Neuronal Form Of AP3 Is Essential For SV Reformation From Activity-dependent Bulk Endocytosis In GABAergic Synapses

The function of the nervous system depends on the exocytotic release of neurotransmitter from synaptic vesicles(SVs).To sustain neurotransmission,SV membranes need to be retrieved,and SVs have to be reformed locally within presynaptic nerve terminals.There are at least four different pathways for the retrieval of SV membranes have been put forward:(1)"kiss-and-run" endocytosis,(2)ultrafast endocytosis,(3)clathrin-mediated endocytosis,CME,and(4)activity-dependent bulk endocytosis,ADBE,which differ with respect to speed,likely molecular components,and maintenance of SV identity.Glutamatergic and GABAergic synapses,representing the major excitatory and inhibitory synapses in the brain,whether they differ presynaptically with regard to morphology,biogenesis,trafficking of synaptic vesicles(SV),and how these differences contribute to their function remain unknown.To comprehensively investigate presynaptic differences between excitatory and inhibitory synapses,we used cryo-electron tomography(Cryo-ET)in situ and endocytic tracing to assess the morphological and recycling properties of SV in hippocampal glutamatergic and GABAergic synapses.We found that excitatory presynaptic boutons contained a morphologically homogenous population of SV,with the majority being typically spherical shape,and the average diameter of SV were around 60 nm.In contrast,inhibitory presynaptic terminals contained a large heterogenous population of pleiomonphic SV that varied in diameter and shape,and that of GABAergic SV reached approximately 80 nm.Our results indicate that glutamatergic and GABAergic synapses use different endocytic modes with different temporal dynamics to retrieve and regenerate SV:glutamatergic synapses directly retrieve individual vesicles of regular sizes,whereas GABAergic synapses preferentially use large endosomes as intermediate stations to regenerate SVIn order to investigate the molecular mechanism involved in SV reformation from endosomes in GABAergic synapses,We used mass spectrum assay to quantify the differential proteins between inhibitory and excitatory neurons which were isolated by flow cytometry and further found that AP3B2,a neuronal specific adaptor protein enrich in GABAergic synaptic terminal and AP3B2 is required for SV reformation from endosomes.Inactivation of AP3B2 results in the accumulation of endosome-like vacuoles(ELY)and a corresponding depletion of properly shaped SV(SVS/SVM)in GABAergic but not glutamatergic synapses.In addition,defective of the AP3B2 decreased the content of characteristic GABAergic,but not glutamatergic synaptic vesicle proteins in isolated synaptosomal fractions.Accordingly,hyperactivity and increased seizure sensitivity were observed in AP3B2 deficient miceTaken together,our results indicated that:(1)GABAergic and glutamatergic synapses use different endocytic modes with different temporal dynamics to retrieve and regenerate SV;(2)AP3B2 is required for SV reformation from endosomes in GABAergic synapses;(3)AP3B2 plays an obligatory role in sorting of SV proteins in GABAergic synapses;(4)Endosomal disfunction in GABAergic synapses may be a new aspect to the pathogenesis of neurological disorders in Ap3b2-/-mice.