Doctoral Degrees (Molecular Biology and Human Genetics)

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

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    Analysis of copy number variation and disease mechanisms underlying Parkinson’s disease
    (Stellenbosch : Stellenbosch University, 2016-03) Van der Merwe, Celia; Bardien, Soraya; Loos, Ben; 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 movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. Although the aetiology of PD is still not fully understood, it is thought to involve a combination of environmental and genetic factors. To date, a number of PD-causing genes have been found. The PINK1 gene is of particular interest for this study, and mutations in this gene result in autosomal recessive inheritance of early onset PD. PINK1 plays a vital role in mitochondrial quality control and homeostasis, and in its absence it is thought to result in an accumulation of dysfunctional mitochondria in neurons, culminating in neuronal cell death. Whilst pharmacological and surgical interventions are available for PD, the current options exhibit adverse side effects with long term treatment. There is a great need to develop new treatments with i. less side effects and ii. that can simultaneously target the multiple pathways associated with this disorder. One molecule is curcumin, the core component of the curry spice turmeric, which is well known for its antioxidant and anti-inflammatory properties and has already been studied for its possible neuroprotective role in Alzheimer’s disease. The aim of the present study was to create a cellular model of PD by decreasing the expression of PINK1 in SH-SY5Y neuroblastoma cells. Thereafter, we aimed to test the protective effects of curcumin on this model in the presence and absence of a known stressor, paraquat. This study also aimed to detect possible copy number variation (CNV) in PINK1 (and other PD-causing genes) in a cohort of South African patients with PD, in order to obtain patient-derived fibroblasts to verify the results obtained from the original cellular model. PINK1 was knocked down using siRNA (Qiagen, USA) in SH-SY5Y neuroblastoma cells, and the knock down was verified by quantitative real time PCR (qRTPCR) and western blotting. Thereafter, PINK1 siRNA cells and control cells were separated into four treatment groups: i. untreated, ii. treated with 25μM paraquat for 24hours, iii. pre-treated with 2μM curcumin for 1hour then treated with 25μM paraquat for 24hours, or iv. treated with 2μM curcumin for 1hour, and various parameters of cellular and mitochondrial function were measured. Cell viability was measured by an MTT assay. Western blot analysis was performed using cleaved PARP and full-length caspase 3 markers to detect levels of apoptosis, and LC3-II and p62 markers to detect autophagic flux. Mitochondrial respiration experiments were completed on the Seahorse XF Analyser using the Mito Stress Test Kit and the Glycolysis Stress Test. Flow cytometry was utilised to measure mitochondrial membrane potential (MMP) using the JC- 1 fluorochrome, and mitochondrial network was analysed by fluorescent microscopy. For CNV detection, MLPA was performed on 210 South African PD patients and putative mutations were verified by qRTPCR on the Lightcycler 96. PINK1 was successfully knocked down at a gene and protein expression level. The PINK1 siRNA cells exhibited a significant decrease in cell viability (p=0.0036), and an increase in apoptosis (p=0.0144). A decrease in PINK1 expression also resulted in significantly decreased MMP (p=0.0008), mitochondrial respiration (p=0.0015), ATP production (p=0.002) and glycolytic capacity (p=0.0445). No significant changes were observed in the connectivity of the mitochondrial network, but autophagic flux was significantly increased in the PINK1 siRNA cells, as detected by increased LC3-II levels (p=0.0152). As expected, paraquat-treated cells exhibited decreased cell viability, increased apoptosis, decreased MMP, autophagic flux, and a more fragmented mitochondrial network. Paraquat treatment therefore successfully acted as a stressor on the cells. Curcumin pre-treatment followed by paraquat treatment rescued cell viability in control cells (p=0.003), and significantly decreased apoptosis in PINK1 siRNA cells (p=0.0018). Curcumin protected mitochondrial dysfunction in PINK1 