Investigating the role of rilmenidine and spermidine on autophagic flux and cell death in a model of Alzheimer's disease

Lumkwana, Dumisile (2020-04)

Thesis (PhD)--Stellenbosch University, 2020.

Thesis

ENGLISH ABSTRACT: Introduction - Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by multiple cognitive deficits. The neuropathology of AD is underpinned by two molecular hallmarks; intracellular protein aggregates known as neurofibrillary tangles (NFTs), composed of hyper-phosphorylated Tau and extracellular amyloid beta (Ab) plaques, composed of Ab peptides derived from the amyloid precursor protein (APP). Both of these occur as a result of an imbalance in proteostasis, leading to neuronal toxicity. Although, we have advanced our understanding of the molecular machinery that regulates the rate of protein degradation through autophagy at basal levels and increasingly so the many aspects of its dysfunction in AD, the deviation of autophagic activity from basal levels and its change during disease pathogenesis in neuronal tissue remains largely unclear. Over the recent years, substantial progress has been made in modulating autophagy using pharmacological agents in vitro and in vivo and mounting evidence points towards autophagy modulation using pharmacological agents as one of the major therapeutic strategies for neurodegenerative diseases. Although spermidine and rilmenidine both enhance autophagy, the relationship between autophagy activity, the extent of protein clearance and cell death onset remains unclear. Moreover, the impact of their concentration on autophagic flux and subsequent protein clearance as well as neuronal toxicity is unclear. Therefore, this study aimed to unravel the impact of using a high and low concentration of spermidine and rilmenidine on autophagic flux, neuronal toxicity and protein clearance using distinct neuronal injury model systems. Methods - Murine hypothalamus-derived GT1-7 neuronal cells and the mouse neuroblastoma (N2a) cell line stably expressing Swedish double mutation APP695 (Swe) associated with AD pathology were used. GT1-7 cells transfected with mRFP-GFP-LC3 and GFP-LC3-RFP-LC3DG were treated with a low and high concentration of spermidine and rilmenidine in the absence and presence of saturating concentrations of bafilomycin, after which the autophagic flux profile was characterized, assessing cellular viability, autophagosome pool, autolysosome pool, autophagosome flux, transition time, p62 puncta and autophagic vacuoles. Cellular viability assays, western blotting, fluorescence microscopy, transmission electron microscopy and correlative light and electron microscopy linked to quantitative morphometric analysis was performed. In addition, potential protective effects of a low and high concentration of spermidine and rilmenidine were assessed in a paraquat (PQ)-induced neuronal toxicity model and in an APP over-expression model. Cellular viability, ROS damage, cell death onset and autophagic activity were assessed in the PQ-induced toxicity model, while in the APP model, cellular viability and protein clusters were assessed and quantified using cellular viability assays and single molecule imaging, i.e. direct Stochastic Optical Reconstruction Microscopy (d-STORM). Moreover, a Correlative Light and Electron Microscopy (CLEM) method was developed and implemented to morphometrically assess the effects of a low and high concentration of spermidine on the localization of autophagosomes in 3 dimensions. Finally, potential protective effects of spermidine at two distinct concentrations were assessed using transgenic mice expressing GFP-LC3, treated with PQ to induce neuronal toxicity associated with neurodegeneration. Results - Our results indicate a concentration-dependent effect of spermidine and rilmenidine on autophagic flux with the detected change in flux depending on the specificity and sensitivity of the method employed. In addition, in the in vitro model of PQ-induced toxicity, our results revealed that spermidine at a low concentration and not a high concentration protected against cell toxicity, ROS damage, cell death as well as microtubule destabilization. To our surprise, both concentrations of rilmenidine failed to protect against ROS damage and cell death and also failed to robustly upregulate autophagy in the same model. Moreover, in the in vitro model of neuronal toxicity induced by APP over-expression, our results showed that both concentrations of spermidine and rilmenidine protected against cytotoxicity. Here, spermidine at a low concentration effectively cleared APP clusters and reduced their size, especially after 48 h of APP over-expression, while rilmenidine, reduced the number and size of APP clusters in a concentration-dependent manner at the same time point. Taken together, the in vitro results reveal a concentration-dependent effect, that is cell type and injury specific, impacting autophagy and cell death control. Moreover, we have successfully implemented a 3D CLEM protocol and revealed that spermidine, in combination with BafA1 decreases autophagosome volume while increasing their surface area in a concentration-dependent manner. Lastly, in vivo, our results reveal that PQ-induced toxicity impacts the brain regions differentially, with the hippocampus being highly susceptible to PQ-induced injury followed by the cortex. Moreover, our results show that both dosages of spermidine robustly protected against oxidative stress, neuronal damage, microtubule destabilization, and upregulated autophagy, in the hippocampus and cortex. However, the low dose of spermidine resulted in more enhanced protection, although, autophagy was here regionspecifically upregulated in a manner dependent on the dose utilized, with the higher dose of spermidine increasing LC3-II in the hippocampus, and the lower dose increasing LC3-II in the cortex. Conclusion - Our results indicate the critical importance of using multiple tools to assess autophagy and show that a concentration-dependent effect of the two selected drugs on autophagic flux exists. In addition, we provide evidence of the distinct, context dependent protective roles of spermidine and rilmenidine in an in vitro model of APP over-expression as well as the protective roles of spermidine using in vitro and in vivo models of PQ-induced neuronal toxicity. These results suggest that firstly administration of spermidine may represent a favourable therapeutic strategy for the treatment of AD and secondly, concentration / flux screening may be more critical for optimal autophagy control than previously thought. Future studies, using an in vivo model over-expressing APP are warranted to further verify the protective effects of spermidine, to foster clinical translation and therapeutic intervention in neurodegenerative disorders.

