IDENTIFICATION OF MOLECULAR PARTNERS INVOLVED IN ACETYLCHOLINE/GLUTAMATE COTRANSMISSION WITHIN SYNAPTIC VESICLES OF STRIATAL CHOLINERGIC INTERNEURONS

Striatal cholinergic interneurons (CINs) express vesicular transporters for acetylcholine (VAChT) and glutamate (VGLUT3) and regulate striatal network activity through the cotransmission of acetylcholine and glutamate. Synaptic vesicles (SVs) in CINs can store and release acetylcholine and glutamate either simultaneously or independently, allowing fine control of striatal function. However, the molecular mechanisms underlying this cotransmission remain poorly understood. We hypothesized that VAChT and VGLUT3 interact directly or indirectly with partner proteins to regulate acetylcholine/glutamate cotransmission. To test this hypothesis, we employed a multidisciplinary strategy combining computational, molecular and imaging approaches. Using the SENSE-PPI deep-learning framework, we screened approximately 1,500 vesicular proteins and identified about 20 candidate interactors of VAChT and/or VGLUT3. Most candidates belong to the SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) complex, a key component of the vesicle fusion machinery controlling neurotransmitter release. Fluorescent in situ hybridization confirmed the expression of these candidates in CINs, while STED super-resolution microscopy demonstrated their localization on VAChT- and VGLUT3-positive SVs. Nearest-neighbor distance analyses revealed that the SNARE proteins VAMP2 and VAMP7 are both associated with VAChT- and VGLUT3-containing vesicles. Bioinformatic analyses identified interaction domains within VAChT and VGLUT3, enabling the design of interfering peptides. Functional experiments using fluorescent glutamate biosensors showed that VAMP7 interacts with VAChT but not VGLUT3, while VAMP7-dependent mechanisms modulate glutamate release via VGLUT1 in the striatum. Together, these findings reveal novel molecular partners of VAChT and VGLUT3 and provide new insights into the mechanisms governing acetylcholine and glutamate release, with potential implications for disorders involving striatal dysfunction.