Characterization of neuronal SNARE protein amisyn and its role in neurotransmission
by Jialin Jin
Date of Examination:2021-10-29
Date of issue:2022-04-01
Advisor:Prof. Dr. Ira Milosevic
Referee:Prof. Dr. Peter Schu
Referee:Dr. Brett Carter
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Name:Jialin JIN doctoral thesis.pdf
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EnglishNeurotransmission is a complex bioprocess that involves a series of complex interactions between proteins and lipids. Exocytosis, which directly mediates neurotransmitter release, is one of the most important steps in the neurotransmission process and has been intensively studied for the last three decades. Various exocytosis regulators have been reported and described in detail, including Munc18, synaptotagmin, complexin, and tomosyn. Among these regulators, amisyn (known as syntaxin bind protein 6) has been rarely studied due to technical challenges related to its expression and lack of specific antibodies. Although studies on amisyn date back to 2002, only 6 publications have focused on this protein. Thus, the basic structural, functional, and physiological characteristics of amisyn remain unclear. As a member of the SNARE family, amisyn is a 24 kDa protein comprising a C-terminal VAMP2-like SNARE domain and an unknown N-terminal domain. Amisyn can bind with syntaxin-1 and SNAP-25 to form a SNARE complex. The amisyn-SNARE complex is fusion-inactive because amisyn does not contain a transmembrane domain. Thus, amisyn is speculated to be an inhibitor of exocytosis. This thesis work addresses the function of amisyn in neuronal and neurosecretory cells, including the function of amisyn’s N-terminal domain. Furthermore, for the first time, the consequences of amisyn’s ablation in a mammalian model were studied systematically. In the first study, we conducted a sequencing analysis to determine the potential structure of the N-terminal domain of amisyn. In the following experiment, we confirmed this speculation and determined that the N-terminal domain of amisyn is a pleckstrin homology domain. This pleckstrin homology domain could bind with phosphatidylinositol 4,5-bisphosphate lipids. Further experimental results suggested that the pleckstrin homology domain of amisyn was involved in both SNARE complex formation and the exocytosis inhibitory process of amisyn. In chromaffin cells, external full-length amisyn could change the size of the readily releasable pool and slowly releasable pool, whereas the SNARE domain of amisyn failed to achieve this. Based on these data, we constructed a new model to explain the role of amisyn in neurotransmission. In this new model, amisyn associates with the phosphatidylinositol 4,5-bisphosphate in the plasma membrane and competes with VAMP2 to form the fusion-inactive SNARE complex. In this way, amisyn can achieve an inhibitory effect. In the second study, we characterized the physiological properties of amisyn using a newly generated amisyn mutant mouse line. We also studied the behavioral and proteomic properties of amisyn-deficient animals. We first determined which synaptic and signaling proteins are altered in the hippocampi of amisyn mutants; the RNA and protein levels were unaltered for all tested candidates, except for VAMP2, rab3a, and α-synuclein. Next, we conducted electrophysiological and behavioral assays to characterize neurotransmission in amisyn mutant animals. We found increased vesicle release probability and enlargement of the readily releasable pool in amisyn-deficient synapses. Further experiments on plasticity revealed that long-term potentiation was abolished in amisyn mutant animals, which was consistent with the results from the behavioral assays, thereby indicating an impairment of learning and memory formation in amisyn mutant animals. The new findings reported in this doctoral thesis further our understanding of amisyn and its roles in neurotransmission, allow us to characterize exocytosis more precisely, and enhance our understanding of the pathological mechanisms underlying some amisyn-related diseases.
Keywords: amisyn; neurotransmission; synaptic vesicle; SNARE protein