AFRIKAANSE OPSOMMING: Inleiding – Alzheimer se siekte (AS) is ’n progressiewe neurodegeneratiewe versteuring wat gekenmerk word deur veelvuldige kognitiewe tekorte. Die neuropatologie van AS word gekenmerk deur twee molekulêre eienskappe; intrasellulêre proteïenaggregate bekend as neurofibrillêre knope (NFK), saamgestel uit hiper-gefosforileerde Tau, en ekstrasellulêre amiloïede beta (Ab) plake, saamgestel uit Ab-peptiede wat afkomstig is van die amiloïede voorloperproteïen (AVP). Beide kom voor as gevolg van ’n wanbalans in proteostase, wat lei tot neuronale toksisiteit. Alhoewel ons begrip van die molekulêre masjinerie wat die tempo van proteïendegradasie deur outofagie op basale vlakke reguleer gevorder het en dan toenemend ook die vele aspekte van AS wanfunksie, bly die afwyking van outofagiese aktiwiteit vanaf die basale vlakke en die verandering tydens siekte patogenese in neuronale weefsel grootliks onduidelik. In die afgelope paar jaar is aansienlike vordering gemaak met die modulering van outofagie met behulp van farmakologiese middels in vitro en in vivo, en die toenemende bewyse dui daarop dat outofagiese modulering deur middel van farmakologiese middels een van die belangrikste terapeutiese strategieë vir neurodegeneratiewe siektes is. Alhoewel spermidien en rilmenidien beide outofagie verhoog, bly die verband tussen outofagie-aktiwiteit, die omvang van proteïenopruiming en die aanvang van seldood nie duidelik nie. Verder, die impak van hul konsentrasie op outofagiese stroom en daaropvolgende proteïenopruiming, sowel as op neuronale toksisiteit, is onduidelik. Daarom was die oogmerk van hierdie studie om die impak van beide hoë en lae konsentrasies spermidien en rilmenidien op outofagiese stroom, neuronale toksisiteit en proteïenopruiming met behulp van verskillende neuronale beseringsmodelsisteme te ontrafel. Metodes – Die muis GT1-7 neuronale sellyn, afkomstig van die hipotalamus, en die muis neuroblastoom (N2a) sellyn, wat die Sweedse dubbele mutasie AVP695 (Swe) wat met AS patologie verband hou stabiel uitdruk, is gebruik. GT1-7 selle wat met mRFPGFP- LC3 en GFP-LC3-RFP-LC3DG getransfekteer is, is behandel met ’n lae en hoë konsentrasie spermidien en rilmenidien in die afwesigheid en teenwoordigheid van versadigde konsentrasies bafilomisien, waarna die outofagiese stroomprofiel gekarakteriseer was deur die evaluering van sellulêre lewensvatbaarheid, outofagosoom poel, outolisosoom poel, outofagosoom stroom, oorgangstyd, p62 stippels en outofagiese vakuole. Sellulêre lewensvatbaarheidstoetse, westelike klad, fluoressensiemikroskopie, transmissie-elektronmikroskopie en korrelatiewe lig- en elektronmikroskopie (KLEM) gekoppel aan kwantitatiewe morfometriese analise is uitgevoer. Die moontlikke beskermende effekte van ’n lae en hoë konsentrasie spermidien en rilmenidien is ook beoordeel in ’n paraquat- (PQ) geïnduseerde neuronale toksisiteitsmodel en in ’n AVP-ooruitdrukking model. Sellulêre lewensvatbaarheid, RSS-skade, aanvang van seldood en outofagiese aktiwiteit is beoordeel in die PQ-geïnduseerde toksisiteitsmodel, terwyl sellulêre lewensvatbaarheid en proteïentrosse in die AVP-model beoordeel en gekwantifiseer is met behulp van sellulêre lewensvatbaarheidstoetse en enkelmolekule-beelding, d.w.s. d-STORM. Verder is ‘n KLEM-metode geöntwikkel en geïmplementeer om die gevolge van ’n lae en hoë konsentrasie spermidien op die lokalisering van outofagosome in drie dimensies morfometries te beoordeel. Laastens is die moontlikke beskermende effekte van spermidien met twee verskillende