Removal of the calcium-dependent regulation of ATP binding in Synapsin I has distinct effects at excitatory and inhibitory synapses

Synapsins are the most abundant family of neuro-specific phosphoproteins associated with the cytoplasmic surface of the synaptic vesicle membrane. These proteins actively regulate synaptic transmission at the level of the presynaptic terminal by controlling the storage and mobilization of synaptic vesicles within a reserve pool. However, it is hypothesized that synapsins could be involved in other stages of synaptic vesicle dynamics such as trafficking, docking, fusion with the plasma membrane and consequent recycling. Synapsin I (SynI) in particular is expressed two isoforms (Ia and Ib) at the presynaptic compartment of all neurons in the adult brain. Several studies suggest that SynI is also involved in axon elongation and synaptic vesicle fusion kinetics. In human, nonsense and missense mutations of SYN1 gene are related to several diseases such as epilepsy and autism spectrum disorder; in fact, SynI knockout (KO) mice show an epileptic and autism-like phenotype. To carry out its functions, SynI requires to bind ATP in a Ca2+-dependent manner thanks to the coordination of a glutamate residue (E373). As ATP binding regulates SynI oligomerization and SV clustering, we analyzed the effect of E373K mutation on neurotransmitter release and short-term plasticity in excitatory and inhibitory synapses. We coupled electrophysiology (patch-clamp recordings) with electron microscopy in primary SynI KO hippocampal neurons in which either the human wild type or the E373K mutant SynI were re-introduced by infection with lentiviral vectors. Our data indicate that E373K mutation affects predominantly excitatory synapses. The frequency of miniature excitatory postsynaptic currents (mEPSCs) was enhanced, without changes in the amplitude and in the number of excitatory synapses. The increment of mEPSCs frequency was totally abolished after acute injection of BAPTA-AM (a specific Ca2+ chelator), suggesting a possible alteration of Ca2+ homeostasis at the presynaptic terminal. Excitatory E373K-Syn I neurons showed reduced evoked EPSC amplitude attributable to a reduction of the readily releasable pool (RRP), while, on the contrary, inhibitory E373K-Syn I neurons did not show any difference both in miniature, evoked IPSC amplitude and RRP size. While no effects in the dynamics and steady state of depression were detected, both excitatory and inhibitory E373K-Syn I neurons failed to recover after stimulation with long high-frequency trains. No mutation-induced changes were observed in network firing/bursting activity as determined with multi-electrode extracellular recordings. Our data suggest that the Ca2+-dependent regulation of ATP-binding to SynI plays important roles in spontaneous and evoked neurotransmitter release that differentially affect the strength of excitatory and inhibitory transmission.