Dynamics of the neuroproteome: protein signaling and translation in neurological disorders

Abstract

The aim of this thesis was to develop and apply the latest proteomics techniques to study protein translation events in disease models of primary astrocytes and neuronal cells. Since this research involves limited sample amounts such as primary neuronal cultures, we first benchmarked an automated phosphopeptide enrichment method for its sensitivity in protein phosphorylation analysis. We further applied a pulsed labeling technique to study protein translation events in two separate studies on Vanishing White Matter disease (VWM) and synaptic plasticity. The hallmark of VWM disease is a mutation in a subunit of the eIF2B protein, which is involved in initiation of protein translation. The proteomics profiling showed differently regulated proteins in wild type and astrocytes carrying a mutation in eIF2B. The here identified differently translated proteins differ from their "normal" counterparts in aspects of their 5' UTRs, indicative for a deregulated protein synthesis in eIF2B mutated astrocytes. This altered synthesis seems to lead to an increase of proteins in the Endoplasmic Reticulum (ER) and the secretory pathway. Possibly, increased disulfide bridge formation in the ER results in oxidative stress. A small-scale metabolic screen highlighted changes in the metabolites 6-phospho-gluconate, NAD+ and NADPH between wild type and VWM astrocytes, which together with an increased Pros1 expression support the hypothesis of increased oxidative stress in VWM astrocytes. We then combined the benchmarked phosphoproteomcis technique with the pulsed labeling technique to study synaptic plasticity through a specific form of long term depression (mGluR-LTD) in neuronal cells. This form of synaptic plasticity relies on protein synthesis and the internalization of AMPA-type glutamate receptors. Several kinases with important roles in mGluR-LTD could be predicted from the acquired phosphoproteomics data, which were later validated in a separate experiment. Furthermore, changes in protein phosphorylation in the AMPA receptor endocytosis pathway upon mGluR-LTD were identified, whereby Intersectin-1 was validated as a vital player in this pathway. The research in this thesis is concluded with a study into the function of Shank proteins in mGluR-LTD. Shank scaffolding proteins are directly linked to specific forms of autism and are known to play a role in mGluR-LTD. An immunopurification assay in combination with results from chapter 4 pointed towards a role for Shank proteins in proteasome activity modulation. The Shank immunopurification showed the identification of known Shank interactors, such as Homer, but also confirmed the interaction of the Shank proteins with the proteasome. Lastly a gel-based proteasome activity assay showed reduced proteasome activity in Shank knockdown compared to control neurons, highlighting the role of Shank proteins in proteasome regulation.