Alzheimer's disease is a progressive neurodegenerative disorder, in which there is a marked decline in neurotransmitters, especially those of the cholinergic pathways. One of the approaches to the symptomatic treatment of Alzheimer's disease is the inhibition of the breakdown of the neurotransmitter acetylcholine, using an acetylcholinesterase inhibitor. One such drug tested, is the organophosphate, metrifonate. Any drug used for the treatment of neurodegenerative disorders should preferably not induce further neurological damage. Thus, in the present study, we investigated whether or not metrifonate is neuroprotective. The in vivo and in vitro effect of this drug on free radicals generation shows that metrifonate increases the level ofthese reactive species. Lipid peroxidation induced using quinolinic acid (QA) and iron (II) and show that metrifonate increased the peroxidative damage induced by using quinolinic acid. Metrifonate is also able to induce lipid peroxidation both in vivo and in vitro. This was reduced in vitro in the presence of melatonin. Using iron (II), in vi/ro, there was no significant difference in the level of lipid peroxidation in the presence of this drug. An investigation of the activity of the mitochondrial electron transport chain and complex I of the electron transport chain in the presence of metrifonate revealed that metrifonate reduces the activity of the electron transport chain at the level of complex I. The activity of the mitochondrial electron transport chain was restored in the presence of melatonin. Pineal organ culture showed that metrifonate does not increase melatonin production. Histological and apoptosis studies show that tissue necrosis and apoptosis respectively, occur in the presence of this agent, which is reduced in the presence of melatonin. Metal binding studies were performed USing ultraviolet spectroscopy, and electrochemical analysis to examine the interaction of metrifonate with iron (II) and iron (III). No shift in the peak was observed in the ultraviolet spectrum when iron (ll) was added to metrifonate. Electrochemical studies show that there may be a very weak or no ligand formed between the metal and drug. This study shows that while drugs such as metrifonate may be beneficial in restoring cognitive function in Alzheimer's disease, it could also have the potential to enhance neurodegeneration, thus worsening the condition, in the long term. / KMBT_363 / Adobe Acrobat 9.54 Paper Capture Plug-in

Cerebral ischaemia, traumatic injury to the brain, inflammatory neurological disorders and HIV infections are amongst the most prevalent causes of neurodegeneration. Neuroprotective strategies are usually to limit the progressive secondary injury that generally occurs, thus limiting overall tissue damage. Neuroprotective strategies are usually to limit the progressive secondary injury that generally occurs, thus limiting overall tissue damage. Sodium channel blockers have been often used for this matter as they prevent the cascade of events culminating in free radical generation and eventually neuronal apoptosis. Newer compounds, such as antiperoxidants and free radical scavengers, show encouraging experimental results, but their clinical use is still very limited. Phenytoin being a popular drug in the treatment of epilepsy has also been used as a neuroprotectant during certain neurological emergencies and in pharmacological prophylaxis of post-traumatic epilepsy. Furthermore this agent functions by prolonging inactivation of voltage gated sodium channels. In these sets of experiment the neuroprotective properties of phenytoin were examined. The histological study revealed that phenytoin confers protection to the CA1 and CA3 regions of the hippocampus under the insult of QUIN. Cells maintain their characteristic shape and minimal tissue necrosis occurs in the presence of this agent. The in vitro effect of this antiepileptic drug on free radicals generation shows that phenytoin does not reduce or prevent the formation of these reactive species. Lipid peroxidation was induced using QUIN and iron (II), two known neurotoxins. The study reveals that only lipid peroxidation induced using iron (II) is reduced by phenytoin. These experiments were carried out in whole rat brain homogenate. These studies show that phenytoin possesses poor free radical scavenging properties. However, the dose-related reduction of iron-induced lipid peroxidation allows for speculation that phenytoin interacts with iron in order to reduce neuronal damage. Metal binding studies were performed using UV, IR and electrochemical analysis to examine the interaction of phenytoin with iron (II) and iron (III). Phenytoin, when added to iron (II) in solution, first oxidises the latter to iron (III) and maintains it in that form. A shift in the peak was observed in the UV spectrum when iron was added to phenytoin. Moreover, electrochemical studies indicate that the interaction between the metal and the ligand is very weak. The IR analysis it shows that phenytoin may be coordinating with iron through the Nitrogen atom on the phenytoin molecule. These studies show that phenytoin maintains iron in its oxidised form, which is a good property to possess as a neuroprotectants. Pineal organ culture showed that phenytoin does not increase melatonin production but slightly and non-significantly reduces the levels of this pineal hormone. However there is a significant rise in precursor NAS levels. As melatonin is known to possess antioxidant and free radical scavenging properties, this could mean that this drug can cause the CNS to become more susceptible to attacks by reactive oxygen species.

