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

Neethling, Annika (2017-03)

Thesis (PhD)--Stellenbosch University, 2017.

Thesis

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.

AFRIKAANSE OPSOMMING: Parkinson se siekte (PS) is ‘n ongeneesbare neurodegeneratiewe versteuring wat gekenmerk word deur die progressiewe verlies van dopaminergiese neurone in die brein van geaffekteerde individue. Beide omgewings- en genetiese faktore dra by tot die etiologie van PS, met meer as ‘n dosyn gene wat geïmpliseer word by die ontwikkeling van hierdie siektetoestand. Desondanks moet die presiese meganisme waardeur elke geen (en mutasie) bydra tot die patofisiologie van PS nog uitgeklaar word. Mitochondriale disfunksie is ‘n herhalende tema wat verband hou met neurodegenerasie en die translokase van die buitenste mitochondriale membranekompleks (TOM), wat onlangs geïdetifiseer is as ‘n belangrike rol-speler in die instandhouding van gesonde mitochondria. Die TOM kompleks wat hoofsaaklik bestaan uit TOM20, TOM22, TOM40 en TOM70 is betrokke by die vervoer van kern-geënkodeerde proteïene tot binne in die mitochondria waar dit benodig word vir normale mitochondriale funksionering. Mutasies in die LRRK2 geen is die mees algeneme oorsaak van outosomale dominante PS en die LRRK2 proteïen word geassosieer met talle sellulêre funksies insluitend mitochondriale homeostase, die autophagy/lisosomale pad weg, sellulêre seine en sinaptiese vesikulêre vervoer. Die mees algemene PS-veroorsaakende mutasie, G2019S, is geleë in die kinase domein en verhoog kinase aktiwiteit van LRRK2. Lede van hierdie navorsingsgroep het onlangs ‘n nuwe variant (Q2089R) geïdentifiseer in LRRK2. Hierdie variant is ook geleë in die kinase domein van LRRK2 en vereis verdere ondersoek ten einde vas te stel of dit wel patogenies is. Die doel van die huidige studie was om funksionele kenmerke van wilde tipe (WT) en gemuteerde LRRK2 (G2019S en Q2089R) te ondersoek onder normale asook stremmings [Carbonyl cyanide m-chlorophenyl hydrazone (CCCP)] kondisies en ook om te bepaal of WT LRRK2 interaksie toon met die subeenhede van die TOM kompleks. Die frekwensie van LRRK2 Q2089R is vasgestel in Suid-Afrikaanse PS pasiënte en kontroles deur die gebruik van n Taqman™ SNP genotipeerings toets. In silico analise van die aminosuur verandering van Glutamien (Q) tot Arginien (R) is uitgevoer met behulp van verskeie voorspellings algoritmes. Twee sellulêre modelle van PS, insluitend (1) HEK293 selle wat getransfekteer is met WT en gemuteerde LRRK2 vektore sowel as (2) pasiënt dermale fibroblaste, is gebruik vir fuksionele studies. LRRK2 mutante vektore was gegenereer deur gebruik te maak van plek-gerigte mutagenese in pcDNA-DEST53, ‘n soogdier uitdrukkings vektor. Velbiopsies van individue wat die G2019S, Q2089R of WT LRRK2 dra is gebruik om fibroblaste te kweek en te gebruik as ‘n ex vivo model vir die siekte. Die kinase aktiwiteit van LRRK2 is ondersoek deur gebruik te maak van outofosforilasie van Serien 1292 en Westerse klad analise. Sel-metaboliese aktiwiteit is gemeet met behulp van ‘n 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromied (MTT) toets en die mitochondriale membraan potentiaal (MMP) is gemeet deur die JC-1 fluorochroom en vloei-sitometrise ontleding. Mitochondriale en glikolitiese respirasie eksperimente was uitgevoer met behulp van die Seahorse XF Analyzer en die mitochondriale DNA kopiegetal is bepaal deur kwantitatiewe “real-time PCR (qRT-PCR)”. Merkers van autophagy, LC3 II en P62, is opgespoor met behulp van Westerse klad analise. Co-lokaliserings