konsentrasies beoordeel met behulp van transgeniese muise wat GFP-LC3 uitdruk en behandel is met PQ om neuronale toksisiteit wat verband hou met neurodegenerasie te induseer. Resultate - Ons resultate dui op ’n konsentrasie-afhanklike effek van spermidien en rilmenidien op outofagiese stroom waar die waargenome verandering in stroom afhanklik is van die spesifisiteit en sensitiwiteit van die metode wat toegepas word. Boonop het ons resultate in die in vitro model van PQ-geïnduseerde toksisiteit aan die lig gebring dat spermidien teen ’n lae konsentrasie maar nie teen ’n hoë konsentrasie beskermend is teen seltoksisiteit, RSS-skade, seldood en mikrotubulie-destabilisasie. Tot ons verbasing kon beide konsentrasies rilmenidien nie teen RSS-skade en seldood beskerm nie, en het ook nie daarin geslaag om outofagie in dieselfde model te opreguleer nie. Verder, in die in vitro model van neuronale toksisiteit wat deur AVP-ooruitdrukking veroorsaak word, het ons resultate getoon dat beide konsentrasies spermidien en rilmenidien beskermend is teen sitotoksisiteit. Hier het ‘n lae konsentrasie spermidien effektief AVP-trosse skoongemaak en hul grootte verminder, veral na 48 uur van AVP-ooruitdrukking, terwyl rilmenidien die aantal en grootte van die AVP-trosse op ’n konsentrasie-afhanklike manier op dieselfde tydstip verminder het. Saamgevat toon die in vitro resultate ’n konsentrasie-afhanklike effek, wat ook afhanklik is van seltipe en besering, wat outofagie en die beheer van seldood beïnvloed. Daarbenewens het ons ’n 3D KLEM-protokol suksesvol geïmplementeer en het aan die lig gebring dat spermidien, in kombinasie met BafA1, die volume van die outofagosoom verlaag terwyl die oppervlakte op ’n konsentrasie-afhanklike manier vergroot. Laastens, in vivo toon ons resultate dat PQ-geïnduseerde toksisiteit die breinstreke tot verskillende mates beïnvloed, met die hippokampus wat die mees vatbaarste is vir PQ-geïnduseerde skade gevolg deur die korteks. Daarbenewens toon ons resultate dat beide dosisse spermidien kragtige beskerming gebied het teen oksidatiewe stress, neuronale skade en mikrotubulie-destabilisasie en het outofagie opgereguleer in die hippokampus en korteks. Die lae dosis spermidien het egter meer beskerming gebied, alhoewel outofagie in ‘n streek- en dosis-afhanklike manier opgereguleer was, met die hoër dosis spermidien wat LC3-II in die hippokampus verhoog en die laer dosis wat LC3-II in die korteks verhoog. Gevolgtrekking - Ons resultate dui op die kritiese belang van die gebruik van veelvuldige metodes om outofagie te evalueer en toon dat daar ’n konsentrasie-afhanklike effek van die twee geselekteerde middels op outofagiese stroom is. Daarbenewens lewer ons bewyse vir die eiesoortige, konteks-afhanklike beskermende rol van spermidien en rilmenidien in ’n in vitro model van AVP-ooruitdrukking, sowel as die beskermende rol van spermidien in in vitro en in vivo modelle van PQ-geïnduseerde neuronale toksisiteit. Hierdie resultate dui daarop dat eerstens, die toediening van spermidien ’n gunstige terapeutiese strategie vir die behandeling van AS verteenwoordig en tweedens, dat konsentrasie / stroomsifting belangriker is vir optimale behering van outofagie as wat voorheen gedink was. Toekomstige studies, met behulp van ’n in vivo model van AVP-ooruitdrukking, word benodig om die beskermende effekte van spermidien verder te bevestig om sodoende kliniese vertaling en terapeutiese ingryping in neurodegeneratiewe versteurings te ondersteun.

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