Wong, Siu Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 129-141). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgements --- p.iv / List of Abbreviations --- p.v / List of Tables --- p.vii / List of Figures --- p.viii / Chapter 1. --- INTRODUCTION / Chapter 1.1 --- Neurodegenerative disorders - a brief overview --- p.1 / Chapter 1.2 --- Polyglutamine diseases --- p.2 / Chapter 1.3 --- Microscopically visible polyglutamine protein aggregates and its relation to toxicity --- p.7 / Chapter 1.4 --- Polyglutamine protein conformers and their relation to toxicity --- p.10 / Chapter 1.5 --- Modeling polyglutamine diseases in Drosophila / Chapter 1.5.1 --- GAL4/UAS spatial transgene expression system in Drosophila --- p.14 / Chapter 1.5.2 --- Temporal control of GAL4/UAS transgene expression system in Drosophila --- p.16 / Chapter 1.5.3 --- Drosophila as a model to study human pathologies --- p.19 / Chapter 1.5.4 --- Drosophila as a model to study polyglutamine diseases --- p.21 / Chapter 1.6 --- Aims of study --- p.26 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Drosophila culture and manipulation / Chapter 2.1.1 --- Drosophila culture --- p.27 / Chapter 2.1.2 --- Phenotypic examination of adult external eye degeneration --- p.27 / Chapter 2.1.3 --- Pseudopupil assay of adult retinal degeneration and observation of green fluorescent protein in adult eyes --- p.28 / Chapter 2.2 --- Semi-quantitative Reverse Transcription-Polymerase Chain Reaction / Chapter 2.2.1 --- RNA extraction from adult Drosophila heads --- p.30 / Chapter 2.2.2 --- DNase treatment of extracted RNA --- p.31 / Chapter 2.2.3 --- Reverse transcription-Polymerase Chain Reaction (RT-PCR) --- p.31 / Chapter 2.2.4 --- Agarose gel electrophoresis --- p.33 / Chapter 2.3 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) / Chapter 2.3.1 --- Protein extraction from adult Drosophila heads --- p.33 / Chapter 2.3.2 --- Preparation of SDS-polyacrylamide gel and electrophoresis --- p.34 / Chapter 2.3.3 --- Western blotting --- p.35 / Chapter 2.3.4 --- Immunodetection --- p.36 / Chapter 2.4 --- Immunoprecipitation --- p.38 / Chapter 2.5 --- Filter retardation assay --- p.39 / Chapter 2.6 --- Isolation and solubilization of SDS-insoluble protein --- p.40 / Chapter 2.7 --- Sucrose gradient sedimentation --- p.41 / Chapter 2.8 --- Preparation of Drosophila tissues for immunofluorescence analysis / Chapter 2.8.1 --- Dissection and immunostaining of Drosophila larval imaginal eye discs --- p.42 / Chapter 2.8.2 --- Cryosectioning and immunostaining of adult Drosophila heads --- p.44 / Chapter 2.9 --- Atomic force microscopy --- p.47 / Chapter 2.10 --- Reagents and buffers / Chapter 2.10.1 --- Reagents for Drosophila culture --- p.48 / Chapter 2.10.2 --- Reagents for RT-PCR --- p.52 / Chapter 2.10.3 --- Reagents for SDS-PAGE --- p.54 / Chapter 2.10.4 --- Reagents for immunoprecipitation --- p.57 / Chapter 2.10.5 --- Reagents for filter retardation assay --- p.57 / Chapter 2.10.6 --- Reagents for isolation and solubilization of SDS-insoluble protein --- p.58 / Chapter 2.10.7 --- Reagents for sucrose gradient sedimentation --- p.58 / Chapter 2.10.8 --- Reagents for immunofluorescence --- p.59 / Chapter 3. --- RESULTS / Chapter 3.1 --- Establishment of an inducible transgenic Drosophila model of polyglutamine diseases / Chapter 3.1.1 --- Introduction --- p.60 / Chapter 3.1.2 --- Results / Chapter 3.1.2.1 --- GAL80ts-mediated inducible expression of expanded polyglutamine protein in Drosophila / Chapter 3.1.2.1.1 --- GAL80ts controls GAL4/UAS-mediated polyQ protein expression --- p.61 / Chapter 3.1.2.1.2 --- Inducible expression of SDS-soluble expanded polyglutamine protein --- p.64 / Chapter 3.1.2.1.3 --- Inducible expression of expanded polyglutamine protein accumulates gradually in form of SDS-insoluble protein --- p.66 / Chapter 3.1.2.1.4 --- Inducible expression of expanded polyglutamine protein results in progressive accumulation of microscopically visible aggregates --- p.68 / Chapter 3.1.2.2 --- Inducible expression of expanded polyglutamine protein causes late-onset progressive neuronal degeneration in Drosophila / Chapter 3.1.2.2.1 --- Inducible expression of expanded polyglutamine protein leads to late-onset progressive deterioration of photoreceptor neurons --- p.68 / Chapter 3.1.2.2.2 --- Inducible expression of expanded polyglutamine protein neither causes external eye degenerative phenotype nor disrupts gross retinal morphology despite deterioration of photoreceptor neurons --- p.72 / Chapter 3.1.2.3 --- Co-expression of caspase inhibitor P35 suppresses polyglutamine-induced neuronal degeneration --- p.72 / Chapter 3.1.2.4 --- Co-expression of molecular chaperone Hsp70 suppresses polyglutamine-induced neuronal degeneration --- p.74 / Chapter 3.1.2.5 --- Inducible expression of expanded polyglutamine protein results in biphasic expression of molecular chaperone Hsp70 in Drosophila --- p.76 / Chapter 3.1.3 --- Discussion --- p.76 / Chapter 3.2 --- Involvement of microscopically visible polyglutamine aggregates in neurodegeneration / Chapter 3.2.1 --- Introduction --- p.83 / Chapter 3.2.2 --- Results / Chapter 3.2.2.1 --- Effect of Hsc70-K71S on microscopically visible polyglutamine aggregates and neuronal degeneration / Chapter 3.2.2.1.1 --- Co-expression of Hsc70-K71S reduces the level of microscopically visible polyglutamine aggregates --- p.83 / Chapter 3.2.2.1.2 --- Co-expression of Hsc70-K71S does not alter polyglutamine transgene expression --- p.84 / Chapter 3.2.2.1.3 --- Co-expression of Hsc70-K71S does not modify polyglutamine-induced neuronal degeneration --- p.87 / Chapter 3.2.2.2 --- Microscopically visible polyglutamine aggregates do not correlate with neuronal degeneration --- p.90 / Chapter 3.2.3 --- Discussion --- p.93 / Chapter 3.3 --- Detection of small