Synergistic SNARE Modulators of Neurotransmission: Complexins and SNAP-29

Synergistic SNARE Modulators of Neurotransmission: Complexins and SNAP-29

Sivakumar, Nandhini

Dissertation

Angenommen am: 2015-05-07

Erschienen: 2016-05-04

Betreuer: Rhee, Jeong Seop Dr.

Gutachter: Dresbach, Thomas Prof. Dr.

Gutachter: Moser, Tobias Prof. Dr.

Zum Verlinken/Zitieren: http://hdl.handle.net/11858/00-1735-0000-0028-8746-7

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Name: PhD EDiss_Nandhini Sivakumar.pdf

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Beschreibung: PhD Dissertation - ...

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Zusammenfassung

Englisch

Neurotransmitter release is a synergistic multistep process occurring at synapses in the brain. Synaptic vesicles containing neurotransmitters tether to the presynaptic active zones, upon which, they are docked and primed to fusion competence by a number of proteins. When an action potential arrives at the terminal, a concomitant rise in the intracellular calcium concentration causes the vesicles to fuse to the presynaptic membrane and release their contents into the synaptic cleft. This process of neuronal excitation-secretion is tightly coupled and mediated by cascades of protein-protein interactions. The native SNAREs Syntaxin-1, SNAP-25 and Synaptobrevin-2 form a core complex that effectively facilitates the vesicle docking-priming-fusion process. Two proteins namely Complexins and SNAP-29 that have been shown to interact with the native SNAREs or their paralogs are detailed in the present study. The SNAP-29 protein belongs to the SNAP-25 family and is believed to be a Golgi-interacting SNARE. It shares only 17% sequence identity with SNAP-25 due to substantial variations in its structure. SNAP-29 was previously shown to interact with multiple Syntaxins localized on various subcellular organelles and was claimed to negatively modulate synaptic transmission in neurons by preventing SNARE disassembly. In the present study, it is proved that conditional loss of SNAP-29 from the forebrain glutamatergic neurons as well as constitutive loss of SNAP-29 in mice did not cause any deficits in transmitter release at glutamatergic or GABAergic synapses. Although constitutive knockout of SNAP-29 in animals caused perinatal lethality, the normal functioning of glutamatergic and GABAergic synapses in the brain could not account for such a dramatic effect. Rather, the lethality seen in these mice was likely due to perturbations in other secretory pathways. The Complexin family comprises of four proteins - Cplx1, Cplx2 are expressed in the brain, while Cplx3 and Cplx4 are prominently expressed in the retina. Previously, it was shown that loss of Cplx1, 2 and 3 in hippocampal neurons (TKO) caused profound deficits in the vesicular release probability and synchronicity of transmitter release. In the present study, this claim was confirmed and also shown that the reintroduction of each of the four wild type Complexins in the TKO via lentiviruses could facilitate synchronous neurotransmission, to variable extents. The strong binding of Cplx1 and Cplx2 to the SNARE complex corroborated their higher efficacy to mediate synchronous transmitter release. In case of Cplx3 and Cplx4, although hardly any binding to the SNARE complex was detectable, they could facilitate transmitter release only if their SNARE binding domain was intact and when targeted to membranes due to their C-terminal CAAX farnesylation. These results prove that all mammalian Complexins are indeed facilitators of synchronous synaptic transmission, and exert their function via SNARE complex binding and additional farnesylation for Cplx3 and Cplx4.

Keywords: SNARE; Complexin; SNAP-29; Glutamatergic