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Abstract

Presynaptic terminals release neurotransmitter in response to incoming electrical impulses, control information transfer between neurons in neural networks, and mediate directly or indirectly most brain functions. In humans, dysfunction of presynaptic terminals contributes to devastating brain disorders such as Alzheimer, schizophrenia, and autism. However, despite of its obvious significance, how defects in the function of presynaptic terminals contribute to the etiology of brain disorders, remains largely unknown. This is because not even the most fundamental operation principles of normal human nerve terminals are well-understood, let alone their dysfunction in disease. Here, I study the basic biology of human presynaptic terminals, as well as their dysregulation in autism-spectrum disorder (ASD). I focus on a protein family called RIMs (Rab interacting molecules), which are central components of the presynaptic active zones. I capitalized on emerging human pluripotent stem-cell technologies to generate human neurons, on CRISPR/Cas9 genome editing to genetically engineer human neurons and create disease models, and on advanced physiological and microscopy technologies to uncover the basic biology of human presynaptic terminals as well as their dysfunction in ASD. Remarkably, I found that compound genetic removal of both RIM1 and RIM2, the main brain RIM isoforms, disassembles human active zones (AZ), prevents synaptic vesicle docking and priming, and blocks synaptic vesicle fusion, rendering human presynaptic terminals functionally silent. Genetic experiments in which either RIM1 or RIM2 are selectively deleted, revealed that RIM1 is the functionally dominant isoform in human neurons. As RIM1 is also a common target of mutations in autistic patients, I generated a panel of knock-in lines comprising all currently described ASD-linked mutations in RIM1, and analyzed systematically their impact on human neuron structure and function. I found that all these disease-linked variants dysregulated human synaptic communication via two convergent mechanisms: impairing either vesicle priming or calcium channel coupling to synaptic vesicles. Importantly, this translates into enhanced or reduced information flow across neural human networks, highlighting the critical role of balanced neurotransmission in the pathogenesis of neuropsychiatric disorders. My work offers a unifying view on the basic biology of human active zones and their dysregulation in autism. To the best of my knowledge, this study represents the first effort aiming to understand how normal human presynaptic terminals work, and how brain disease affects them. Along this line, here I contribute to future efforts aiming to the development of new therapeutic strategies to reverse common presynaptic mechanisms disrupted in autistic patients.