SDS-insoluble expanded polyglutamine protein species and its association with neurodegeneration / Chapter 3.3.1 --- Introduction --- p.97 / Chapter 3.3.2 --- Results / Chapter 3.3.2.1 --- Accumulation of SDS-soluble expanded polyglutamine protein does not correlate with neuronal degeneration --- p.98 / Chapter 3.3.2.2 --- Identification of small SDS-insoluble expanded polyglutamine protein species / Chapter 3.3.2.2.1 --- Accumulation of total SDS-insoluble expanded polyglutamine protein positively correlates with progressive neuronal degeneration --- p.99 / Chapter 3.3.2.2.2 --- Accumulation of large SDS-insoluble expanded polyglutamine protein does not correlate with neuronal degeneration --- p.99 / Chapter 3.3.2.2.3 --- Accumulation of small SDS-insoluble expanded polyglutamine protein correlates with neuronal degeneration --- p.104 / Chapter 3.3.3 --- Discussion --- p.107 / Chapter 3.4 --- Biophysical characterization of small SDS-insoluble expanded polyglutamine protein species / Chapter 3.4.1 --- Introduction --- p.109 / Chapter 3.4.2 --- Results / Chapter 3.4.2.1 --- Separation of expanded polyglutamine protein species by sucrose gradient sedimentation --- p.110 / Chapter 3.4.2.2 --- Morphological studies of small SDS-insoluble expanded polyglutamine protein species by atomic force microscopy --- p.112 / Chapter 3.4.3 --- Discussion --- p.118 / Chapter 4. --- GENERAL DISCUSSION --- p.124 / Chapter 5. --- CONCLUSION --- p.127 / Chapter 6. --- REFERENCES --- p.129

Huntington's Disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the Huntingtin gene IT15. In this study we demonstrated that Bcl-2 interacting mediator of cell death Extra Long (BimEL) protein expression was significantly increased in cells expressing mutant Huntingtin (mHtt). Moreover, striatal BimEL expression remained high in an R6/2 HD mouse model throughout the disease progression. Utilizing novel BimEL phospho-mutants we demonstrated the phosphorylation of Ser65 to be important for the stabilization of BimEL. We provided evidence that impaired proteasome function, increased JNK activity and reduced striatal BDNF lead to changes in the phosphorylation of BimEL, thereby promoting its stabilization specifically within the striatum of R6/2 mice. Furthermore, knocking down BimEL expression prevented mHtt-induced cell death in a HD cell culture. Taken together, these findings suggest that BimEL may contribute to the selective neurodegeneration and pathogenesis of HD. / by Rebecca Leon. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Ataxia telangiectasia (AT) is a well-characterized neurodegenerative disease resulting from a genetic defect in the Atm gene causing an absence or very low expression of the ATM protein. As AT patients are prone to the development of insulin resistance and atherosclerosis, the aim or the current study was to investigate the importance of the ATM protein in the endothelium and its role in the signalling pathways of nitric oxide (NO) production. To accomplish this, the first objective was to establish an in-house endothelial cell isolation technique harvested from normal and insulin resistant animals. Unfortunately, these cultures, although staining positive with an endothelial cell specific stain, were not pure enough and did not express endothelial NO synthase (eNOS), the central enzyme in NO production. The remainder of the study utilized commercial aortic endothelial cells (AECs) and found that there was a significant increase in NO production when the ATM protein was inhibited by the specific inhibitor, Ku-60019. The beneficial impact of increased NO production includes maintaining vascular homeostasis, promoting angiogenesis, initiating DNA repair by activating p53 and inhibiting smooth muscle cell proliferation. On the other hand, reactive oxygen species (ROS) and reactive nitrogen species (RNS) also generated by high levels of NO, can exert both protective and harmful effects. Examples of these include cell death due to high concentrations of ROS. However, Ku-60019 did not result in increased cell death of AECs. We demonstrated for the first time, a relationship between endothelial ATM protein kinase and the generation of NO. The signalling pathways involved in NO production and glucose utilization form a network of interrelationships. Central to both pathways is the activity of two protein kinases, PKB/Akt and AMPK. Both these kinases are known to phosphorylate the eNOS enzyme to produce NO on the one hand and AS160 to induce GLUT 4 translocation and glucose uptake on the other hand. Activation of the ATM protein is postulated to be a prerequisite for PKB/Akt activation and it may also result in activation of AMPK. However, using insulin to stimulate ATM, we could not show that inhibition of ATM in endothelial cells affected expression or insulin-stimulated activation of PKB/Akt while the PI3-K inhibitor wortmannin, inhibited the latter. In addition, inhibition of ATM negatively regulated the phospho/total ratio of AMPK. We therefore postulate that the NO production elicited by inhibition of ATM, may not be as result of eNOS activity. A second important observation was that inhibition of ATM significantly enhanced phosphorylation of the p85 regulatory subunit of PI3-K. This would imply that ATM normally has an inhibitory effect on p85 phosphorylation and therefore PI3-K activation. We base this assumption on previous publications showing that Ku-60019 does not inhibit PI3K. This again indicates that ATM has a hitherto unexplored regulatory role in endothelial function. / AFRIKAANSE OPSOMMING: Ataxia telangiectasia (AT) is a goed-gekarakteriseerde neurodegeneratiewe siekte a.g.v. ‘n genetiese afwyking in the Atm geen wat lei tot ‘n afwesige of lae uitdrukking van die ATM proteïen. Aangesien AT pasiënte geneig is om insulienweerstandigheid en