Long-term maintenance of adult neural stem cells (NSCs) is an intricate process of activation, expansion, and differentiation while preserving the stem cell pool. Several regulatory mechanisms underlie the delicate balance in the choice between quiescence versus activation for lifelong NSC maintenance and continuous neurogenesis. Perturbations in this dynamic process result in disease manifestation. The quiescence/activation of NSC was shown to be regulated by the Rb/E2F axis through a molecular program mediated by REST (RE1 Silencing Transcription Factor). Loss of Rb family increased NSC activation at the expense of quiescence through activator E2F transcription factors. The activation and neurogenesis of NSCs were impaired by the loss of effector E2Fs, as well as loss of Opa1, the latter indicating that mitochondrial dynamics is important to maintain stem cell state. Single-cell transcriptome analysis from NSC lineages isolated from adult mouse hippocampus revealed that stem cell progenies are uniquely affected in Opa1-KO leading to impairments in NSC activation and differentiation. Unbiased transcriptional profiling suggested a mitochondrial dysfunction in Opa1-KO that results in activation of classic cellular stress response pathway genes (Atf4, Slc7a11 and Chac1). Thus, the regulatory gene network comprising quiescence (Rb) and activation (E2Fs) programs, and mitochondrial metabolism (Opa1) and their interplay ensures the maintenance of the molecular program of NSC, particularly revealing how it enables stem cells survive stress.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction: Doxorubicin (DOX), infamously known as the “red devil,” is considered the most effective antineoplastic drug utilized in oncologic practice today. However, its clinical use is hampered due to cumulative, dose-dependent cardiotoxicity, which can lead to reduced quality of life, irreversible heart failure and death. The mechanisms involved in the pathogenesis of cardiotoxicity have not been fully elucidated, but have previously been demonstrated to involve oxidative stress, calcium dysregulation and mitochondrial dysfunction. Since the mitochondria play a critical role in generation of reactive oxygen species, the maintenance of calcium homeostasis and are the most extensively damaged by DOX, they have become the main focus of novel therapeutic interventions. The morphology and function of these dynamic organelles are regulated in part by mitochondrial fission and fusion events, as well as mitochondrial quality control systems. Since mitochondrial morphology is often associated with crucial cellular functions, this study aimed to investigate the long-term effects of DOX on mitochondrial dynamics and the mitochondrial quality control systems, mitophagy and the ubiquitin-proteasome pathway (UPP). Additionally, since the mitochondria and the endoplasmic reticulum (ER) are two interconnected organelles, and both play a role in maintaining calcium homeostasis, this study further assessed the effects of chronic DOX treatment on ER function and calcium status. Materials and Methods: In order to fully establish the effect of chronic DOX treatment in vitro, two cardiac cell lines were utilized in this study. H9C2 cardiomyoblasts and humanderived Girardi heart cells were cultured under standard culture conditions until ± 70-80% confluency was reached, where after treatment commenced. Cells were treated daily with 0.2 and 1.0 μM of DOX for 96 and 120 hours in order to simulate chronic, cumulative cardiotoxicity. Cell viability and apoptotic cell death were assessed with the MTT assay and Caspase Glo 3/7 assays, respectively. The expression of proteins involved in mitochondrial dynamics, mitochondrial biogenesis, the ubiquitin-proteasome pathway, mitophagy and ER stress were determined with Western blotting. Organelle morphology was visualized with fluorescence microscopy, and flow cytometry was used to assess mitochondrial and ER load. In order to determine the oxidative capacity, stress and status within the cells following treatment, the Oxygen radical absorbance capacity (ORAC), Thiobarbituric acid reactive substances (TBARS) and Glutathione (GSH) assays were employed respectively. Finally, intracellular