Investigation of Neurexin 2 as a candidate for Parkinson's Disease

Date
2022-09
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
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.
AFRIKAANS OPSOMMING: Parkinson se siekte (PS) is 'n neurodegeneratiewe versteuring wat hoofsaaklik beweging beïnvloed en word gekenmerk deur die verlies van dopaminergiese neurone in die substantia nigra pars compacta (SNpc). Daar is geen geneesmiddel vir die siekte nie en huidige dwelmbehandelings het dikwels ernstige newe-effekte. Verskeie patogene variante is in PS geïmpliseer, in verskeie gene, waaronder SNCA, LRRK2, PRKN en PINK1. Hierdie variante is egter hoofsaaklik gevind in individue van Europese afkoms. In Afrika suid van die Sahara (SSA) het studies wat oor die genetiese etiologie van PS gedoen is, getoon dat hierdie bekende patogene variante slegs geringe bydraers tot die etiologie is. Aangesien SSA na verwagting 'n toename in ouderdomsverwante afwykings, soos PS, in die gesig staar as gevolg van die geleidelike verbetering in lewensgehalte en verhoogde lewensverwagting, is dit belangrik om die wanorde in hierdie bevolkings te bestudeer. Vir hierdie doel het ons individue met PS van die Suid-Afrikaanse bevolking gewerf vir genetiese studies. Een van die probande wat gewerf is, het 'n familiegeskiedenis van PS gehad en het ook verskeie PS geaffekteerde en onaangeraakte familielede gehad. Hierdie familie is as ZA253 aangewys. Daarom het ons besluit om volledige eksome volgordebepaling op drie van die geaffekteerde individue uit te voer in 'n poging om die genetiese etiologie van hul wanorde te verduidelik. Variante wat nuut of skaars was (MAF < 1%), nie-sinoniem, heteroseries en gedeel onder die drie individue is geprioritiseer. Dit is gevind in die CCNF, CFAP65, NRXN2, RTF1 en TEP1 gene. Na die sifting van onaangeraakte lede en etnies-ooreenstemmende kontroles, sowel as die uitvoering van pad- en uitdrukkingsanalise en funksionele voorspellings van die effek van die variant op die vertaalde proteïen, is die p.G849D variant in NRXN2 (neurexin 2) geprioritiseer vir verdere studie. Eerstens het ons molekulêre dinamiese (MD) simulasies uitgevoer nadat ons 'n homoloë model van die menslike NRXN2α proteïen gebou het. Hierdie simulasies het getoon dat die variant 'n destabiliserende effek op die proteïenstruktuur gehad het en gelei het tot 'n uitgebreide konformasie van die laminin / neurexin / sex hormone bindende domein 6 (LNS6), wat verantwoordelik is vir die binding van ander proteïene. Daarna het ons 'n literatuursoektog op die neurexin-geenfamilie gedoen om vas te stel of dit goeie kandidaatgene vir PS is. Ons het bevind dat daar 'n gevestigde rol van neurexins in neuropsigiatriese afwykings is, soos outisme spektrumversteurings en skisofrenie, asook bewyse van 'n rol vir neurexins in neurodegeneratiewe afwykings, soos Alzheimer se siekte en PS. Daarom het ons tot die gevolgtrekking gekom dat NRXN2 'n goeie kandidaatgen is vir verdere ondersoek met behulp van funksionele studies. Funksionele studies is dan uitgevoer met behulp van 'n cDNA ooruitdrukkingsmodel in SH-SY5Y neuroblastoma selle om die effek van die variant in 'n in vitro model van PD te analiseer. Eerstens het ons toetsings gebruik om die effek van die mutant NRXN2α proteïen op seldood, mitochondriale funksie en reaktiewe suurstofspesies (ROS) produksie te ondersoek. Ons het bevind dat ooruitdrukking van die mutant proteïen 'n negatiewe uitwerking op al hierdie aspekte gehad het en daarom tot die gevolgtrekking gekom het dat die mutant NRXN2α 'n giftige terugvoerlus van mitochondriale disfunksie, verhoogde ROS generasie en verhoogde neuronale seldood kan veroorsaak. Gevolglik het ons proteomiese analise op dieselfde model uitgevoer om te bepaal hoe ooruitdrukking van NRXN2α sellulêre paaie beïnvloed. Interessant genoeg, ooruitdrukking van die wilde-tipe proteïen het gelei tot die verryking van proteïene wat betrokke is by neurodegeneratiewe paaie, bied voorlopige bewyse dat NRXN2α by hierdie paaie betrokke kan wees. Ooruitdrukking van die mutant proteïen het gelei tot die afname in proteïene wat betrokke is by ribosomale funksionering. Aangesien NRXN2α 'n sinaptiese proteïen is, is dit moontlik dat die variant sinaptiese vertaling beïnvloed. Inderdaad, disreguleerde sinaptiese vertaling is gekoppel aan veranderde mitochondriale fisiologie. Daarom het ons veronderstel dat gedisreguleerde sinaptiese vertaling en mitochondriale disfunksie gekoppel is en saamwerk om neuronale dood tot gevolg te hê. Die laaste deel van die studie het die effek van die variant op die sinaptiese funksie van NRXN2α ondersoek. Ons het eers MD simulasies gebruik om die variant se effek op die binding van NRXN2α aan 'n bekende bindende vennoot, neuroligin 1 (NLGN1) te ondersoek. In sinapse bind neurexins aan neuroligins om sinaptiese oordrag en instandhouding te vergemaklik. Die resultate van die simulasies dui daarop dat die variant hierdie interaksie kan ontwrig. Daarna het ons sinaptiese merkers in vitro, in gedifferensieerde SH-SY5Y selle, gevlek om te bepaal of ooruitdrukking van die mutant proteïen sinapsevorming en sinaptiese oordrag beïnvloed. Ons het 'n toename in die vlakke van beide merkers gevind wat moontlik aandui dat daar verhoogde sinapsevorming is wat verhoogde oordrag tussen sinapse tot gevolg het. Aangesien die MD simulasies getoon het dat die variant neurexin-neuroligien sein kan ontwrig, stel ons voor dat hierdie toename in oordrag 'n kompenserende meganisme is en stel voor dat hierdie reaksie mettertyd die sinaptiese instandhoudingstelsels sal spanning en uiteindelik tot neurodegenerasie sal lei. Ten slotte het ons bevindings aangedui dat 'n variant in NRXN2α gekoppel kan word aan mitochondriale en sinaptiese disfunksie wat uiteindelik tot neurodegenerasie kan lei. Verdere geteikende eksperimente in ander PD-modelle is egter nodig om hierdie bevindings te bewys. Nietemin is dit belangrik om na die genetika van PD in onderstudiede bevolkings te kyk, aangesien dit kan lei tot die ontdekking van nuwe gene en siektemeganismes onderliggend aan hierdie versteuring. Daarom kan studies soos hierdie help om lig te werp op hierdie aftakelende versteuring.
Description
Thesis (PhD) -- Stellenbosch University, 2022.
Keywords
Parkinson's disease -- Genetic aspects, Human genetics, Nervous system -- Degeneration, UCTD
Citation