aterosklerose te ontwikkel, was die doel van hierdie studie om die belang van die ATM proteïen in die endoteel, en sy rol in die seintransduksiepaaie betrokke by stikstofoksied (NO) produksie, te ondersoek. Om dit te bereik, was die eerste mikpunt om ‘n eie endoteelsel isolasie-tegniek (ge-oes van normale en insulienweerstandige diere) te vestig. Ongelukkig was hierdie selkulture nie suiwer genoeg nie.Ten spyte daarvan dat hulle positief getoets het met ‘n endoteelsel-spesifieke kleurstof kon geen uitdrukking van eNOS, die sentrale ensiem verantwoordelik vir NO produksie, waargeneem word nie. Die res van die studie het van kommersiële aorta endoteelselle (AES) gebruik gemaak, en daar is gevind dat die inhibisie van die ATM proteïen met die spesifieke inhibitor, Ku-60019, tot ‘n beduidende toename in NO produksie gelei het. Die voordelige impak van verhoogde NO produksie sluit die handhawing van vaskulêre homeostase, bevordering van angiogenese, inisiëring van DNA herstel deur p53 aktivering en inhibisie van gladdespiersel proliferasie in. Reaktiewe suurstofspesies (ROS) en reaktiewe stikstofspesies (RNS) wat ook a.g.v.verhoogde NO gegenereer word, kan egter beide beskermende sowel as skadelike effekte uitoefen. Voorbeelde sluit seldood a.g.v. hoë ROS konsentrasies in. Ku-60019 het egter nie tot ‘n toename in seldood van die AES gelei nie. Hierdie studie het vir die eerste keer aangetoon dat daar ‘n verwantskap tussen die endoteel ATM proteïen kinase en die produksie van NO bestaan. Die seintransduksie paaie betrokke by NO produksie en glukose verbruik vorm ‘n interafhanklike netwerk. Die aktiwiteit van twee proteïen kinases, PKB/Akt en AMPK, is sentrale rolspelers in beide paaie. Albei hierdie kinases is daarvoor bekend dat hulle die eNOS ensiem fosforileer om NO te produseer, maar terselfdertyd ook lei tot AS160 fosforilering, wat tot GLUT 4 translokering en glukose opname lei. Dis is voorgestel dat aktivering van die ATM proteïen ‘n voorvereiste vir PKB/Akt aktivering mag wees en verder kan dit ook tot aktivering van AMPK lei. Ons kon nie aantoon dat inhibisie van ATM in endoteelselle die uitdrukking of insulien-geïnduseerde aktivering van PKB/Akt onderdruk nie, terwyl die PI3-K inhibitor, wortmannin, wel laasgenoemde geïnhibeer het. Verder het die inhibisie van ATM die fosfo/totale AMPK verhouding negatief gereguleer. Ons postuleer dus dat die NO produksie waargeneem tydens ATM inhibisie, moontlik nie die gevolg van eNOS aktiwiteit was nie. ‘n Tweede belangrike waarneming was dat die inhibisie van ATM die fosforilering van die p85 regulatoriese subeenheid van PI3-K beduidend laat toeneem het. Dit impliseer dat ATM normaalweg ‘n inhibitoriese effek op p85 fosforilering, en dus PI3-K aktivering, het. Hierdie aanname word gemaak n.a.v. vorige publikasies wat getoon het dat Ku-60019 nie PI3-K inhibeer nie. Dit dui weer eens daarop dat ATM ‘n tot nog toe onbekende regulatoriese rol in endoteelfunksie het.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

多聚谷氨酰胺疾病 (Polyglutamine diseases) 是一類在各自的致病基因編碼區的CAG重複編碼擴張造成的顯性遺傳神經退退化疾病。已擴大的CAG訊息核醣核酸 (Expanded CAG transcripts) 在多聚谷氨酰胺蛋白疾病作出細胞毒性作用。從基因減弱篩查中，我發現U2AF50能修飾已擴大的CAG訊息核醣核酸的毒性。並發現U2AF50能與已擴大的CAG訊息核醣核酸作實體互動，能參與已擴大的CAG訊息核醣核酸的核出口 (Nuclear export)。U2AF50的基因減弱增強已擴大CAG訊息核醣核酸在細胞核的累積和毒性。這突出核醣核酸的核出口在多聚谷氨酰胺疾病的重要性，並暗示細胞核是已擴大的CAG訊息核醣核酸毒性的起源地。此外，我鑑定已擴大的CAG訊息核醣核酸在亞細胞的分佈，並發現它們特別累積在核仁 (Nucleolus) 內。核仁是核糖體核醣核酸（rRNA）的轉錄場所。我發現已擴大的CAG訊息核醣核酸減弱rRNA基因啟動子 (rRNA promoter) 的活性並且抑制核糖體核醣核酸的轉錄。 核糖體核醣核酸基因轉錄的抑制，促進核糖體蛋白RpL23和E3連接酶MDM2蛋白作實體互動，從而增強p53的穩定性導。穩定的p53能夠轉移至線粒體 (Mitochondria)。我還發現，線粒體內的p53能打亂Bcl-xL與 Bak的實體互動，導致細胞色素C釋放到細胞質，這導致凋亡蛋白酶 (Caspases) 的活化和細胞凋亡。我的研究，首次證明核仁參與在多聚谷氨酰胺疾病的發病機制中，揭示了在多聚谷氨酰胺疾病中的新致病機制。 / Polyglutamine (polyQ) diseases are a class of dominantly inherited neurodegenerative disorders caused by the expansion of CAG-repeat encoding glutamine within the coding region of the respective disease genes. Expanded CAG transcripts have been reported to contribute to cytotoxicity in polyQ diseases. From a candidate gene knockdown screen, I identified U2AF50 as a modifier of RNA toxicity. U2AF50 has been reported to be involved in RNA nuclear export, and I showed that it interacted specifically with expanded CAG transcripts. Knockdown of U2AF50 expression enhanced nuclear accumulation of expanded CAG transcripts and neurotoxicity. This part of my work highlights the role of RNA nuclear export in polyQ degeneration and implies that the nucleus is a major site for RNA toxicity. In addition, I determined the subcellular distribution of expanded CAG transcripts and found that they particularly localized in the nucleolus. The nucleolus is a critical sub-nuclear compartment for ribosomal RNA (rRNA) transcription. I discovered that expanded CAG transcripts in nucleolus inhibited rRNA transcription by inactivating the rRNA gene promoter activity. Inhibition of rRNA transcription promoted the interaction between ribosomal protein L23 and the ubiquitin E3 ligase MDM2, which led to the stabilization of p53 and its accumulation in mitochondria. I also found that mitochondrial p53 disrupted the interaction between the anti-apoptotic protein, Bcl-xL, and pro-apoptotic protein, Bak, subsequently causing Cytochrome c release, caspase activation, and apoptosis. In summary, my study first describes the involvement of nucleolar function in polyQ pathogenesis and uncovers a new pathogenic mechanism in polyQ diseases. / Detailed summary in vernacular field only. / Tsoi, Ho. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 220-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Thesis Committee --- p.ii / Declaration --- p.iii / Acknowledgement --- p.iv / Abstract --- p.v / Abstract in Chinese --- p.vii / List of Abbreviations --- p.viii / List of Figures --- p.x / List of Tables --- p.xvi / Table of Contents --- p.xvi / Chapter 1 --- Introduction / Chapter 1.1 --- Introduction to Polyglutamine Diseases --- p.1 / Chapter 1.1.1 --- Etiology of Polyglutamine Diseases --- p.1 / Chapter 1.1.2 --- Common Features of Different Types of Polyglutamine Disease --- p.1 / Chapter 1.2 --- Pathogenic Mechanisms of Expanded Polyglutamine Proteins --- p.4 / Chapter 1.2.1 --- Pathogenesis of Polyglutamine Diseases --- p.4 / Chapter 1.2.1.1 --- Loss-of-function toxicity --- p.4 / Chapter 1.2.1.2 --- Gain-of-function toxicity --- p.4 / Chapter 1.3 --- Expanded CAG Transcript-mediated Pathogenic Mechanism --- p.6 / Chapter 1.3.1 --- Identification of the Toxic Role of Expanded CAG Transcripts --- p.6 / Chapter 1.3.2 --- Nuclear Foci Formation of Expanded CAG Transcripts and Polyglutamine Pathogenesis --- p.8 / Chapter 1.4 --- Receptor-mediated RNA nuclear export Transport --- p.9 / Chapter 1.4.1 --- Introduction to RNA Nuclear Export --- p.9 / Chapter 1.4.2 --- Regulation of RNA Nucleocytoplasmic Transport and Human Diseases --- p.11 / Chapter 1.5 --- Function of Nucleolus --- p.12 / Chapter 1.5.1 --- Ribosomal RNA Transcription --- p.12 / Chapter 1.5.2 --- Nucleolar Stress and Apoptosis --- p.15 / Chapter 1.6 --- Research Plan --- p.17 / Chapter 1.6.1 --- Project Objective --- p.17 / Chapter 1.6.2 --- Experimental Model --- p.17 / Chapter 1.6.2.1 --- In vivo Drosophila Model --- p.17 / Chapter 1.6.2.2 --- In vitro Cell Culture Model --- p.19 / Chapter 1.6.2.3 --- Transgenic Mouse Model --- p.20 / Chapter 1.6.3 --- Significance of the Present Study --- p.21 / Chapter 2 --- Materials and Methods / Chapter 2.1 --- Molecular Cloning --- p.22 / Chapter 2.1.1 --- Polymerase Chain Reaction (PCR) --- p.22 / Chapter 2.1.2 --- Primers Used for PCR --- p.29 / Chapter 2.1.3 --- Restriction Digestion --- p.31 / Chapter 2.1.4 --- Agarose Gel Electrophoresis --- p.32 / Chapter 2.1.5 --- Preparation of genomic DNA from A Single Adult Fly --- p.34 / Chapter 2.1.6 --- Removal of 5' Phosphate Groups on Linearized Plasmids --- p.35 / Chapter 2.1.7 --- Addition of 5' Phosphate Group to Linker Oligonucleotides --- p.35 / Chapter 2.1.8 --- Ligation Reaction --- p.37 / Chapter 2.1.9 --- Bacterial Transformation --- p.37 / Chapter 2.2 --- Mammalian Cell Culture --- p.40 / Chapter 2.3 --- Drosophila Culture --- p.44 / Chapter 2.4 --- Semi-quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.48 / Chapter 2.5 --- Microscopy --- p.51 / Chapter 2.6 --- Protein Sample Preparation and Concentration Measurement --- p.53 / Chapter 2.7 --- Co-immunoprecipitation --- p.57 / Chapter 2.8 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting --- p.62 / Chapter 2.9 --- Bacterial Protein Purification --- p.65 / Chapter 2.1 --- DNA Methylation Assay --- p.68 / Chapter 2.11 --- Mitochondrial Fraction Isolation --- p.79 / Chapter 3 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50 Modulates Polyglutamine Diseases Toxicity by Altering the Subcellular Localization of Expanded CAG Transcripts in vivo / Chapter 3.1 --- The Nuclear Accumulation of Expanded CAG Transcripts Correlates with the Neurodegeneration in vivo --- p.72 / Chapter 3.1.1 --- Expanded CAG Transcripts Predominantly Localize in the Nucleus in Drosophila Model of Machado-Joseph Disease --- p.72 / Chapter 3.1.2 --- Nuclear Accumulation of Expanded CAG Transcripts Correlates with the Neurodegeneration in an Inducible Model of Machado-Joseph Disease --- p.73 / Chapter 3.1.3 --- Nuclear Accumulation of Expanded CAG Transcripts Correlates with the Neurodegeneration in Inducible DsRed[subscript CAG100] Model. --- p.76 / Chapter 3.1.3.1 --- Expanded CAG Transcripts Induce the Expression of Pro-apoptotic Genes --- p.77 / Chapter 3.1.3.2 --- Co-expression of p35 Suppresses the Toxicity Induced by the Expanded CAG Transcripts --- p.80 / Chapter 3.2 --- A Candidate-gene RNA Interference Approach was Employed to Identify Genetic Factors Involved in Nuclear Export of Expanded CAG Transcripts --- p.80 / Chapter 3.3 --- Confirmation of the Modulatory Effect of U2 Small Nuclear Riboprotein Auxiliary Factor 50 on Machado-Joseph Disease in vivo --- p.84 / Chapter 3.4 --- The Modulatory Effect of U2 Small Nuclear Riboprotein Auxiliary Factor 50 on Different Drosophila Models of Polygultamine Diseases --- p.84 / Chapter 3.5 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50 Specifically Modulates Expanded CAG Transcript-induced Toxicity in vivo --- p.87 / Chapter 3.5.1 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Enhances Expanded CAG Transcript-induced Toxicity --- p.87 / Chapter 3.5.2 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Does Not Modulate Expanded PolyQ Protein-induced Toxicity --- p.89 / Chapter 3.5.3 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Does Not Alter the Expression Level of Expanded CAG Transcripts in vivo --- p.89 / Chapter 3.5.4 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Does Not Modulate the Toxicity in Fragile X syndrome in vivo --- p.91 / Chapter 3.6 --- Over-expression of Human U2 Small Nuclear Riboprotein Auxiliary Factor 65 Does Not Modulate Expanded CAG Transcript-induced Toxicity in Drosophila --- p.91 / Chapter 3.7 --- Expanded CAG Transcripts Does Not Compromise Endogenous Function of U2 Small Nuclear Riboprotein Auxiliary Factor 50 --- p.94 / Chapter 3.8 --- A Correlation between Nucleocytoplasmic Localization of Expanded CAG Transcripts and Its Induced Toxicity --- p.97 / Chapter 3.8.1 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Enriched DsRedCAG100 Transcripts in the Nucleus in vivo --- p.99 / Chapter 3.8.2 --- Knockdown of U2 Small Nuclear Riboprotein Auxiliary Factor 50 Enriched MJDCAG78 Transcripts in the Nucleus in vivo --- p.99 / Chapter 3.9 --- Expanded CAG-repeat on the Transcripts Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 50/65 in vivo and in vitro --- p.102 / Chapter 3.9.1 --- Expanded CAG