and mitochondrial calcium was assessed and quantified with superresolution structured illumination microscopy (SR-SIM) and flow cytometry respectively. Results: DOX significantly reduced cell viability and increased apoptosis in both in vitro cardiac cell models. This study further demonstrated that the expression of mitochondrial fusion proteins, Mfn 1 and Mfn 2 were significantly downregulated, whilst the regulators of fission, Drp1 and hFis1, were significantly elevated, therefore shifting the balance of mitochondrial dynamics towards fission. Unopposed and elevated mitochondrial fission was clearly evident from the morphology of these organelles, which displayed short, highly fragmented mitochondria with a dispersed network following treatment. Chronic DOX also downregulated the regulator of mitochondrial biogenesis, PGC-1α, thus inhibiting the formation of new, functional mitochondria. The E3 ligases, MARCH5 and Parkin were highly upregulated following treatment, indicating activation of the UPP and mitophagy. Although chronic DOX stimulated K48 ubiquitination following treatment, it inhibited the catalytic activity of the 26S proteasome, therefore blocking proteasomal degradation. Although the antioxidant capacity (measured as ORAC) was significantly enhanced by both concentrations of DOX, an increase in oxidative stress status was shown following DOX treatment. In this regard lipid peroxidation significantly increased, while redox status of the endogenous antioxidant, glutathione, significantly decreased. Additionally chronic DOX treatment induced ER stress, which lead to an increase in cytosolic and mitochondrial calcium. In response to ER stress, the unfolded protein response (UPR) was then stimulated. Discussion: Results from this study indicate that chronic DOX treatment disrupts the balance of mitochondrial dynamics, favouring mitochondrial fission. Mitochondrial fragmentation is mediated by the downregulation of fusion proteins regulated by the E3 ubiquitin ligase, MARCH5 as well as by the increase in mitochondrial calcium. Mitochondrial fission results in mitophagy, an adaptive response to protect the cardiac cell against damaged mitochondria. This study also indicates that during chronic DOX-induced cardiotoxicity ER stress and the UPR are induced, which is possibly responsible for the disruption in calcium homeostasis. The inhibition of mitochondrial biogenesis coupled with elevated mitophagy as observed in this chronic study, elucidates a plausible mechanism whereby DOX induces mitochondrial dysfunction. Unregulated mitochondrial fragmentation and inhibited mitochondrial biogenesis are known to regulate various cardiomyopathies, therefore since both these effects are induced by chronic DOX treatment suggests a mechanism whereby cardiotoxicity, and ultimately heart failure are produced. This study provides new insight into the role of chronic DOX plays in altering mitochondrial dynamics and mitochondrial quality control systems. Further investigations targeted at limiting mitochondrial fission may reduce the cardiovascular side effects associated with DOX. / AFRIKAANSE OPSOMMING: Inleiding: Doksorubisien (DOX), ook bekend as die “rooiduiwel,” word beskou as die mees effektiewe anti-neoplastiese middel wat tans in onkologie praktyke gebruik word. Die kliniese gebruik hiervan word gerem deur die kumulatiewe dosis-afhanklike kardiotoksisiteit wat tot verlaagde lewenskwaliteit, onomkeerbare hartversaking, en tot die dood kan lei. Die meganismes wat by die kardiotoksiese patogenese betrokke is, is nog onbekend, maar die meganisme het moontlik te doen met oksidatiewe stres, kalsiumwanregulering en mitochondriale wanfunksionering. Omrede die mitochondria ‘n kritieke rol in die vorming van reaktiewe suurstofspesies speel, asook die handhawing van kalsiumhomeostase en die mees beskadigde organelle deur DOX, het die hooffokus na nuwe terapeutiese intervensies verskuif. Die morfologie en funskie van hierdie dinamiese organelle word gereguleer deels deur mitochondriale fragmentering en fussie, asook mitochondriale kwaliteitsbeheersisteme. Omrede mitochondriale morfologie geassosieer is met noodsaaklike sellulêre funksies, het hierdie studie gepoog