Investigating Tau pathology in an in vitro model for Alzheimer’s disease

Powrie, Yigael Samuel Louis (2016-12)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Introduction Alzheimer’s disease is a neurodegenerative disease of the brain and the leading cause of dementia globally. Severe cognitive and short term memory deficits are commonly associated with this disease. The pathology is characterised by two molecular hallmarks that manifest in brain tissue, which are intercellular plaques composed of β-amyloid, and intracellular protein aggregates known as neurofibrillary tangles (NFTs) composed of phosphorylated Tau, a microtubule (MT) associated protein (MAP). Under homeostatic conditions Tau facilitates the dynamic polymerisation of the microtubule network, which acts as part of the cytoskeleton and platform for vesicular transport. Tau is generally phosphorylated to modulate its binding affinity to the network. However, under pathological conditions it becomes hyperphosphorylated, leading to dissociation from the MT. Dissociated Tau is thought to form NFT aggregates, which causes the MT to become susceptible to cleavage by the severing proteins, Katanin p60 and Spastin. However, it has not been determined when this process occurs in disease progression and whether it is indeed a confounding factor leading to the onset of neuronal cell death. Moreover, although dysfunction of the autophagic lysosomal pathway, an inherent proteolytic process for long-lived proteins and organelles, has been shown to be implicated in the onset of protein aggregation, its role in the context of MT dysfunction remains unclear. Aims and Methods The aims of this study were to assess microtubulin and Tau dynamics in an in vitro model of autophagic dysfunction that is similar to the Alzheimer’s disease pathology. It was hypothesized that a disruption in the autophagy process would lead to maladaptive changes in the microtubulin and Tau dynamics prior to the onset of cell death. GT1-7 neuronal cells were cultured under standard conditions and treated with Chloroquine diphosphate (CQ), a lysosome deacidifying agent, to induce an autophagic dysfunctional state. Two time points of exposure to CQ were established using a WST-1 assay to assess the molecular changes occurring prior to and during the onset of cell death. Western blot analysis was utilised to quantify protein levels of acetylated α-tubulin, Tau, pTau, Katanin p60 and Spastin in response to CQ-induced autophagy dysfunction. Furthermore, cells were transfected with a GFP-Tau DNA construct, using the Neon® transfection system. Additionally, cells were fixed and stained post-transfection with fluorescent Alexa® Fluor secondary antibodies against primary antibodies recognising acetylated α-tubulin, pTau, Katanin p60 and Spastin. Fluorescent microscopy analysis was performed using Super Resolution Structured Illumination Microscopy (SR-SIM), Stochastic Optical Reconstruction Microscopy (STORM), Correlative Light and Electron Microscopy (CLEM) and confocal microscopy techniques on the LSM-780 Elyra PS.1 system to assess protein localisation in response to CQ treatment. Moreover, co-localisation was assessed between acetylated α-tubulin and Tau, pTau, Spastin and Katanin p60 respectively. Results Fluorescent microscopy analysis revealed that CQ-induced autophagy dysfunction caused acetylated α-tubulin protein structure to become progressively impacted, which manifested as breakages in the network. Tau protein levels decreased non-significantly, but fluorescent microscopy revealed the formation intracellular Tau aggregates. In addition, Tau co-localised with acetylated α-tubulin under control conditions and remained co-localised in response to CQ treatment. Phosphorylated Tau protein levels did increase non-significantly, but fluorescent microscopy revealed no aggregate formation. Katanin p60 protein levels significantly increased, however, the protein did not co-localise with acetylated α-tubulin under control conditions or in response to CQ-induced autophagy dysfunction. Spastin protein levels increased non-significantly, however, Spastin co-localised with acetylated α-tubulin under control conditions, which significantly increased in response to autophagy dysfunction. Discussion and Conclusion Our results indicate that CQ-induced autophagy dysfunction causes Tau aggregation, but no dissociation from the microtubule network. Furthermore, the microtubulin network becomes unstable, despite its continuous association with Tau, which may be caused by increased Spastin-mediated severing. To conclude, the data clearly demonstrate that these pathological perturbations occur prior to the onset of cell death, which not only highlights novel therapeutic targets, but also the lack of optimal timing in the therapeutic interventions utilised in Alzheimer’s disease treatment.

