Myelin is a protein complex which plays an integral role in developing and maintaining proper brain function. Due to the plasticity of the brain, and the dynamic nature of myelin, it is critical to develop methods that allow for the investigation of changes in myelin in vivo, to further our understanding of the brain. A substantial amount of myelin is found in the grey matter (GM) of the cerebral cortex – the outermost structure of the brain that supports higher order functions including cognition and more fundamental functions, such as sensation and motor control. While in vivo investigations have traditionally used imaging to focus on myelin in the deep white matter (WM) tracts in the brain, advances in magnetic resonance imaging (MRI) are now allowing investigations of intracortical myelin (ICM). The research in this thesis presents methodology for investigating intracortical myelin levels using magnetic resonance imaging (MRI) in humans, with the aim of developing a better understanding of how myelin contributes to healthy cortical function, and how it may be disrupted in disease. To characterize intracortical myelin, a novel MRI analysis technique was developed early in this work to report the thickness of the heavily myelinated and lightly myelinated layers of the cortex. This measure of myelinated cortical thickness uses a clustering algorithm to separate the layers of the cortex based on voxel intensity in a T1- weighted (T1W) MRI with strong intracortical contrast. The resulting myelinated thickness maps match known myelin profiles of the brain, with cortical regions such as the primary visual and motor cortices displaying proportionally thicker, heavily myelinated layers. The utility of the myelinated cortical thickness for answering clinical questions was tested in bipolar disorder, where a preferential loss of the more myelinated layers in the dorsal lateral prefrontal cortex was found. This study provided the first in vivo evidence of ICM disruptions in bipolar disorder. Later in the thesis work, after surface-based analysis techniques became available, an alternative approach to investigate intracortical myelin was developed that sampled the T1W image intensity at a calculated depth of the cortex as a measure of myelin content. This methodology was used for studying the association of ICM with age in healthy adults ranging from late adolescence to middle-adulthood. It was found that three cortical depths followed a similar trajectory through this age-span, reaching their peak between 35 and 40 years of age. This study contributes to a picture of ICM amounts increasing well into middle age in healthy adults and provides a baseline for studies investigating how this may be disrupted in disease Up to this point, the analysis in the thesis used a specialized T1W MRI that had been optimized to provide strong intracortical contrast, but a question remained of how useful the technique would be if more commonly collected clinical MRIs were used as inputs. This analysis was thus applied to standard T1W and T2-weighted (T2W) anatomical MRIs to test its clinical applicability. 360 participants were investigated from the TRACK-HD dataset to test if intracortical signal analysis could follow the progression of Huntington’s disease. A significant increase in intracortical T1W/T2W signal was found in the most advanced disease group in several cortical regions. This increase in intracortical signal is likely tracking a known increase in iron and/or myelin levels in the Huntington’s disease brain. However, this work suggests that ICM studies would best be conducted with optimized imaging to better be able to characterize the subtle ICM variations within the GM. Overall, the work in this thesis presents two techniques for whole-brain mapping of the distribution of intracortical myelin using MRI. The clinical applicability of the techniques was demonstrated in examples of mental and neurodegenerative disorders. The future directions of this work include developing imaging specific to either myelin or iron as well as revisiting these problems while imaging at greater resolution to better characterize the laminar profile across the cortex. / Thesis / Doctor of Philosophy (PhD)

Neurodegenerative diseases are characterized by selective neuronal vulnerability and subsequent degeneration in specific areas of the brain. Huntington’s Disease (HD) is inherited as an autosomal dominant mutation that primarily affects the cells of the striatum and the cerebral cortex, leading to a triad of symptoms that include the progressive loss of motor function, defects in cognitive ability and psychiatric manifestations. HD is caused by a CAG repeat expansion that exceeds 37 repeats in Exon1 of the ​HTT​ gene, manifesting as a pathogenic polyglutamine (polyQ) amino acid tract expansion in the huntingtin protein. HD is a late onset disorder, with disease onset around 40-50 years of age and symptoms that worsen over 10-20 years. Only a few symptomatic treatments are available and there is currently no cure for the disease. Therapeutics to target the huntingtin gene itself have only been in clinical trial in the past 2 years. The length of the expansion has an inverse relationship with the age of disease onset. Most patients that have repeats between 40-45 CAG, however, have varying age of disease onset. Recent genome-wide association studies (GWAS) have implicated DNA handling and repair pathways as modifiers of age of disease onset up to 6 years. Therapeutic approaches to modify and delay onset indefinitely through other genetic targets will require identification of pathological mechanisms that precede disease onset. Several hallmark phenotypes have been identified in cell and animal models, including pathogenic aggregate formation. These models are not reflective of human biology, using excessively large CAG repeats (>100) associated with the more aggressive, juvenile HD, overlooking the importance of GWAS results and the progression of disease with lower pathogenic CAG repeats (40-50 CAG). We have therefore generated novel, clinically-relevant human patient fibroblast cell lines and have characterized several disease phenotypes. My thesis presents a culmination of several projects that focus on disease modelling, primarily outlining phenotypic differences between wildtype and HD cells that will benefit our understanding of disease pathogenesis. / Thesis / Doctor of Philosophy (PhD)

Mitochondrial dysfunction has been identified as a key factor in the progression of Parkinson's disease. Mitochondrial dysfunction has been shown to induce stress pathways, leading to neuronal dysfunction and cell death. Our lab has previously identified that, in neurons, reconfiguring the mitochondria using supercomplex assembly factors is protective against excitotoxic stress. For this thesis, we sought to characterize the stress pathways and synaptic impairment in an in vitro mitochondrial dysfunction model. Then, to determine if we can rescue the deficits shown, we manipulated mitochondrial integrity using the inner mitochondrial membrane targeted isoform of MCL1, which has previously been shown to regulate cristae structure and mitochondrial supercomplex assembly. We demonstrate that the integrated stress response is activated upon mitochondrial dysfunction. Next, we show mitochondrial dysfunction leads to a downregulation of synaptic genes involved in neurotransmission. Finally, our results show that both the antiapoptotic outer mitochondrial membrane-targeted isoform, and MCL1-Matrix are able to prevent cell death in response to mitochondrial dysfunction; however, MCL1-Matrix confers greater reduction in ISR activation and reactive oxygen species production. These data suggest that manipulating mitochondrial integrity, using MCL1-Matrix, confers a broad protective effect against neuronal stressors and may be used as a novel approach to preventing Parkinson's disease.