om die langtermyneffkte van DOX op mitochondriale dinamika en die mitochondriale kwaliteitsbeheersisteme, mitofagie en die ubikwitien-proteosoomweg (UPW) te ondersoek. Siende dat die mitochondria en die endoplasmiese retikulum (ER) twee interverweefde organelle is, en beide ‘n rol speel in die handhawing van kalsiumhomeostase, het hierdie studie verder die effekte van chroniese DOX behandeling op ER funksie en kalsiumstatus ondersoek. Materiaal en Metodes: Om die effek van chroniese DOX behandeling in vitro te verstaan in hierdie studie, is twee hartsellyne gebruik. H9C2 kardiomioblaste en menslike Girardi hartselle is onder standaardtoestande tot ± 70-80% konfluensie bereik is gekweek, waarna behandeling begin is. Selle is daagliks met 0.2 en 1.0 μM DOX vir 96 en 120 uur behandel om chroniese en kumulatiewe kardiotoksisiteit n ate boots. Selvatbaarheid en apoptotiese seldood is onderskeidelik ondersoek deur middel van die MTT en Caspase Glo 3/7 toetse. Die proteïenuitdrukking betrokke by mitochondriale dinamika, mitochondriale biogenese, die ubikwitien-proteosoom weg, mitofagie en ER stres is deur middel van westerse afblatting bepaal. Organelmorfologie is deur middel van fluoresensie mikroskopie gevisualiseer, en vloeisitometrie was gebruik om die aantal mitochondria en ER lading te bepaal. Om die oksidatiewe kapasiteit, stres en status binne die selle na behandeling te bepaal, is die ORAC, TBARS en GSH toetse onderskeidelik gebruik. Laastens was die intrasellulêre en mitochondriale kalsium ondersoek en gekwantifiseer met superresolussie gestruktureerde illuminasie mikroskopie (SR-SIM) en vloeisitomerie. Resultate: DOX het selvatbaarheid betekenisvol verlaag en apoptose in beide in vitro kardiale selmodelle verhoog. Hierdie studie het verder aangetoon dat die uitdrukking van mitochondriale fussie proteïene, Mfn 1 en Mfn 2 betekenisvol afgereguleer is, terwyl die reguleerders van fragmentering, Drp1 en hFis1, betekenisvol verhoog is en daardeur die balans van mitochondriale dinamika na fussie verskuif. Onverhinderde en verhoogde mitochondriale fragmentering is duidelik sigbaar deur die morfologie van die organelle, wat as kort, hoogsgefragmenteerde mitochondria met ‘n verspreide netwerk na behandeling vertoon. Chroniese DOX het ook die mitochondriale biogenese reguleerder, PGC-1α, afgereguleer en daardeur die vorming van nuwe, funksionele mitochondria geinhibeer. Die E3 ligase, MARCH5 en Parkin is hoogs opgereguleer na behandeling, wat aktivering van UPW en mitofagie aantoon. Alhoewel chroniese DOX K48 ubikwitinering na behandeling gestimuleer het, het dit die katalitiese aktiwiteit van die 26S proteasoom geinhibeer en dus die proteosomale degradasie geblokkeer. Antioksidantkapasiteit en oksidatiewe status was betekenisvol na behandeling wat gevolglik tot hoë vlakke oksidatiewe skade binne die selle gelei het. Addisioneel het chroniese DOX behandeling ER stres geïnduseer wat tot ‘n toename in sitosoliese en mitochondriale kalsium gelei het. In reaksie op die ER stres is die UPW gestimuleer. Bespreking: Resultate van hierdie studie het aangetoon dat chroniese DOX behandeling die balans van mitochondriale dinamika onderbreek en sodoende mitochondriale fragmentering bevoordeel. Mitochondriale fragmentering word gemediëer deur die afregulering van fussie proteïene wat deur die E3 ubikwitienligase, MARCH5, gereguleer word, en ook deur die toename in mitochondriale kalsium. Mitochondriale fragmentering induseer mitofagie, ‘n aanpassingsreaksie om die hartselle teen beskadigde mitochondria te beskerm. Hierdie studie toon verder ook dat gedurende chroniese DOX-geïnduseerde ER stres, word die UPW ook geïnduseer, wat moontlik dan verantwoordelik is vir die ontwrigting van kalsiumhomeostase. Die inhibering van mitochondriale biogenese gekoppel met verhoogde mitofagie soos waargeneem in hierdie studie, verklaar ‘n moontlike meganisme waardeur DOX mitochondriale wanfunksionering veroorsaak. Ongereguleerde mitochondriale fragmentering en geinhibeerde mitochondriale biogenese is bekend om verskeie kardiomiopatieë te reguleer. Omrede beide hierdie effekte geinduseer word deur chroniese DOX behandeling kan dit moontlik ‘n meganisme voorstel waarby kardiotokiese en uiteindelik hartversaking ontwikkel. Hierdie studie bied nuwe insig in die rol wat chroniese DOX speel in die wysiging van mitochondriale- dinamika en kwaliteitskontrole sisteme. Verdere ondersoeke wat die mitochondriale fragmentering kan verminder mag moontlik die kardiovaskulêre newe-effekte wat met DOX behandeling geassosieer is, verlaag. / National Research Foundation (NRF)