siRNA cells by increasing MMP (p=0.0472) and maximal respiration (p=0.0014), as well as significantly increasing MMP (p=0.0307) and maximal respiration (p=0.032) in control cells. Additionally, curcumin treatment resulted in increased autophagic flux (p=0.0017) in stressed control cells. These results highlight a protective effect of curcumin against paraquat and against the damaging effects on the mitochondria in cells with decreased PINK1 expression. Lastly, MLPA analysis did not reveal any PINK1 CNV mutations in a total of 210 South African PD patients, and fibroblasts were therefore not obtained. A number of false positive mutations were identified that were not verified by qRTPCR. A common polymorphism M192L resulting in a false positive PARK2 exon 5 deletion was found in a number of patients, all of whom were of Black or Mixed Ancestry ethnic groups. One patient was shown to harbour a heterozygous deletion in PARK2 exon 4. In conclusion, PINK1 siRNA-mediated knock down in SH-SY5Y neuroblastoma cells can be used as a model of PD to study aspects of mitochondrial dysfunction. Furthermore, curcumin should be considered as a possible therapeutic target for PD, as it exhibits protective effects against paraquat at a mitochondrial level. Given the low toxicity of curcumin, and the fact that it is already part of a dietary regimen in most populations worldwide, further studies on elucidating its biochemical and cellular properties are therefore warranted. The use of natural compounds such as curcumin as therapeutic agents is currently a topical and fast-growing area of research, and holds much promise for clinical application in various diseases including neurodegenerative disorders such as Alzheimer’s disease and PD.
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    Functional characterization of sequence variants in leucine-rich repeat kinase 2 (LRRK2) and its possible interaction with the translocase of outer mitochondrial membrane (TOM) protein complex
    (Stellenbosch : Stellenbosch University, 2017-03) Neethling, Annika; Bardien, Soraya; Williams, Monique Joy ; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Parkinson’s disease (PD) is an incurable neurodegenerative disorder, characterized by the progressive loss of dopaminergic neurons in the midbrain of affected individuals. Both environmental and genetic factors contribute to the aetiology of PD, with more than a dozen genes implicated in disease development. Yet, the exact mechanisms by which each gene (and mutation) contribute to the pathophysiology of PD remain to be elucidated. Mitochondrial dysfunction is a recurring theme associated with neurodegeneration and recently the translocase of outer mitochondrial membrane (TOM) complex, which plays a role in the maintenance of healthy mitochondria, has been implicated in PD pathogenesis. The TOM complex, consisting primarily of TOM20, TOM22, TOM40 and TOM70, is involved in the translocation of nuclear-encoded proteins into the mitochondria where they are needed for normal mitochondrial function. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of autosomal dominant PD and the LRRK2 protein has been associated with numerous cellular functions including mitochondrial homeostasis, the autophagy/lysosomal pathway, cell signalling and synaptic vesicle trafficking. The most common PD-causing mutation, G2019S, is located in the kinase domain of LRRK2 and has consistently been shown by various researchers to increase kinase activity. Recently, members of our group identified a novel variant (Q2089R) in LRRK2. This variant is also located in the kinase domain of LRRK2 and requires further investigation to determine its pathogenicity. The aim of the present study was to functionally characterize wild type (WT) and mutant LRRK2 (G2019S and Q2089R) under basal and stress [Carbonyl cyanide m-chlorophenyl hydrazone (CCCP)] conditions and also to determine whether WT LRRK2 interacts with the TOM complex. The frequency of LRRK2 Q2089R in South African PD patients and controls was determined using a custom Taqman™ SNP genotyping assay. In silico analysis of the effect of the amino acid substitution from Glutamine (Q) to Arginine (R) was performed using various prediction tools. Two cellular models of PD including (1) HEK293 cells transfected with WT and mutant LRRK2 constructs and (2) patient-derived dermal fibroblasts were