Doctoral Degrees (Molecular Biology and Human Genetics)

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Browsing Doctoral Degrees (Molecular Biology and Human Genetics) by browse.metadata.advisor "Cloete, Ruben"

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    Investigation of Neurexin 2 as a candidate for Parkinson's Disease
    (Stellenbosch : Stellenbosch University, 2022-09) Cuttler, Katelyn; Bardien, Soraya; Cloete, Ruben; Farrer, Matthew; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Parkinson’s disease (PD) is a neurodegenerative disorder which primarily affects movement and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). There is no cure for the disorder and current drug treatments often have severe side effects. Several pathogenic variants have been implicated in PD, in various genes including SNCA, LRRK2, PRKN, and PINK1. However, these variants have mainly been found in individuals of European ancestry. In Sub-Saharan Africa (SSA), studies done on the genetic aetiology of PD have shown that these known pathogenic variants are only minor contributors to the aetiology. Since SSA is expected to face a surge in age-related disorders, such as PD, due to the gradual improvement in quality of life and increased life expectancy, it is important to study the disorder in these populations. To this end, we have recruited individuals with PD from the South African population for genetic studies. One of the probands recruited had a family history of PD and also had several PD affected and unaffected family members. This family was designated ZA253. Therefore, we decided to perform whole exome sequencing on three of the affected individuals in an attempt to elucidate the genetic aetiology of their disorder. Variants that were novel or rare (MAF < 1%), non synonymous, heterozygous, and shared amongst the three individuals were prioritised. These were found in the CCNF, CFAP65, NRXN2, RTF1, and TEP1 genes. After screening unaffected members and ethnic-matched controls, as well as performing pathway and expression analysis and functional predictions of the effect of the variant on the translated protein, the p.G849D variant in NRXN2 (neurexin 2) was prioritised for further study. First, we performed molecular dynamic (MD) simulations after constructing a homologous model of the human NRXN2α protein. These simulations showed that the variant had a destabilizing effect on the protein structure and resulted in an extended conformation of the laminin/neurexin/sex-hormone binding domain 6 (LNS6), which is responsible for binding to other proteins. Thereafter, we performed a literature search on the neurexin gene family to determine if they are good candidate genes for PD. We found that there is a well-established role of neurexins in neuropsychiatric disorders, such as autism spectrum disorders and schizophrenia, as well as evidence of a role for neurexins in neurodegenerative disorders, such as Alzheimer’s disease and PD. Therefore, we concluded that NRXN2 is a good candidate gene for further examination using functional studies. Functional studies were then performed using a cDNA overexpression model in SH-SY5Y neuroblastoma cells to analyze the effect of the variant in an in vitro model of PD. First, we used assays to examine the effect of the mutant NRXN2α protein on cell death, mitochondrial function, and reactive oxygen species (ROS) production. We found that overexpression of the mutant protein had a negative effect on all of these aspects and therefore concluded that the mutant NRXN2α could induce a toxic feedback loop of mitochondrial dysfunction, increased ROS generation and increased neuronal cell death. Consequently, we performed proteomics analysis on the same model to determine how overexpression of NRXN2α affects cellular pathways. Interestingly, overexpression of the wild type protein led to the enrichment of proteins involved in neurodegenerative pathways, providing preliminary evidence that NRXN2α could be involved in these pathways. Overexpression of the mutant protein led to the decline in proteins involved in ribosomal functioning. Since NRXN2α is a synaptic protein, it is possible that the variant affects synaptic translation. Indeed, dysregulated synaptic translation has been linked to altered mitochondrial physiology. Therefore, we hypothesized that dysregulated synaptic translation and mitochondrial dysfunction are linked and act together to result in neuronal death. The last part of the study examined the effect of the variant on the synaptic function of NRXN2α. We first used MD simulations to examine the variant’s effect on the binding of NRXN2α to a known binding partner, neuroligin 1 (NLGN1). In synapses, neurexins bind to neuroligins to facilitate synaptic transmission and maintenance. The results of the simulations suggest that the variant may be able to disrupt this interaction. Thereafter, we stained synaptic markers in vitro, in differentiated SH-SY5Y cells, to determine whether overexpression of the mutant protein affects synapse formation and synaptic transmission. We found an increase in the levels of both markers possibly indicating that there is increased synapse formation resulting in increased transmission between synapses. Since the MD simulations showed that the variant could disrupt neurexin neuroligin signalling, we propose that this increase in transmission is a compensatory mechanism and suggest that, over time, this response would strain the synaptic maintenance systems and eventually lead to neurodegeneration. In conclusion, our findings have indicated that a variant in NRXN2α may be linked to mitochondrial and synaptic dysfunction that may eventually lead to neurodegeneration. However, further targeted experiments in other PD models are required in order to prove these findings. Nevertheless, it is important to look at the genetics of PD in understudied populations as this may lead to the discovery of new genes and disease mechanisms underlying this disorder. Therefore, studies such as these can help to shed light on this debilitating disorder.