Characterization of the Complexin - SNARE Protein Network in Different Synaptic SystemsDoctoral thesis
Date of Examination:2022-05-02
Date of issue:2023-01-24
Advisor:Prof. Dr. Nils Brose
Referee:Prof. Dr. Nils Brose
Referee:Prof. Dr. Tiago Fleming Outeiro
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EnglishAt neuronal synapses, the exocytotic SNARE complex is formed by Syntaxin 1, SNAP25 and Synaptobrevin 2. Complexin (Cplx) regulates the SNARE function to achieve the high speed and spatial precision of synaptic vesicle fusion. All four known mammalian Cplxs share a short central α helix, which was shown to be necessary for SNARE complex binding of Cplx1. The high degree of conservation in this central domain suggests that probably all Cplxs exert their function via an interaction with SNARE complexes, raising the question whether different Cplxs act upon different SNARE complex types. Peptides representing this domain bind to reconstituted SNARE complexes with submicromolar affinities in vitro. An affinity purification approach was developed with short synthetic peptides covering the central α-helical SNARE-binding domain of Cplxs1 to 4. After incubation of the immobilized Cplx peptides with detergent extracts of mouse cortex or retina, multiple Cplx-binding proteins were detected using a quantitative mass spectrometry approach. The detailed analysis of these proteins shows that basically there are differences in the Cplx interactomes. In the cortex samples, a variety of possible regulators and effectors were identified, among them different members of the of SNARE protein family including Syntaxin 1, SNAP25 and Synaptobrevin 2. Moreover, the samples also contained the complete set of SNAREs which are known to form complexes of the endosomal and lysosomal pathway, respectively. Surprisingly, these SNARE proteins were found to be Cplx isoform independent. A co-enrichment with neuronal SNARE proteins was excluded by repeating the affinity purification approach with HEK cells, which do not contain neuronal SNARE proteins. A functional effect of Cplx on non-exocytotic pathways was shown with a transferrin uptake assay. Cplx1 and Cplx2 are expressed in conventional synapses of almost all neuron types of the brain, while Cplx3 and Cplx4 are preferentially expressed in ribbon synapses of retinal photoreceptors and bipolar cells. The different distribution pattern of the two Cplx subgroups raises the question whether Cplx3 and 4 contribute to the highly specialized mode of neurotransmitter release found in ribbon synapses. Therefore, analysis of proteins which were detected in the course of experiments using retina material was rather directed to the extended interaction network. Interestingly, RIBEYE and some Transducin subunits were identified as specific interactors of Cplx3 and 4. The results of this work suggest that Cplx may be involved in processes beyond the regulation of synaptic exocytosis.
Keywords: Neurobiology; SNARE proteins; Complexin; Endocytosis; Cortex; Retina