used for the functional studies. LRRK2 mutant constructs were generated using site-directed mutagenesis in pcDNA-DEST53, a mammalian expression vector. We obtained skin biopsies from individuals harbouring G2019S, Q2089R or WT LRRK2 and cultured dermal fibroblasts as an ex vivo model of the disorder. We investigated the kinase activity of LRRK2 using autophosphorylation of Serine 1292 and Western blot analysis. Metabolic activity was measured using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and mitochondrial membrane potential (MMP) was measured using the JC-1 fluorochrome and flow cytometric analysis. Mitochondrial and glycolytic respiration experiments were performed using the Seahorse XF Analyzer and mitochondrial DNA copy number was determined by quantitative real-time PCR (qRT-PCR). Autophagic markers, LC3 II and P62, were detected using Western blot analysis. Co-localization experiments of WT LRRK2 and the TOM complex was performed using confocal and super resolution structured illumination microscopy (SR-SIM), while protein interactions were investigated using co-immunoprecipitation and Western blot analysis. The frequency of Q2089R was found to be 0.2% (1/493) in PD patients and 0.1% (1/776) in controls. Multiple in silico tools predicted the Q to R substitution to possibly be pathogenic [‘deleterious’ (CADD score=24.1, ‘possibly damaging’ (Polyphen) and ‘disease causing’ (Mutation Taster)]. The LRRK2 constructs were successfully generated and fibroblasts were successfully cultured. Notably, in HEK293 cells, we found that Q2089R almost completely abolished autophosphorylation activity of LRRK2 (p=0.026). Q2089R-carrying cells also exhibited a decrease in metabolic activity in HEK293 cells (p=0.016) and fibroblasts (p<0.05). In addition, in both cell types a significantly decreased MMP was observed [p=0.043 and p=0.009 for HEK293 cells and fibroblasts (under stress), respectively]. Furthermore, Q2089R-carrying fibroblasts showed an increase in basal respiration (p=0.012), proton leak respiration (p=0.0001), maximal respiration (p<0.0001) and spare respiratory capacity (p<0.0001), while ATP-coupling efficiency (p=0.0014), glycolytic reserve (p=0.006) and glycolytic capacity (p=0.007) was significantly reduced. In both models, Q2089R cells exhibited an increase in autophagosome pool size (p<0.05 for LC3 II and p<0.05 for P62). In the case of G2019S, a marked increase in autophosphorylation activity (p=0.019) was observed in HEK293 cells, which is in accordance with many previous studies. Decreased metabolic activity (p=0.021) and MMP (p=0.038) were also observed in these cells. G2019S-carrying fibroblasts displayed reduced metabolic activity (p<0.05) and increased basal respiration (p=0.029), ATP-linked respiration (p=0.029), glycolysis (p=0.001) and autophagosome pool size (p=0.022 for LC3 II). The MMP of these fibroblasts showed a non-significant trend for a decrease under stress conditions (p=0.057). Interestingly, WT LRRK2 was shown to co-localize and co-immunoprecipitate with a protein complex containing subunits TOM22, TOM40 and TOM70 but not TOM20 under basal conditions. Under stress conditions, an association between LRRK2 and TOM20 was observed while the association between LRRK2 and the complex containing TOM22 and TOM70 increased. Finally, from our findings and the published literature, we propose a model for the involvement of LRRK2 (WT and Q2089R) in cellular functioning and cell death. This involves the loss of kinase activity and association with the TOM complex, which ultimately links LRRK2 with mitochondrial (dys)function, mitochondrial biogenesis and the autophagy/lysosomal pathway. In conclusion, we characterized a functional variant in the kinase domain of LRRK2 and propose additional functions for this large multi-domain protein. This study also provides evidence for a novel association between LRRK2 and the TOM complex. Interestingly, our findings challenge the notion that it is only increased LRRK2 kinase activity that is implicated in PD pathogenesis. We acknowledge, however, that our findings are preliminary and that further validation studies are necessary to validate our results and hypothesis. Future targeted experiments on LRRK2 are needed in order to unravel the complex pathobiology and to decipher the sequence of events that lead to development of PD in susceptible individuals.