Transcripts Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 50 in vivo --- p.102 / Chapter 3.9.2 --- Expanded CAG Transcripts Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 65 in vitro --- p.103 / Chapter 3.9.3 --- Expanded CAG Transcripts Directly Interact with U2 Small Nuclear Riboprotein Auxiliary Factor 65 in vitro --- p.103 / Chapter 3.10 --- Identification of Expanded CAG Transcripts Interacting Domain on U2 Small Nuclear Riboprotein Auxiliary Factor 65 --- p.107 / Chapter 3.10.1 --- Generation of Different Myc-tagged U2 Small Nuclear Riboprotein Auxiliary Factor 65 Expression Constructs --- p.107 / Chapter 3.10.2 --- RNA Recognition Motif 3 on U2 Small Nuclear Riboprotein Auxiliary Factor 65 Is Essential for the Interaction with Expanded CAG Transcripts --- p.109 / Chapter 3.11 --- Nuclear RNA Export Factor 1 is Involved in U2 Small Nuclear Riboprotein Auxiliary Factor 65-mediated Nuclear Export of Expanded CAG Transcripts --- p.113 / Chapter 3.11.1 --- The Effect of Full Length U2 Small Nuclear Riboprotein Auxiliary Factor 65 and its Corresponding Deletion Mutants on Nuclear Export of Expanded CAG Transcripts --- p.113 / Chapter 3.11.2 --- Formation of Complexes Composed of Nuclear RNA Export Factor 1/U2 Small Nuclear Riboprotein Auxiliary Factor 65/Expanded CAG Transcripts in HEK293 Cells --- p.115 / Chapter 3.12 --- The Nuclear Export of Expanded CAG Transcripts is Mediated by U2 Small Nuclear Riboprotein Auxiliary Factor 65 and Nuclear RNA Export Factor 1 --- p.120 / Chapter 3.13 --- Aging Compromises the Nuclear Export of Expanded CAG Transcripts in Polyglutamine Disease Mouse Model --- p.123 / Chapter 3.13.1 --- Expanded CAG Transcripts Accumulate in the Nucleus of Polyglutamine Disease Mouse Model --- p.123 / Chapter 3.13.2 --- Expression Level of U2 Small Nuclear Riboprotein Auxiliary Factor 65 Declines with Age in Mice --- p.124 / Chapter 3.14 --- Discussion --- p.127 / Chapter 3.14.1 --- Expanded CAG Transcripts Induce Nuclear Toxicity through a Mechanism Independent on Pathogenic Mechanism Mediated by Other Trinucleotide Repeats Expansion --- p.127 / Chapter 3.14.2 --- Nuclear Accumulation of Expanded CAG Transcripts Leads to Neurodegeneration --- p.128 / Chapter 3.14.3 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50 Modulates Expanded CAG Transcript-induced Toxicity by Mediating the Subcellular Localization of Expanded CAG Transcripts --- p.129 / Chapter 3.14.4 --- U2 Small Nuclear Riboprotein Auxiliary Factor 65 and Nuclear RNA Export Factor 1 Regulate the Nuclear Export of Expanded CAG Transcripts --- p.130 / Chapter 3.14.4.1 --- U2 Small Nuclear Riboprotein Auxiliary Factor 50/65 Interacts with Expanded CAG Transcripts and Mediates the Subcellular localization of Expanded CAG Transcripts --- p.130 / Chapter 3.14.4.2 --- U2 Small Nuclear Riboprotein Auxiliary Factor 65 Requires Nuclear RNA Export Factor 1 to Mediate the Nuclear Export of Expanded CAG Transcripts --- p.131 / Chapter 3.14.4.3 --- Developmental Decline of U2 Small Nuclear Riboprotein Auxiliary Factor 65 Protein Level is a Factor That Leads to Progressive Neurodegeneration in Polyglutamine Diseases --- p.134 / Chapter 4 --- Expanded CAG Transcripts Induce Nucleolar Stress / Chapter 4.1 --- Expanded CAG-repeat Sequence Mediates the Nucleolar Localization of RNA Transcripts in vitro --- p.135 / Chapter 4.1.1 --- Machado-Joseph Disease Cell Model --- p.135 / Chapter 4.1.2 --- EGFPCAG Cell Model --- p.137 / Chapter 4.2 --- Expanded CAG Transcripts Suppress Nucleolar Function in vitro and in vivo --- p.140 / Chapter 4.2.1 --- Expanded CAG Transcripts Suppress Ribosomal RNA Transcription in vivo --- p.140 / Chapter 4.2.1.1 --- Drosophila Model of Machado-Joseph Disease --- p.140 / Chapter 4.2.1.2 --- Drosophila Model of DsRedCAG --- p.142 / Chapter 4.2.1.3 --- Transgenic Mouse Model of PolyQ Disease --- p.142 / Chapter 4.2.2 --- Expanded CAG Transcripts Suppress rRNA Transcription in vitro --- p.145 / Chapter 4.2.2.1 --- Machado-Joseph Disease Patient-derived Fibroblast Cell Lines --- p.145 / Chapter 4.2.2.2 --- Expanded CAG Transcript-expressing HEK293 Cells --- p.145 / Chapter 4.3 --- Expanded CAG Transcripts Disrupt the Interaction between RNA Polymerase I and rRNA Promoter in vitro --- p.148 / Chapter 4.4 --- Expanded CAG Transcripts Disrupt the Interaction between Upstream Binding Factor and Upstream Control Element in vitro and in vivo --- p.149 / Chapter 4.4.1 --- Expanded CAG Transcripts Compromise the Interaction between Upstream Binding Factor and Upstream Control Element in vitro --- p.149 / Chapter 4.4.2 --- Expanded CAG Transcripts Compromise the Interaction between Upstream Binding Factor and Upstream Control Element in vivo --- p.151 / Chapter 4.5 --- Expanded CAG Transcripts Induce DNA Hyper-methylation on Upstream Control Element in vitro and in vivo --- p.151 / Chapter 4.5.1 --- The HpaII-PCR Assay for DNA Methylation --- p.154 / Chapter 4.5.2 --- Expanded CAG Transcripts Lead to DNA Hyper-methylation of Upstream Control Element in vitro --- p.154 / Chapter 4.5.2.1 --- Expanded CAG Transcript-expressing HEK293 Cells --- p.154 / Chapter 4.5.2.2 --- Machado-Joseph Disease Patient-derived Fibroblast Cell Lines --- p.156 / Chapter 4.5.3 --- Expanded CAG Transcripts Lead to DNA Hyper-methylation of Upstream Control Element in vivo --- p.156 / Chapter 4.5.4 --- Expanded CAG Transcripts Disrupt the Regulatory Mechanism of Upstream Control Element Methylation in vitro --- p.159 / Chapter 4.6 --- Expanded CAG Transcripts Induce Nucleolar Stress and Apoptosis --- p.161 / Chapter 4.6.1 --- Expanded CAG Transcripts Induce Nucleolar Stress in vitro and in vivo --- p.162 / Chapter 4.6.1.1 --- Expanded CAG Transcript-expressing HEK293 Cells --- p.162 / Chapter 4.6.1.2 --- Transgenic Mouse Model of PolyQ Disease --- p.162 / Chapter 4.6.2 --- Expanded CAG Transcripts