Fast neurotransmitter release regulated by the endocytic scaffold intersectin

Abstract

Sustained fast neurotransmission requires the rapid replenishment of release-ready synaptic vesicles (SVs) at presynaptic active zones. Although the machineries for exocytic fusion and for subsequent endocytic membrane retrieval have been well characterized, little is known about the mechanisms underlying the rapid recruitment of SVs to release sites. Here we show that the Down syndromeassociated endocytic scaffold protein intersectin 1 is a crucial factor for the recruitment of release-ready SVs. Genetic deletion of intersectin 1 expression or acute interference with intersectin function inhibited the replenishment of release-ready vesicles, resulting in short-term depression, without significantly affecting the rate of endocytic membrane retrieval. Acute perturbation experiments suggest that intersectin-mediated vesicle replenishment involves the association of intersectin with the fissioning enzyme dynamin and with the actin regulatory GTPase CDC42. Our data indicate a role for the endocytic scaffold intersectin in fast neurotransmitter release, which may be of prime importance for information processing in the brain.endocytosis | synaptic transmission | synaptic vesicle recruitment N eurotransmission depends on the exocytosis of synaptic vesicles (SVs) at active zones (AZs) and the subsequent retrieval of SV membranes by endocytosis. Sustained fast neurotransmission requires rapid replenishment of release-ready SVs composing the readily releasable pool (RRP) (1-3). Whereas the machinery for SV fusion is well understood, the mechanisms controlling the number of SVs within the RRP and the rate of replenishment remain enigmatic (4, 5). It has been postulated that the rate of replenishment of release-ready SVs in addition to molecular priming reactions is regulated by the availability of SV release sites (6-8). The reuse of release sites appears to depend on components of the endocytic machinery, including dynamin (9-12). However, considering that the rate of endocytosis is ∼10-100 times slower than that of replenishment of release-ready SVs, the question arises as to how endocytic proteins affect rapid neurotransmitter release.Data from dynamin mutants can be interpreted as a deficit in SV recycling (e.g., loss of the SV reserve pool; ref. 13). Alternatively, endocytic proteins may regulate exocytosis more directly, independent of membrane retrieval. If so, interference with select endocytic factors might impair replenishment of release-ready SVs without affecting the rate of SV membrane retrieval.A possible candidate for such regulation is the early-acting multidomain endocytic protein intersectin (14-18), a protein overexpressed in Down syndrome, which also associates with components of the exocytic machinery [i.e., synaptosomal-associated protein 25 (SNAP-25)] (19). Although a role for intersectin in endocytosis has been established in Drosophila (16,17), the precise function of intersectin 1 in mammalian central synapses has not yet been analyzed extensively. Optical measurements of endocytosis in cultured ...

Intersectin 1 is a component of the Reelin pathway to regulate neuronal migration and synaptic plasticity in the hippocampus

“…To explore the physiological functions of ITSN1 in vivo, we analyzed the overall brain architecture of KO mice deficient in ITSN1 (25,26). Nisslstained sections of hippocampi from ITSN1 KO mice showed a gross dispersion of pyramidal neurons in the CA1 and CA3 areas and an altered shape of the dentate gyrus (DG) (Fig.…”

“…S4C). In contrast, Reelin failed to augment TBS-induced LTP in hippocampal slices from ITSN1 KO mice ( (25,35).…”

“…cell signaling (30), exocytosis/endocytosis (25,27,28,(31)(32)(33), development of dendritic spines (29,34), cortical midline connectivity (35), and spatial learning (35), its precise physiological function in the mammalian central nervous system in vivo remains incompletely understood. Here we identify ITSN1 as a component of Reelin signaling that acts predominantly by facilitating the VLDLR-Dab1 axis to direct neuronal migration in the forebrain and to augment synaptic plasticity.…”

“…In the present study, we analyzed KO mice deficient in intersectin 1 (ITSN1) (25,26), a scaffold protein highly expressed in neurons (27). ITSN1 is a multidomain protein comprising two Eps15 homology (EH) domains and five SH3 domains (A-E) connected via a central helical region and a C-terminal DH-PH domain with guanine nucleotide-exchange activity toward Cdc42 (Fig.…”

Tissue-specific dynamin-1 deletion at the calyx of Held decreases short-term depression through a mechanism distinct from vesicle resupply

“…Genetic perturbation of endocytic proteins often leads to a delay in vesicle resupply, as shown in mutants of endophilin (54)(55)(56), synaptojanin (57,58), and dap160/intersectin-1 (59). Intersectin-1 KO synapses in mice showed deficient vesicle replenishment even without a detectable change in capacitance decays (60). We tested the vesicle resupply rate by examining the recovery of transmission evoked by an AP at different intervals following each high-frequency stimulation (30 APs at 300 Hz) (Fig.…”