADBE via its depletion in primary neuronal cultures. Kinase-dead PI4KIIα rescues ADBE in these neurons but not a phosphomimetic form mutated at the GSK3β site, Ser-47. Ser-47 phosphomimetic peptides inhibit ADBE in a dominant-negative manner, confirming that Ser-47 phosphorylation is essential for ADBE. Phosphomimetic PI4KIIα interacts with a specific cohort of presynaptic molecules, two of which, AGAP2 and CAMKV, are also essential for ADBE when depleted in neurons. Thus, PI4KIIα is a GSK3β-dependent interaction hub that silos essential ADBE molecules for liberation during neuronal activity.

Following exocytosis, synaptic vesicles (SVs) have to be reformed with the correct complement of proteins in the correct stoichiometry to ensure continued neurotransmission. Synaptophysin is a highly abundant, integral SV protein necessary for the efficient retrieval of the SV SNARE protein, synaptobrevin II (sybII). However the molecular mechanism underpinning synaptophysin-dependent sybII retrieval is still unclear. We recently identified a male patient with severe intellectual disability, hypotonia, epilepsy and callosal agenesis who has a point mutation in the juxtamembrane region of the fourth transmembrane domain of synaptophysin (T198I). This mutation had no effect on the activity-dependent retrieval of synaptophysin that was tagged with the genetically-encoded pH-sensitive reporter (pHluorin) in synaptophysin knockout hippocampal cultures. This suggested the mutant has no global effect on SV endocytosis, which was confirmed when retrieval of a different SV cargo (the glutamate transporter vGLUT1) was examined. However neurons expressing this T198I mutant did display impaired activity-dependent sybII retrieval, similar to that observed in synaptophysin knockout neurons. Interestingly this impairment did not result in an increased stranding of sybII at the plasma membrane. Screening of known human synaptophysin mutations revealed a similar presynaptic phenotype between T198I and a mutation found in X-linked intellectual disability. Thus this novel human synaptophysin mutation has revealed that aberrant retrieval and increased plasma membrane localisation of SV cargo can be decoupled in human disease.

Huntington's disease (HD) is caused by CAG repeat expansion within the HTT gene, with the dysfunction and eventual loss of striatal medium spiny neurons a notable feature. Since medium spiny neurons receive high amounts of synaptic input, we hypothesised that this vulnerability originates from an inability to sustain presynaptic performance during intense neuronal activity. To test this hypothesis, primary cultures of either hippocampal or striatal neurons were prepared from either wild-type mice or a knock-in HD mouse model which contains 140 poly-glutamine repeats in the huntingtin protein (httQ140/Q140). We identified a striatum-specific defect in synaptic vesicle (SV) endocytosis in httQ140/Q140 neurons that was only revealed during high frequency stimulation. This dysfunction was also present in neurons that were heterozygous for the mutant HTT allele. Depletion of endogenous huntingtin using hydrophobically-modified siRNA recapitulated this activity-dependent defect in wild-type neurons, whereas depletion of mutant huntingtin did not rescue the effect in httQ140/Q140 neurons. Importantly, this SV endocytosis defect was corrected by overexpression of wild-type huntingtin in homozygous httQ140/Q140 neurons. Therefore, we have identified an activity-dependent and striatum-specific signature of presynaptic dysfunction in neurons derived from pre-symptomatic HD mice, which is due to loss of wild-type huntingtin function. This presynaptic defect may render this specific neuronal subtype unable to operate efficiently during high frequency activity patterns, potentially resulting in dysfunctional neurotransmission, synapse failure and ultimately degeneration.

Summary: Compensatory endocytosis of released synaptic vesicles (SVs) relies on coordinated signaling at the lipid-protein interface. Here, we address the synaptic function of C-terminal binding protein 1 (CtBP1), a ubiquitous regulator of gene expression and membrane trafficking in cultured hippocampal neurons. In the absence of CtBP1, synapses form in greater density and show changes in SV distribution and size. The increased basal neurotransmission and enhanced synaptic depression could be attributed to a higher vesicular release probability and a smaller fraction of release-competent SVs, respectively. Rescue experiments with specifically targeted constructs indicate that, while synaptogenesis and release probability are controlled by nuclear CtBP1, the efficient recycling of SVs relies on its synaptic expression. The ability of presynaptic CtBP1 to facilitate compensatory endocytosis depends on its membrane-fission activity and the activation of the lipid-metabolizing enzyme PLD1. Thus, CtBP1 regulates SV recycling by promoting a permissive lipid environment for compensatory endocytosis. : Ivanova et al. demonstrate a dual role of CtBP1 in synaptic transmission. Nuclear CtBP1 restricts synaptogenesis and vesicular release probability, whereas presynaptic CtBP1 promotes compensatory endocytosis via activation of the lipid enzyme PLD1. Phosphorylation by Pak1 controls the redistribution of CtBP1 from active zones toward endocytic sites linking presynaptic exo- and endocytosis. Keywords: compensatory endocytosis, CtBP1, Bassoon, PLD1, synaptic vesicle recycling, membrane fission, synaptic vesicle pools, presynapse, exo-endocytosis coupling, Pak1