eksperimente van LRRK2 en die TOM kompleks is uitgevoer met behulp van konfokale en super resolusie gestruktureerde verligtings mikroskopie (SR-SIM), terwyl proteïen interaksies ondersoek is met behulp van mede-immunopresipitasie en Westerse klad analise. Die frekwensie van Q2089R was 0.2% (1/493) in PS pasiënte en 0.1% (1/776) in kontrole individue. Verskeie in silico toetse het voorspel dat die Q na R vervanging moontlik patogenies sal wees [‘nadelig’ (CADD telling=24.1), ‘moontlik skadelik’ (PolyPhen) en ‘siekte-veroorsakend’ (Mutation Taster)]. Die LRRK2 vektore was suksesvol gegenereer en die fibroblaste is suksesvol gekweek. In HEK293 selle is bevind dat Q2089R byna heeltemal die outofosforilasie aktiwiteit van LRRK2 afgeskaf het (p=0.026). Q2089R-draende selle het ook ‘n afname in sel-metaboliese aktiwiteit getoon in HEK293 selle (p=0.016) en fibroblaste (p<0.05). Verder, in beide seltipes het die MMP aansienlik afgeneem [(p=0.043 en p=0.009 vir HEK293 selle en fibroblaste (onder stres), onderskeidelik)]. In Q2089R-fibroblaste was n toename in basale respirasie (p=0.012), proton lek respirasie (p=0.0001), maksimale respirasie (p<0.0001) en vrye respiratoriese kapasiteit (p<0.0001) waargeneem, terwyl ATP-koppelings doeltreffendheid (p=0.0014), glikolitiese reserwe (p=0.006) en glikolitiese kapasiteit (p=0.007) aansienlik verminder is. In beide modelle van Q2089R is die autophagosoom poel grootte verhoog (p<0.05 vir LC3 II en p<0.05 vir P62). In die geval van G2019S, in ooreenstemming met veskeie vorige studies, is n verhoging in outofosforilasie aktiwiteit waargeneem in HEK293 selle. Verminderde sel lewensvatbaarheid (p=0.021) en MMP (p=0038) is ook opgemerk in hierdie selle. G2019S-draende fibroblaste het n afname getoon in selproliferasie terwyl basale respirasie (p=0.029), ATP-gekoppelde respirasie (p=0.029), glikoliese (p=0.001) en autophagosoom poel grootte (p=0.022 vir LC3 II) toegeneem het. Die MMP van hierdie fibroblaste het 'n nie-beduidende tendens van afname onder stremming getoon (p=0.057). Interessant genoeg is dit bevind dat WT LRRK2 co-lokaliseer en mede-immunopresipiteer met ‘n proteïen kompleks wat TOM22, TOM40 en TOM70 maar nie TOM20 bevat onder normale toestande nie. Onder stremming is 'n assosiasie tussen LRRK2 en TOM20 waargeneem terwyl die assosiasie tussen LRRK2 en die kompleks wat TOM22 en TOM70 bevat toegeneem het. Laastens, uit ons bevindinge en vanuit die gepubliseerde literatuur, stel ons 'n model voor vir die betrokkenheid van LRRK2 (WT en Q2089R) in sel funksionering en seldood. Dit behels die verlies van kinase aktiwiteit en assosiasie met die TOM kompleks, wat uiteindelik LRRK2 heg met mitochondriale (dis)funksie, mitochondriale biogenese en die autophagy/lisosomale pad weg. Ten slotte is daar ‘n funksionele variant in die kinase domein van LRRK2 gekarakteriseer en bykomende funksies vir hierdie groot multi-domein proteïen voorgestel. Hierdie studie bied ook bewyse van 'n nuwe assosiasie tussen LRRK2 en die TOM kompleks. Daarenteen bevraagteken hierdie studie die idee dat dit net verhoogde LRRK2 kinase aktiwiteit is wat betrokke is by PS ontwikkeling. Die navorsers van hierdie studie erken egter dat die bevindinge voorlopig is en dat verdere verifiering studies nodig is om die resultate asook hipotese te bevestig. Toekomstige geteikende eksperimente op LRRK2 is nodig om die komplekse patobiologie te ontrafel en om die volgorde van gebeure te ontsyfer wat aanleiding gee tot die ontwikkeling van PS in vatbare individue.

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