Parkinson's disease (PD) is an age-related neurodegenerative disease, which is characterized by severe loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and consequent dopamine depletion in its projecting area. In this dissertation, I evaluated the neurotoxicity of several classes of insecticides/drugs/neurotoxins to the striatal dopaminergic pathway and their potential relationship to Parkinsonism in the C57BL/6 mouse model, using biochemical and molecular biology methods. In the first objective, I investigated the neurotoxicity in striatal dopaminergic pathways following co-application of permethrin (PM), chlorpyrifos (CPF) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The study was done because pyrethroid and organophosphorus compounds are widely used insecticides and they have been implicated in Gulf War Syndrome. We found that short-term, high-dose exposure to PM or CPF had no significant effects on the expression of dopamine transporter (DAT), tyrosine hydroxylase (TH), or α-synuclein protein in striatal nerve terminals, but the insecticides slightly enhanced the neurotoxicity of MPTP in C57BL/6 mice at 28 days post-treatment. This finding indicates a slowly developing neurotoxicity may occur after termination of high-dose exposure. Long-term, low-dose exposure to PM did not show significant neurotoxicity to striatal dopaminergic pathways when given alone, nor did this injection of PM enhance the neutotoxicity of MPTP in C57BL/6 mice. In addition, experiments with pure cis or trans isomers of permethrin showed that both cis and trans isomers contributed equally to the neurotoxicity of PM in the short-term high dose study. Previous studies demonstrated a deficiency in mitochondrial function in PD, and a high density of K⁺ATP channels are present in substantia nigra, which play an important role in the maintenance of the membrane potential under metabolic stress. Therefore, in the second objective, I investigated the effect of K⁺ATP channel blockage on the neurotoxicity of mitochondrial-directed neurotoxins to striatal dopaminergic pathways. I found that mitochondrial inhibitors are potent releasers of preloaded dopamine from striatal nerve terminals, with the most potent compounds active in the nanomolar range. Co-application of the K⁺ATP channel blocker glibenclamide selectively increased the dopamine-releasing effect by complex I inhibitors in vitro, and potentiated the neurotoxicity of MPTP (a complex I inhibitor) on DAT and TH expression, in vivo. Mechanistic studies demonstrated that mitochondrial inhibitor-induced dopamine release is Ca²⁺-dependent. In addition, the selectivity of glibenclamide is not correlated to ATP depletion, but associated with the generation of excessive reactive oxygen species at the site of complex I. In the third objective, I conducted comparative studies on the mode of action of rotenone-/reserpine-/tetrabenzaine (TBZ)-induced depletion, in vitro, as these three compounds share some similarities in their chemical structures. I found that rotenone, reserpine and TBZ selectively released preloaded dopamine and serotonin (5-HT), with the rank order as rotenone>reserpine>TBZ. Mechanistic studies demonstrated more than one mechanism was involved in both rotenone- and reserpine-induced neurotransmitter release. Ca²⁺-stimulated vesicular release and neurotransmitter transporter-mediated release are the common mechanisms involved in rotenone- and reserpine-induced dopamine release. Overall, the insecticides/drugs/neurotoxins tested in the above experiments all exhibited some effect on the nigrastrital dopaminergic pathway, either alone or by enhancing the toxicity of other chemicals in combination treatment. / Ph. D.

