The synapse is a highly specialized and dynamic structure, which is involved in regulating neurotransmission. As the vehicles of neurotransmitters, synaptic vesicles (SVs) are essential for passing of neural signals from one neuron to another. Disruptions in SV trafficking and release will lead to various forms of neurological disorders. The process of synaptic vesicles priming, docking and fusion with presynaptic membrane has been described thoroughly, and a lot of molecules involved in this process have been identified, such as Synaptotagmins, Synapsins, Synaptobrevins and Munc18. It was also reported that SVs can cluster in specific synaptic compartment to form distinct pools and this well-regulated form is significant for normal neurotransmitter release. However, how SV cluster to particular region and maintain normal neurotransmitter release is not very clear yet.
Previous studies have showed that Neurexin (NRX) and Neuroligins (NLGs) can form heterodimer at synaptic membrane and it is essential for the formation, development, maturity and plasticity of synapse. Medical research has also found that the mutation of nrx and nlg are associated with mental disorders, such as autism. Previous studies have showed that NRX is involved in the regulation of the structure and function of synapses, however, the detailed molecular mechanism is not very clear. We used Drosophila NMJ as a model system and revealed the significant role of Drosophila NRX (DNRX) in modulating SV clustering and release at synaptic terminals.
NRX is a kind of synaptic adhesion molecule, it is widely expressed in the central and peripheral nervous system. Our results, for the first time, demonstrated that DNRX regulates the clustering and spontaneous release frequency of SVs through presynaptic F-actin. Mechanistically, we employed integrative approaches and demonstrated that DNRX can directly bind to Scribble, a tumor suppressor protein, via its c terminal PDZ-binding motif. Following, we have also revealed that Scribble bridges DNRX to Dpix, a Rac/Cdc42-specific guanine nucleotide exchange factor (GEF). Dpix can activate Rac1, which is involved in pathways that promote actin polymerization and a well-regulated function of the brain. To determine the specific motif of DNRX that participate in the regulation of F-actin and SV, according to the previous result we constructed the transgenic fly with the deletion of PDZ-binding motif (DNRXΔPDZ), after driving DNRXΔPDZ at presynapse in dnrx mutant we have found that DNRXΔPDZ could not rescue the defeat of F-actin assembly and SV release observed in dnrx mutant. Finally, we have found that the level of Rac1-GTP is decreased after reducing the expression of DNRX or Scribble, it indicates DNRX regulates Rac1-GTP level via Scribble/Dpix complex physiologically. To further confirm the function of Rac1 in this process, we got the donimat nagetive Rac1 (Rac1DN) and intergrated it into dnrx mutant background, after inducing Rac1DN at presynapse in dnrx mutant we still could not find any rescue effect in F-actin assembly and SV release.
In summary, we have provided important new insights into the function of DNRX in modulating pre-synaptic F-actin through the Scribble/Dpix complex, and how it regulates presynaptic SV clustering and release via activating Rac1. On the other hand, the findings will deepen the understanding for the pathological mechanism of neurological disorders such as autism.