Proteomics analysis of the p.G849D variant in neurexin 2 alpha may reveal insight into Parkinson's disease pathobiology

Abstract

Parkinson's disease (PD), the fastest-growing neurological disorder globally, has a complex etiology. A previous study by our group identified the p.G849D variant in neurexin 2 (NRXN2), encoding the synaptic protein, NRXN2α, as a possible causal variant of PD. Therefore, we aimed to perform functional studies using proteomics in an attempt to understand the biological pathways affected by the variant. We hypothesized that this may reveal insight into the pathobiology of PD. Wild-type and mutant NRXN2α plasmids were transfected into SH-SY5Y cells. Thereafter, total protein was extracted and prepared for mass spectrometry using a Thermo Scientific Fusion mass spectrometer equipped with a Nanospray Flex ionization source. The data were then interrogated against the UniProt H. sapiens database and afterward, pathway and enrichment analyses were performed using in silico tools. Overexpression of the wild-type protein led to the enrichment of proteins involved in neurodegenerative diseases, while overexpression of the mutant protein led to the decline of proteins involved in ribosomal functioning. Thus, we concluded that the wild-type NRXN2α may be involved in pathways related to the development of neurodegenerative disorders, and that biological processes related to the ribosome, transcription, and tRNA, specifically at the synapse, could be an important mechanism in PD. Future studies targeting translation at the synapse in PD could therefore provide further information on the pathobiology of the disease.

Keywords: Parkinson’s disease; mass spectrometry; mitochondrial dysfunction; neurexin 2α (NRXN2); p.G849D; proteomics; ribosomal functioning; synaptic translation.

Figure 1

Unique and differentially abundant proteins identified in this experiment. (A) Scatterplot of the standard deviation (log₁₀) vs mean (log₁₀) shows good clustering of all samples along the linear regression line. Graph generated by Scaffold 1.4.4. (B) Venn diagram of the total proteins identified in each treatment group shows the distribution of shared proteins and numbers of proteins unique to each group. Diagram generated with Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny; Oliveros, 2015). Volcano plots showing differentially abundant proteins when comparing EV vs NT (C), WT vs NT (D), MUT vs NT (E), and MUT vs WT (F). The gray line indicates the significance threshold. Significant proteins (p ≤ 0.05, Students t-test) are colored purple. Where possible, individual proteins have been labelled. Proteins found within the red funnel may become statistically insignificant with increased sample sizes. Graphs generated with GraphPad Prism^® 5.02. Abbreviations: EV: empty vector transfected cells, MUT: mutant transfected cells; NT: non-transfected cells; WT: wild-type transfected cells.

Figure 2

STRING protein–protein interaction diagrams of the differentially abundant proteins. (A) EV vs NT, (B) WT vs NT, (C) MUT vs NT, (D) MUT vs WT. Proteins which do not form part of an interaction network have been excluded. STRING: https://string-db.org (Szklarczyk et al., 2019). EV: empty vector transfected cells, MUT: mutant transfected cells; NT: non-transfected cells; WT: wild-type transfected cells.