AFRIKAANSE OPSOMMING: Inleiding Alzheimer’s siekte is ‘n neurodegeneratiewe siekte van die brein en die hoofoorsaak van demensie ter wêreld. Hewige kognitiewe en korttermyngeheue gebreke word algemeen geassosiëer met hierdie siekte. Die patologie word gekenmerk deur twee molekulêre kenmerke wat manifesteer in die breinweefsel. Dit sluit in intersellulêre plaakvorming wat bestaan uit amiloïed-β, en intrasellulêre aggregate, ook bekend as neurofibrillêre knope (NFK), wat uit Tau, ’n mikrotubulêre (MT) geassosiëerde proteïn (MAP) bestaan. Onder homeostatiese toestande fasiliteer Tau die dinamiese polimerisasie van die mikrotubulêre netwerk, wat dien as deel van die sitoskelet, en ook as ’n platform vir vesikulêre vervoer funksioneer. Tau is normaalweg gefosforileer om die bindingsaffiniteit vir die mikrotubulêre netwerk te moduleer. Nieteenstande, onder patologiese toestande word Tau gehiperfosforileer wat veroorsaak dat dit van die mikrotubulêre netwerk dissosiëer. Gedissosieerde Tau vorm vermoedelik neurofibrillêre knope, wat veroorsaak dat die mikrotubulêre netwerk vatbaar is vir degradasie deur die proteïne, katanin p60 en spastin. Nieteenstaande, is dit nog nie vasgestel waneer hierdie proses in die siekte progressie plaas vind nie, en of dit ’n bepalende faktor is wat lei tot die aanslag van neuronale seldood. Hoewel disfunksionering van die autofagiese lisosoomsisteem, ’n proteolitiese proses vir verouderde en beskadigde proteïne en organelle, geimpliseerd is in die aanslag van die vervorming van proteïn aggregate, is dit in die konteks van mikrotubulêre netwerk destabilisasie steeds onduidelik. Doel en Metodes Die doel van hierdie studie was dus om die dinamika van Tau en die mikrotubulêre netwerk in ’n in vitro model van autofagiese dusfunksionering, wat soortgelyk is aan die Alzheimer’s siekte patologie, te ondersoek. Die hipotese wat gestel is, is dat ’n ontwrigting in die outofagiese proses tot wanaangepaste veranderinge in die dinamika van Tau en die mikrotubulêre netwerk voor die aanslag van seldood sal aanleiding gee. GT1-7 neuronale selle was deur middel van selkultuur geweek onder standaardtoestande met ’n lisosomiese onversuringsmiddel, chlorokiendifosfaat (CQ) behandel, om ’n outofagiese disfunksionele toestand te veroorsaak. Twee CQ blootstellingsperiodes was vasgestel met ’n WST-1 toets om molekulêre veranderinge, voor en gedurende die aanvang van seldood, te ondersoek. Western blot analiese is gebruik om die proteïnvlakke van geasetileerde α-tubulien, Tau, pTau, katanin p60 en spastin in reaksie op CQ-geïnduseerde autofagiese disfunksie, te kwantifiseer. Selle was getransfekteer met ’n GFP-Tau DNS plasmied met die Neon® transfekteringsstelsel. Verder, was selle chemies gepreseveer na transfektering en met fluoresserende Alexa® Fluor sekondêre teenliggaampies teen die primêre teenliggaampies wat geasetileerde α-tubulien, pTau, katanin p60 en spastin erken, gekleur. Fluoressensie mikroskopie is gebruik met behulp van Super Resolusie Gestruktureerde Verligtings Mikroskopie (SR-SIM), Stogastiese Optiese Rekonstruksie Mikroskopie (STORM), Korrelatiewe Lig en Elektron Mikroskopie (CLEM), en konfokale mikroskopiese tegnieke op die LSM-780 Elyra PS.1 stelsel, om proteïenlokalisering te evalueer in reaksie op CQ behandeling. Verder, was ko-lokalisering tussen geasetileerde α-tubulien en pTau, katanin p60 en spastin onderskeidelik geëvalueer. Resultate Fluoressensie mikroskopie analiese het gewys dat CQ-geïnduseerde outofagiese disfunksie ’n progressiewe impak op die geasetileerde α-tubulien struktuur gehad het, wat gemanifesteer as gebreke in die netwerk. Tau proteïenvlakke het nie betekenisvol afgeneem nie. Fluoressensie mikroskopie analiese het ook gewys dat Tau aggregate in reaksie op CQ behandling gevorm het. Benewens, het Tau geko-lokaliseer met geasetileerde α-tubulien onder onbehandelde toestande, en só gebly tydens outofagiese disfunksie. Gefosforileerde Tau proteïenvlakke het nie betekenisvol toegeneem nie, en geen proteïenaggregate het gevorm nie. Katanin p60 proteïenvlakke het beduidenlik toegneem, maar het nie met die geasetileerde α-tubulien die in onbehandelde toestande òf gedurende outofagiese disfunksie geko-lokaliseer nie. Spastienproteïenvlakke het nie betekensvol toegneem nie. Spastien het geko-lokaliseer met die geasetileerde α-tubulien onder onbehandelde toestande, en het progressief en beduidenlik geko-lokaliseer tydens outofagiese disfunksie. Bespreking en gevolgtrekking Ons resultate toon dat CQ-geïnduseerde outofagiese disfunksie veroorsaak dat Tau aggregate vorm, maar nie van die mikrotubulêre netwerk dissosiëer nie. Verder, word die mikrotubulêre netwerk onstabiel ten spyte van die gedurende assosiëering met Tau, wat mag aandui dat daar ’n toename in spastien bemiddelde degradasie van die netwerk is. Opsommend demonstreer hierdie data dat die patologiese versteurings voor die aanslag van seldood plaasvind. Dit beklemtoon nie net moontlike nuwe terapeutiese teikens nie, maar ook die gebrek aan optimale tydsberekening in terapeutiese intervensies wat gebruik word in Alzheimer’s siekte behandeling.

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