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    Identification of novel Parkinson’s disease genes in the South African population using a whole exome sequencing approach
    (Stellenbosch : Stellenbosch University, 2016-03) Glanzmann, Brigitte; Bardien, Soraya; Gamieldien, Junaid; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH SUMMARY: Parkinson’s disease (PD) is a progressive and severely debilitating neurodegenerative disorder that is characterised by a range of motor symptoms and the selective loss of dopaminergic neurons in the substantia nigra. While the aetiology of PD remains poorly understood, it is hypothesised to involve a combination of various environmental, genetic and cellular factors that independently or collectively contribute to neurodegeneration and ultimately disease. To date, a number of genes including Parkin, PINK1, LRRK2, SNCA, DJ-1, ATP13A2 and VPS35 that have been directly associated with disease and investigations of their functions have provided significant insights into the pathobiology of PD. However, these genes do not play a significant role in the South African PD cohort and for this reason, novel genes and pathogenic mutations must be investigated and identified. This will aid in early diagnosis of patients and also ultimately for the design of more effective therapeutic strategies to treat this debilitating and poorly understood chronic systemic disorder. The present study aimed to identify novel PD-causing mutations in the South African Afrikaner population using a genealogical and whole exome sequencing (WES) approach.. The Afrikaner are unique to South Africa and are known to have undergone a bottleneck in the 1800s which has led to genetic founder effects for a number of different disorders in this particular group. Additionally, we further aimed to determine whether the identified putative disease-causing mutation(s) could be attributed to the development of PD in other South African ethnic groups. A total of 458 patients were recruited, of which 148 were self-identified as Afrikaner. From these, a total of 48 Afrikaner probands were subjected to extensive genealogical analyses and 40 of them could be traced back to a single common couple. For this reason, it was hypothesised that the disorder in these patients may be due to a genetic founder effect. The use of a whole genome SNP array confirmed the relatedness of the individuals to varying degrees (8 to 12 generations back) and subsequently three of the probands and one affected sibling were selected for WES. The selected individuals were sequenced using the Illumina Genome Hiseq 2000TM and approximately 78 000 variants were identified for each individual. Numerous bioinformatics tools were used to scrutinize the variants but none were able to produce a candidate list of plausible disease-causing variants. All variants identified were either present at high frequency, did not co-segregate with the disorder or were artefacts. In order to facilitate and expedite the variant prioritisation process, a novel method for the filtration of WES data was designed in-house. This strategy named TAPER™ (Tool for Automated selection and Prioritisation for Efficient Retrieval of sequence variants) implements a set of logical steps by which to prioritise candidate variants that could be pathogenic. It is primarily aimed at the support of resource-constrained scientific environments with limited bioinformatics capacity. As a proof of concept various independent WES datasets for PD, severe intellectual disability and microcephaly as well as ataxia and myoclonic epilepsy were used, and TAPER™ was able to successfully prioritise and identify the causal variants in each case. Through the use of TAPER™, two putative candidate variants in SYNJ1 and USP17 were identified. The homozygous V1405I variant in SYNJ1 was found only in the affected sibling pair and in none of the 458 patients and 690 control individuals that had been screened. This variant is predicted to be deleterious across multiple platforms and has a CADD score of 29.40 and may alter synaptic vesicle recycling. The homozygous C357S variant in USP17 was found in 18/458 probands (12 Afrikaner, two white and four mixed ancestry) but was identified in 0.14% of the controls (1/184 Afrikaner, 0/160 white, 0/180 mixed ancestry and 0/160 black). This variant is also anticipated to be deleterious across multiple platforms and has a CADD score of 34.89. In summary, the results of the present study reveal that PD in the 40 South African Afrikaner patients studied is not due to a founder effect, but highlights two variants of interest for future studies. Further work is necessary to analyse both of these variants and to assess their possible effect on protein structure and function. The discovery of novel PD-causing genes is important as this allows for the generation of disease-linked protein networks, thereby facilitating identification of additional disease genes and subsequently providing insights into the underlying pathobiology. Moreover, this knowledge is critical for the development of improved treatment strategies and drug interventions that will ultimately prevent or halt neuronal cell loss in susceptible individuals. Although the present study did not conclusively identify a novel PD-causing gene, it does provide a solid foundation for future work in our laboratory in the challenging and rapidly evolving research area of WES and bioinformatics, and its application to studies on PD.