Mitochondrial dynamics resulting from competing fusion and fission reactions are required for normal cellular function in eukaryotes. Mgm1, a dynamin related protein, is a key component in yeast mitochondrial fusion and is evolutionarily conserved. Previous in vivo studies suggest that the GTPase domain and oligomerization are required for Mgm1 mediated mitochondrial inner membrane fusion. This work demonstrates that purified Mgm1 forms dynamic low order oligomers, and has GTPase activity and kinetic properties consistent with a mechanoenzyme and with a role in inner membrane mitochondrial fusion. Mutations of key residues in the GTPase domain show diminished GTPase activity, while a mutation in the GTPase effector domain implicated in self-assembly results in a lower propensity to form oligomers. Together these data indicate that Mgm1 mediates fusion through oligomerization and GTP binding/hydrolysis in a manner similar to other dynamin mechanoenzymes.

Mitochondrial dynamics resulting from competing fusion and fission reactions are required for normal cellular function in eukaryotes. Mgm1, a dynamin related protein, is a key component in yeast mitochondrial fusion and is evolutionarily conserved. Previous in vivo studies suggest that the GTPase domain and oligomerization are required for Mgm1 mediated mitochondrial inner membrane fusion. This work demonstrates that purified Mgm1 forms dynamic low order oligomers, and has GTPase activity and kinetic properties consistent with a mechanoenzyme and with a role in inner membrane mitochondrial fusion. Mutations of key residues in the GTPase domain show diminished GTPase activity, while a mutation in the GTPase effector domain implicated in self-assembly results in a lower propensity to form oligomers. Together these data indicate that Mgm1 mediates fusion through oligomerization and GTP binding/hydrolysis in a manner similar to other dynamin mechanoenzymes.

The cell possesses a number of vital mechanisms to respond to different stressors. Mitochondria are dynamic organelles which undergo constant changes in length, transport and inner membrane structure and curvature. Invaginations of this inner membrane, cristae, have been known to respond to the energetic state of mitochondria, but the regulation of these changes as well as the consequences thereof remain undetermined. We find that Optic Atrophy 1 (OPA1), a protein involved in inner membrane fusion and cristae maintenance during cell death, can respond to the energetic state of mitochondria and the cell. Moreover, OPA1-dependent changes in cristae structure are required for resistance to starvation induced cell death, proper functioning of the electron transport chain, for growth in galactose media and for maintenance of ATP synthase assembly. Interestingly, we demonstrate that select members of the mitochondrial solute carriers (SLC25A) interact with OPA1 and affect the response of OPA1 to substrate levels. Taken together, we propose an SLC25A-dependent role for OPA1 in sensing energy substrate availability and responding to alter cristae, bioenergetics and cellular survival. We also identified KIAA0664 as a novel OPA1-interacting protein, describe its subcellular localization and investigate its role in mitochondrial fusion and in mitochondrial localization. Finally, since both known carriers of mitochondrial glutathione were demonstrated to interact with OPA1, we investigated the role of OPA1 in cellular glutathione redox. OPA1 depleted cells demonstrated both increased total cellular glutathione and a shift in redox to its reduced form. The role of OPA1 in glutathione levels and redox ratios required GTPase activity, but surprisingly not fusion. Since glutathione is a master regulator of reactive oxygen species detoxification, these findings may shed light on the role of OPA1 in ROS-induced cell death pathways.