Lead to Stabilization of p53 in vitro and in vivo --- p.165 / Chapter 4.6.2.1 --- Expanded CAG Transcripts Lead to Stabilization of p53 in vitro --- p.165 / Chapter 4.6.2.2 --- Expanded CAG Transcripts Lead to Stabilization of p53 in vivo --- p.167 / Chapter 4.6.3 --- Expanded CAG Transcripts Enrich p53 in Mitochondria in vitro --- p.167 / Chapter 4.6.4 --- Expanded CAG Transcripts Lead to Disruption of interaction between Bcl-xL and Bak by p53 in mitochondria in vitro --- p.169 / Chapter 4.6.5 --- Expanded CAG Transcripts Lead to Release of Cytochrome c in vitro --- p.171 / Chapter 4.6.6 --- Expanded CAG Transcripts Lead to Activation of Caspase 3 in vitro --- p.173 / Chapter 4.7 --- Discussion --- p.176 / Chapter 4.7.1 --- Expanded CAG Transcripts Compromise Nucleolar Function --- p.176 / Chapter 4.7.2 --- Expanded CAG Transcripts Induce Apoptosis via Nucleolar Stress --- p.176 / Chapter 4.7.3 --- The Origin of Nucleolar Stress Induced by Expanded CAG Transcripts --- p.178 / Chapter 5 --- Expanded CAG Transcripts Interact with Nucleolin and Deplete It from Upstream Control Element to Suppress Ribosomal RNA Transcription / Chapter 5.1 --- Nucleolin is an Interacting Partner of Expanded CAG Transcripts --- p.180 / Chapter 5.1.1 --- Nucleolin is Pulled down by S1-tagged Expanded CAG Transcripts in vitro --- p.180 / Chapter 5.1.2 --- Expanded CAG Transcripts Interact with Endogenous Nucleolin in vitro --- p.181 / Chapter 5.1.3 --- Expanded CAG Transcripts Directly Interact with Nucleolin in vitro --- p.184 / Chapter 5.2 --- RNA Recognition Motifs 2 and 3 on Nucleolin Interact with Expanded CAG Transcripts --- p.184 / Chapter 5.2.1 --- Generation of Expression Constructs Carrying Full Length Nucleolin and its Deletion Mutants --- p.184 / Chapter 5.2.2 --- Mapping of Domains on Nucleolin Required for Interacting with Expanded CAG Transcripts --- p.187 / Chapter 5.3 --- Nucleolin Regulates Ribosomal RNA Transcription by Mediating the DNA Methylation of Upstream Control Element in HEK293 Cells --- p.187 / Chapter 5.3.1 --- Nucleolin is involved in Regulating the Interaction between Upstream Binding Factor and Upstream Control Element in vitro --- p.191 / Chapter 5.3.2 --- Nucleolin is Involved in Regulating DNA Methylation Level of Upstream Control Element in vitro --- p.191 / Chapter 5.3.3 --- Nucleolin Associates with Upstream Control Element in vitro --- p.194 / Chapter 5.4 --- Expanded CAG Transcripts Deplete Nucleolin from Upstream Control Element in vitro and in vivo --- p.194 / Chapter 5.4.1 --- Expanded CAG Transcripts Compete Nucleolin with Upstream Control Element in vitro --- p.197 / Chapter 5.4.2 --- Expanded CAG Transcripts Compete Nucleolin with Upstream Control Element in vivo --- p.197 / Chapter 5.4.3 --- Expanded Polyglutamine Proteins does not Interact with Nucleolin in vitro --- p.200 / Chapter 5.5 --- Over-expression of Nucleolin Counteracts the Effect of Expanded CAG Transcripts on Ribosomal RNA Transcription in vitro --- p.200 / Chapter 5.5.1 --- Over-expression of Nucleolin Restores the Methylation Level of Upstream Control Element in Dose-dependent Manner in vitro --- p.200 / Chapter 5.5.1.1 --- The Dosage Effect of Nucleolin on DNA Hyper-methylation of Upstream Control Element Induced by Expanded CAG Transcripts in vitro --- p.202 / Chapter 5.5.1.2 --- Does-dependent Expression of Nucleolin in vitro --- p.202 / Chapter 5.5.1.3 --- The Effect of Nucleolin Over-expression on DNA Hyper-methylation of Upstream Control Element Induced by Expanded CAG Transcripts is Dose-dependent in HEK293 cells --- p.205 / Chapter 5.5.2 --- Over-expression of Nucleolin Does Not Alter the Expression Level of Expanded CAG Transcripts in vitro --- p.205 / Chapter 5.5.3 --- Over-expression of Nucleolin Relieves the Nucleolar Stress induced by Expanded CAG Transcripts in vitro --- p.208 / Chapter 5.6 --- Discussion --- p.212 / Chapter 5.6.1 --- The Physical Interaction between Expanded CAG Transcripts and Nucleolin Leads to Suppression of Ribosomal RNA Transcription --- p.212 / Chapter 5.6.2 --- Expanded CAG Transcripts Deprive Upstream Control Element of Nucleolin to Induce Toxicity --- p.212 / Chapter 5.6.3 --- Nucleolin Suppresses Expanded CAG Transcript-induced Cell Death --- p.213 / Chapter 5.6.4 --- Expanded CAG Transcripts Employ both p53-dependent and p53-independent pathways to Induce Cell Death --- p.214 / Chapter 6 --- Concluding Remarks --- p.216 / References --- p.220

Patients with giant axonal neuropathy (GAN) exhibit loss of motor and sensory function and typically live for less than 30 years. GAN is caused by autosomal recessive mutations leading to low levels of gigaxonin, a ubiquitously-expressed cytoplasmic protein whose cellular roles are poorly understood. GAN pathology is characterized by aggregates of intermediate filaments (IFs) in multiple tissues. Disorganization of the neuronal intermediate filament (nIF) network is a feature of several neurodegenerative disorders, including amyotrophic lateral sclerosis, Parkinson's disease and axonal Charcot-Marie-Tooth disease. In GAN such changes are often striking: peripheral nerve biopsies show enlarged axons with accumulations of neurofilaments; so called "giant axons." Interestingly, IFs also accumulate in other cell types in patients. These include desmin in muscle fibers, GFAP (glial fibrillary acidic protein) in astrocytes, and vimentin in multiple cell types including primary cultures of biopsied fibroblasts. These findings suggest that gigaxonin may be a master regulator of IFs, and understanding its function(s) could shed light on GAN as well as the numerous other diseases in which IFs accumulate. However, an interaction between gigaxonin and IFs has not been detected and how IF accumulation is triggered in the absence of functional gigaxonin has not been determined. To address