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    Identification of parkin interactions: implications for Parkinson’s disease
    (Stellenbosch : Stellenbosch University, 2015-12) Haylett, William Lloyd; Bardien, Soraya; Kinnear, Craig; Carr, Jonathan; 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 progressive and debilitating neurodegenerative disorder, characterized by a distinct motor phenotype and the selective loss of dopaminergic neurons in the substantia nigra. While the etiology of PD is not fully understood, it is thought to involve a combination of different genetic, cellular and environmental factors that independently or concurrently contribute to neurodegeneration. To date, several PD-causing genes have been identified, and investigations of their function have provided novel insights into the pathobiology of disease. Particularly interesting among the known PD genes is parkin, mutations in which are the most common genetic cause of early onset PD. Parkin is an E3 ligase that ubiquitinates protein substrates and targets such substrates for degradation via the ubiquitin proteasome system (UPS). Therefore, the loss of parkin may result in the deleterious accumulation or dysregulation of parkin substrates and neurotoxicity. Parkin’s enzymatic activity has also been implicated in the maintenance of mitochondrial health, and mitochondrial dysfunction is commonly reported in cellular and animal models of parkin deficiency. This study aimed to investigate parkin and its role in PD on various levels. Initially, genetic screening approaches were used to assess the contribution of parkin mutations to PD in a group of 229 South African patients. It was concluded that parkin mutations are rare in the South African PD population, being present in only seven (3.1%) patients in the study group. Interestingly, this study identified two of only three Black African PD patients with mutations in a known PD-causing gene to date. The low frequency of known PD genes raises the interesting possibility that the unique South African ethnic groups may harbor mutations in novel PD-causing genes. Although many parkin-interacting proteins have been identified in the literature, it is anticipated that novel, pathologically-relevant parkin substrates remain to be discovered. Hence, this study used a yeast two-hybrid (Y2H) approach to identify novel parkin interactions. This yielded 29 putative parkin interactors, of which four, namely ATPAF1, SEPT9, actin and 14-3-3η, were prioritized for verification by co-localization and co-immunoprecipitation experiments. Interestingly, two of the parkin interactors (ATPAF1 and SEPT9) were found to accumulate in the absence of parkin, supporting their role as authentic parkin substrates. The identification of these two intriguing proteins implicates parkin in the regulation of mitochondrial ATP synthase assembly and septin filament dynamics, which may be of significant relevance to our understanding of processes underlying neurodegeneration. Moreover, it was aimed to assess various markers of mitochondrial function in a parkin-deficient cellular model, as previous studies had reported conflicting results regarding mitochondrial impairments in patient-derived cells with parkin mutations. Hence, dermal fibroblasts were obtained from PD patients with homozygous parkin mutations, after which cell growth and viability, mitochondrial membrane potential, respiratory rates and the integrity of the mitochondrial network were assessed. Surprisingly, it was found that cell growth was significantly higher in the parkin-mutant fibroblasts compared to wild-type controls fibroblasts under basal conditions (p=0.0001), while exhibiting a greater inhibition of cell growth in the presence of the mitochondrial toxin CCCP (p=0.0013). Furthermore, whereas the mitochondrial networks of patient-derived fibroblasts were more fragmented than controls (p=0.0306), it was found that mitochondrial respiratory rates were paradoxically higher in the patients (p=0.0355). These unanticipated findings are suggestive of a compensatory response to the absence of parkin. The parkin-deficient cellular model was also used in a pilot study of the functional effects of vitamin K2 treatment, which has recently been identified as a promising PD therapeutic modality. It was found that treatment with vitamin K2 resulted in more interconnected mitochondrial networks (p=0.0001) and enhanced respiratory rates (p=0.0459) in both parkin-mutant and wild-type control cells. While these results need to be studied further, it suggests that vitamin K2 supplementation may be of use as a general promoter of mitochondrial integrity and function. In conclusion, this dissertation highlights some novel interactions of the parkin protein and some interesting phenotypes of parkin deficiency. It is hoped that further investigation of parkin and its role in PD will, ultimately, aid in the development of therapeutic strategies to treat this debilitating and poorly-understood disorder.
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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.