No description available.

Cardiovascular disease (CVD) is the leading global cause of death. Coronary Artery Disease (CAD) is a grouping of the most common cardiovascular diseases and is the current leading cause of death in developed countries. Treatments for CAD include pharmaceuticals as well as surgical interventions such as percutaneous coronary intervention (PCI) and coronary artery bypass grafting. However, these treatments do not completely remove the risk of adverse outcomes. Endothelial dysfunction is the underlying cause of CAD and is initiated by the chronic inflammation of the vasculature due to increased oxidative stress and production of reactive oxygen species (ROS). Previous studies have shown that the deletion of caveolin, a signaling molecules abundant within endothelial cells, can enhance inflammatory responses and lead to increased oxidative stress and ROS production. Mitochondrial ROS created from dysfunctional mitochondrial dynamics has also been shown to contribute to the inflammation of the endothelium. We hypothesize that due to the link between caveolin and endothelial dysfunction, and the link between mitochondria and endothelial dysfunction, caveolin has an important function in mitochondrial dynamics and that the loss of caveolin increases the mitochondrial fission via a Drp1-dependent pathway. Our data shows that adenoviral silencing of caveolin-1 in rat aortic endothelial cells increases Drp1 expression but does not significantly alter mitochondrial morphology. Overexpression of caveolin-1 via an adenoviral construct in these cells produces a decrease in Drp1 expression without altering mitochondrial morphology. This data provides insight into the pathophysiology of CAD and could provide us with new therapeutic targets in the future. / Biomedical Sciences

Eukaryotic cells are unique in their ability to form complex multicellular organisms giving rise to distinct physiological systems. However, the ability for such complexity to evolve likely stems from an early event in which endosymbiosis of an aerobic prokaryote by a eukaryotic precursor gave rise to the eukaryotic organelle we now know as mitochondria. Mitochondria are colloquially known as the “power house” of the cell due to their ability to produce ATP through oxidative phosphorylation, but perform numerous other vital functions within the cell including sequestration of cytosolic Ca2+, production and sequestration of reactive oxygen species (ROS), and initiation of various forms of cell death. Mitochondria are especially important in neurons given their high demand for ATP and the importance of Ca2+ signaling in neuron excitability and development. Neurons are highly compartmentalized and plastic cells requiring the ability to control energy supply and Ca2+ signaling locally within given specialized structures such as dendritic spines or synaptic boutons. Therefore, mitochondria must be able to localize to particular sub-cellular locales and respond functionally to signaling occurring in that environment. Mitochondrial transport and function are heavily dependent upon the ability of mitochondria to undergo opposing and reversible fission and fusion events. Mitochondrial fission and fusion are themselves regulated by GTPase enzymes which physically catalyze constriction and fusion of the mitochondrial membranes. Mutations in mitochondrial fission and fusion enzymes specifically cause neurological disease in humans and recent work has illustrated the necessity of a proper balance of mitochondrial fission in neuron development, survival, and plasticity. Despite recognizing the importance of mitochondrial fission and fusion in neuron survival, development, and function we lack a concrete understanding of how changes in the equilibrium of fission and fusion impact these processes in vivo. In this thesis we investigate how promoting or inhibiting mitochondrial fission, through phosphoregulation of the mitochondrial fission enzyme Dynamin related protein 1 (Drp1) at mitochondria, impacts neuron survival and memory in vivo. We find that inhibiting phosphorylation of Drp1 at Serine 656 (S656) at the mitochondria, through deletion of a mitochondrial targeted A kinase anchoring protein (AKAP) known as AKAP1 in mice, increases cerebral infarct volume following transient occlusion of the mid-cerebral artery. Oppositely, promoting phosphorylation of Drp1-S656 at the mitochondria, through deletion of the PP2A regulatory subunit Bβ2 which localizes the PP2A heterotrimer to mitochondria, decreases cerebral infarct volume following occlusion of the mid-cerebral artery. Mechanistic in vitro studies in primary neurons reveal these effects are dependent upon the phosphorylation state of Drp1-S656 and likely due to altered mitochondrial respiratory capacity, ROS production, and Ca2+ homeostasis. Interestingly, we also observe improved hippocampal dependent memory in mice in which AKAP1 has been deleted which also appears dependent upon the phosphorylation state of Drp1-S656 and Ca2+ homeostasis. Ultimately, these findings provide insight into how phosphoregulation of Drp1 at the mitochondria alters neuron survival and function through shifting the mitochondrial fission/fusion equilibrium and consequently mitochondrial function.

