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Functional analysis of candidate orthologues of Synapse-Detective-1 (SYD-1) in mice

Wentzel, Corinna. Functional analysis of candidate orthologues of Synapse-Detective-1 (SYD-1) in mice. 2013, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_10382

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Abstract

Synapses are central processing units for neuronal communication. Therefore, the investigation of synapse development and the mechanisms underlying formation of pre- and postsynaptic structures are crucial for understanding directional information flow in the brain. The differentiation of presynaptic boutons encompasses a local re-organization of the actin cytoskeleton, recruitment of synaptic vesicles, and the assembly of vesicle release sites, the so-called active zones. Several trans-synaptic adhesion complexes, have been identified that can instruct this differentiation process, such as receptor tyrosine phosphatases and the neuroligin/neurexin complex. However, the intracellular mechanisms linking adhesion to the formation of mature active zones in mammalian cells have remained obscure.
Genetic screens have led to the identification of two cytoplasmic molecules, Synapse-Defective-1 and -2 (SYD-1, SYD-2) that are essential for active zone assembly downstream of cell surface receptors in C.elegans and Drosophila (Hallam et al, 2002; Owald et al, 2010; Zhen & Jin, 1999). In invertebrates, SYD-1 is required for the concentration of SYD-2/liprin-a at the presynapse where it interacts with the active zone protein ELKS-1/Bruchpilot (Dai et al, 2006). We have identified two mouse orthologues of SYD-1, which we named mSYD1A and mSYD1B (mouse Synapse-Defective-1A/B). As the invertebrate proteins, mSYD1A and mSYD1B contain a C2 and a GTPase activating (GAP) domain. mSYD1A is expressed in neurons during embryonic and postnatal development. The protein is present in synaptosomal membrane fractions, indicating that a pool of mSYD1A is associated with synapses.
Knockdown of mSYD1A in primary cultures of cerebellar granule cells leads to a decrease in synaptic vesicle clusters, which can be rescued presynaptically by re-expression of mSYD1A. Overexpression of mSYD1A increases synapse density. Using a FRET-based assay, we discovered that mSYD1A exhibits GAP activity towards the small GTPase RhoA. Intra-molecular interactions between the N-terminal domain and the GAP domain of mSYD1A inhibit GAP activity, whereas this inhibition can be released by targeting full-length mSYD1A to the plasma membrane. Using biochemical and Yeast-2-Hybrid assays, we identified mSYD1A interacting proteins that associate with the N-terminal domain and might serve as endogenous activators of mSYD1A function. Amongst these binding partners is the active zone protein liprin-a2. In a complex with liprin-a2, mSYD1A is recruited into plasma membrane clusters downstream of the synaptic receptor protein tyrosine phosphatase LAR in COS cells. Furthermore, we generated knockout mice for mSYD1A and mSYD1B and are beginning to analyze them.
Thus, we identified a novel regulator of presynaptic assembly called mSYD1A that is evolutionary conserved from C.elegans to mouse.
Advisors:Scheiffele, Peter
Committee Members:Barde, Yves-Alain
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Neurobiology > Cell Biology (Scheiffele)
Item Type:Thesis
Thesis no:10382
Bibsysno:Link to catalogue
Number of Pages:147 Bl.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:53
Deposited On:27 Jun 2013 09:53

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