these questions I undertook a proteomic screen to identify the normal binding partners of gigaxonin. Prominent among them were several classes of IFs, including the neurofilament subunits whose accumulation leads to the axonal swellings for which GAN is named. Strikingly, human motor neurons (MNs) differentiated from GAN iPSCs recapitulate this key phenotype. Accumulation of nIFs can be rescued by reintroduction of gigaxonin, by viral delivery or genetic correction. GAN iPS-MNs do not display survival vulnerability in the presence of trophic factors, but do display increased cell death in the presence of oxidative stress. Preliminary experiments suggest that in iPS-MNs nIFs are degraded by contributions from both the proteasome and lysosome. Gigaxonin interacts with the autophagy protein p62 which has been implicated in the clearance of ubiquitin aggregates by the lysosome, and this interaction is greatly enhanced in conditions of oxidative stress. My data provide the first direct link between gigaxonin loss and IF aggregation, and suggest that gigaxonin may be a substrate adaptor for the degradation of IFs by autophagy, pointing to future approaches for reversing the phenotype in human patients.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the degeneration of cortical and spinal motor neurons. Animal models of ALS based on known ALS-causing mutations are instrumental in advancing our understanding of the pathophysiology of motor neuron degeneration. Recent identification of mutations in the genes encoding RNA-binding proteins TDP-43 and FUS has suggested that aberrant RNA processing may underlie common mechanisms of neurodegeneration in ALS and focused attention on the normal activities of TDP-43 and FUS. However, the role of the normal functions of RNA-binding proteins in ALS pathogenesis has not yet been established. In this thesis I present my work on novel FUS-based mouse lines aimed at clarifying the relationships between ALS-causing FUS mutations, normal FUS function and motor neuron degeneration. Experiments in mutant FUS knock-in mice show evidence of both loss- and gain-of-function effects as well as misfolding of mutant FUS protein. Characterization of mice expressing ALS-mutant human FUS cDNA in the nervous system reveals selective, early onset and slowly progressive motor neuron degeneration that is mutation dependent, involves both cell autonomous and non-cell autonomous mechanisms and models key aspects of ALS-FUS. Using a novel conditional FUS knockout mutant mouse, I also demonstrate that postnatal elimination of FUS selectively in motor neurons or more broadly in the nervous system has no effect on long-term motor neuron survival. Collectively, our findings suggest that a novel toxic function of mutant FUS, and not the loss of normal FUS function, is the primary mechanism of motor neuron degeneration in ALS-FUS.

Neurodegenerative disorders constitute a class of diseases that express characteristic misfolded proteins that aggregate and induce neuronal toxicity and death. Huntington’s disease is one such fatal protein misfolding disease. Currently no therapeutic avenue can delay or stop the progression of the disease. In this context, there is a need to identify therapeutic pathways and drug targets that can prevent or delay pathogenesis in neurodegenerative diseases involving protein misfolding. This dissertation describes how our search for new drug targets have led us to identify protein disulfide isomerase and three unique small molecules that modulate its activity as a means to protect neuronal cells from neurodegenerative protein misfolding diseases, such as Huntington’s disease. Protein disulfide isomerase is a thiol-oxidoreductase in the endoplasmic reticulum that has garnered increased attention because of its implicated role in numerous human diseases, including cancer, human immunodeficiency virus pathogenesis, and thrombosis. Validating protein disulfide isomerase as target for neurodegenerative disorders may open up new therapeutic strategies to understand and treat these diseases. First, I describe the identification and validation of protein disulfide isomerase as a target of the neuroprotective small molecule, 16F16. I show that 16F16 is an irreversible inhibitor of protein disulfide isomerase that binds covalently to both cysteines in the active site. This inhibition is protective in cell and brain-slice models of Huntington’s disease, as well as in the brain-slice model of Alzheimer’s disease. Next, I describe the neuroprotective small molecule IBS141 that was originally incorrectly annotated with a chemical structure. I elucidate the correct structure of the active compound using analytical chemistry, revealing it to be the natural product securinine. Furthermore, I identify the binding site of securinine to protein disulfide isomerase and show that the inhibition of the protein is protective in cell and brain-slice models of neurodegenerative diseases. In addition to finding this unexpected activity of securinine, I provide a systematic roadmap to those who encounter compounds with incorrect structural annotation in the course of screening campaigns. Last, I describe the discovery of LOC14, a nanomolar, reversible, modulator of protein disulfide isomerase that protects cells and medium spiny neurons from the toxic mutant huntingtin protein. I find that this protection results from LOC14 binding adjacent to the active site and inducing protein disulfide isomerase to adopt an oxidized conformation. LOC14, has dramatically improved potency for protein disulfide isomerase over previously identified inhibitors and displays favorable pharmaceutical properties, making it an idea compound to evaluate the therapeutic potential of modulating protein disulfide isomerase in in vivo models of neurodegenerative diseases.