Alzheimer’s disease (AD) is characterized pathologically by proteinaceous aggregates composed primarily of amyloid \(\beta (A \beta)\) and tau. Diseases characterized by abnormal deposition of tau are collectively termed “tauopathies.” \(A \beta\) acts upstream of tau in the AD pathogenesis pathway, but tau expression is required for the neurodegenerative effects of \(A \beta\). Mitochondrial abnormalities have been documented in Alzheimer’s disease and related tauopathies, but the causal relationship between mitochondrial changes and neurodegeneration, as well as specific mechanisms promoting mitochondrial dysfunction, are unclear. Mitochondrial morphology is regulated by fission and fusion events within and between individual mitochondria, and misregulation of this process has been observed in several neurodegenerative diseases. The contribution of mitochondrial dynamics to the pathogenesis of Alzheimer’s disease and tauopathy has not yet been determined. We have found that expression of tau promotes elongation of mitochondria in Drosophila and vertebrate neurons. Elongation is followed by mitochondrial dysfunction, aberrant cell cycle reactivation, and cell death, which can be rescued in vivo by genetically restoring the proper balance of mitochondrial fission and fusion. Tau induces mitochondrial elongation by inhibiting mitochondrial localization of DRP1, the primary effector of fission. We have previously demonstrated that direct tau-mediated stabilization of filamentous (F)-actin is critical for neurotoxicity. Here we show that actin stabilization is responsible for the mislocalization of DRP1 following tau expression. Additionally, we identify regulatory roles for F-actin and myosin II in DRP1 localization. Similarly to overexpression of human tau, loss of endogenous Drosophila tau (dtau) induces mitochondrial elongation, but through distinct mechanisms. Expression of human \(A \beta\)in Drosophila induces mitochondrial fragmentation and neuronal toxicity, which are reversed by depletion of dtau. Together, we demonstrate that human disease-associated tau induces neurotoxicity through disruption of mitochondrial dynamics, which can be mediated by enhanced actin stabilization. We also observe a novel role for dtau in the regulation of mitochondrial dynamics, a function critical to the ability of endogenous tau to mediate the effects of \(A \beta\). These findings offer new insights into the contribution of mitochondrial dysfunction to AD and tauopathy, and highlight the emerging role of mitochondrial dynamics in the pathogenesis of neurodegenerative disease.

Although pathological alterations in gene expression and mitochondria function in response to cardiac ischemia are well recognized, the mechanisms driving these changes are incompletely understood. Nuclear to mitochondrial communication regulating gene expression and mitochondrial function is a critical process following cardiac ischemic injury. Here we determine that cyclin C, a component of the transcriptional regulator, Mediator complex, directly regulates cardiac and mitochondrial function by modifying mitochondrial fission. We tested the hypothesis that cyclin C has a binary function as a transcriptional cofactor in the nucleus and acute regulation of cardiac energetics in ischemia by enhancing mitochondrial fission in the cytoplasm. In response to stress, cyclin C translocates to the cytoplasm enhancing mitochondria fission in part through interactions with Cdk1. Using cardiac specific cyclin C knockout and overexpression mouse models, we determined cyclin C regulates mitochondria morphology under basal and ischemic conditions in vivo. Furthermore, pretreatment with a Cdk1 inhibitor followed by ischemia in vivo results in reduced mitochondrial fission. Together, our study reveals that cyclin C regulates both hypertrophic gene expression and mitochondrial fission providing new insights into the regulation of cardiac energy metabolism following acute ischemic injury.