Synaptic proteins and their interactors form crucial molecular machineries for brain functioning, and reveal high molecular and functional diversity. Understanding their diverse structural organization is essential to understand complex synaptic processes. In this thesis, I applied several (interaction) proteomics strategies to further specify the protein complex organization of major players in synaptic functioning. The Glycine-receptor (GlyR) mediates inhibitory neurotransmission, is involved with locomotion, respiration and nociception, and implicated in startle disease/hyperekplexia. In chapter 2 I determined the interactome of the GlyR in the brainstem using an immunopurification mass spectrometry (IP-MS) strategy with multiple antibodies against the GlyR and its major interactor Gephyrin. The excitatory synapse protein IQ motif and Sec7 domain 3 (IQSEC2), and known Gephyrin interactor IQ motif and Sec7 domain 3 (IQSEC3) were shown to be part of GlyR containing complexes here. Additional separation of GlyR complexes with an IP-blue native-PAGE/mass spectrometry (IP-BN-PAGE/MS) approach, demonstrated that this novel GlyR: Gephyrin: IQSEC2/3 assembly forms a small and distinct high molecular weight population of GlyRs. The scaffolding protein Gephyrin has multiple splice isoforms with different biochemical properties. In chapter 3 I designed specific antibodies against the major Gephyrin-C3 and C4 splice isoforms, and validated their specificity. With these novel antibodies, I determined Gephyrin-C3 and C4 cellular expression, their interaction profiles with overlapping and specific interactors and subcomplex compositions. The neuronally expressed Gephyrin-C4 revealed strong binding to the GlyR, IQSEC3 and Nitric oxide synthase 1 (NOS1), whereas Gephyrin-C3 revealed high expression in astrocytes, reduced binding to the GlyR and specific interaction with NLGN2. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) is the major synaptic excitatory ionotropic receptor in the brain. The most abundant AMPAR subtypes in the hippocampus are GluA1/2 and GluA2/3 heterotetramers, which contribute differentially to mechanisms of synaptic plasticity. Their functional differences may be in part caused by regulation through specific associated proteins. In chapter 4 I analyzed the complex compositions of the two most abundant AMPAR subtypes in the hippocampus. I performed quantitative and interaction proteomics on wildtype and GluA1- and GluA3 gene deleted mice. Whereas GluA1/2 co-purified TARP-γ8, PRRT1 and CNIH2 with highest abundances, GluA2/3 receptors revealed strongest co-purification of CNIH2, TARP-γ2, and OLFM1. Additional IP-MS, IP-BN-PAGE/MS and microscopy analysis revealed a direct interaction between TARP-γ8 and PRRT1 and their co-assembly into an AMPAR subcomplex especially near the synapse. Alzheimer’s disease, one of the most well-known disorders of the brain, is characterized by early hippocampal memory deficits and dysfunctional synapses. In chapter 5 I studied the proteome of a synapse enriched fraction obtained from the hippocampus of the APP/PS1 mouse model of AD at 6 and 12 months of age using data independent acquisition (DIA) mass spectrometry. I first assessed the usefulness of a recently improved directDIA analysis workflow as an alternative to conventional DIA analysis using a project specific library. I subsequently applied this workflow to our datasets, and revealed most regulation at 12-months. In particular proteins involved in Aβ homeostasis and microglial-dependent processes.
|18 May 2022
|Published - 18 May 2022
- Proteomics, Synapse, Glycine-receptor, Gephyrin, AMPA-receptor, protein-protein interactions, APP/PS1, Alzheimer's Disease,