β-Carboline sind heterozyklische Indolalkaloide, die ubiquitär in unserer Umwelt und Nahrung vorkommen, aber auch endogen aus Tryptophan gebildet werden können. Aufgrund der strukturellen Verwandtschaft bestimmter β-Carboline zu dem dopaminergen Neurotoxin MPP+ wird ein möglicher Beitrag zur Pathogenese der Parkinson-Krankheit diskutiert. MPP+ ist seit langem für seine selektive Toxizität gegenüber dopaminergen Neuronen und das Auslösen von Parkinsonsymptomen bekannt. Insbesondere 2,9-DiMe-BC wurde in erhöhter Konzentration in der lumbalen cerebrospinalen Flüssigkeit von Parkinsonpatienten detektiert, jedoch nicht in Kontrollprobanden. Eine Inhibierung von Komplex I der mitochondrialen Atmungskette und eine selektive Toxizität auf DA Neurone konnten nachgewiesen werden. Die genauen Mechanismen des Zelltodes bleiben jedoch ungeklärt. Im Rahmen dieser Arbeit wurden die Mechanismen des Zelltodes, ausgelöst durch 2,9-DiMe-BC, in dopaminergen Primärzellkulturen des Mesencephalons von C57Bl/6-Mäusen untersucht. Drei weitere BC 2-Me-BC, 9-Me-BC und 1,9-DiMe-BC standen für Untersuchungen zur Verfügung. In ersten Experimenten wies 9-Me-BC und 1,9-DiMe-BC keine Toxizität gegenüber DA Neuronen auf. Aufgrund der höheren Toxizität von 2,9-DiMe-BC verglichen mit 2-Me-BC wurden nachfolgende Experimente mit dem zweifach methylierten BC durchgeführt. Durch die Behandlung mit 2,9-DiMe-BC konnte ein höherer Verlust der DA Neurone gegenüber anderen neuralen Zellen festgestellt werden. Eine selektive Aufnahme über den Dopamintransporter und damit verbundene Schädigung der DA Neurone, wie bei MPP+, konnte nicht nachgewiesen werden. Für 2,9-DiMe-BC wurde eine LC50 der DA Neurone von 14,1 µM und für MPP+ von 4,4 µM bestimmt. 2,9-DiMe-BC verursachte in der Gesamtkultur eine erhöhte Entstehung von reaktiven Sauerstoffspezies und eine gesteigerte Laktatproduktion. In diesem Zusammenhang kann eine Hemmung von Komplex I der Atmungskette vermutet werden. Des Weiteren konnte eine Verringerung des mitochondrialen Membranpotentials und des ATP-Gehaltes gemessen werden. Eine Aktivierung des apoptotischen Zelltodes wurde mit einer erhöhten Aktivität von Caspase-3 nachgewiesen. Durch die Behandlung mit 2,9-DiMe-BC wurde in der Primärzellkultur jedoch auch in erhöhtem Maß Nekrose ausgelöst. Dabei wurde eine höhere Sensitivität von jüngeren Kulturen (8. DIV) gegenüber älteren (10. DIV) festgestellt. Genexpressionsanalysen konnten das Auslösen von oxidativem Stress und Apoptose durch 2,9-DiMe-BC bestätigen, da mehrere Gene dieser Prozesse hochreguliert wurden. Des Weiteren wurden Gene reguliert, die im Zusammenhang mit der Hitzeschock-Antwort, Entzündungsprozessen, DNA-Schädigung und Reparatur, Zellalterung und Proliferation stehen. Zusammenfassend lässt sich sagen, dass 2,9-DiMe-BC die Mitochondrienaktivität hemmt, sowohl nekrotische als auch apoptotische Prozesse in der dopaminergen mesencephalen Primärzellkultur auslöst und die Entstehung von oxidativem Stress eine zentrale Rolle spielt. Der zweite Teil dieser Arbeit beschäftigte sich mit der Untersuchung von unerwarteten neuroprotektiven Effekten von 9-Me-BC in der Primärzellkultur. Durch die Behandlung mit 9-Me-BC verringerte sich die LDH-Freisetzung und reduzierte sich die Anzahl der nekrotischen Zellen um 50 %. Nach 24 h konnte eine verminderte Caspase 3-Aktivität gemessen werden, die allerdings nach 48 h im Vergleich zur Kontrolle wieder zunahm. Hier wären längerfristige Untersuchungen zur Klärung dieser Frage anzuschließen. Des Weiteren erhöhte sich der intrazelluläre ATP-Gehalt. Möglicherweise fand eine energieabhängige Verschiebung von Nekrose zu Apoptose statt. Genexpressionsanalysen zeigten, dass verschiedene Gene von inflammatorischen und apoptotischen Signaltransduktionswegen herrunterreguliert wurden. Überraschenderweise erhöhte sich nach der Behandlung mit 9-Me-BC die Anzahl DA Neurone konzentrationsabhängig um bis zu 20 %. Diese Beobachtung ist neu und wurde über noch kein anderes BC berichtet. Der Effekt wurde durch die Inhibierung des DAT aufgehoben und lässt eine DAT-abhängige Aufnahme von 9-Me-BC vermuten. Die signifikante Erhöhung der Anzahl beschränkte sich nur auf DA Neurone, während sich der Gesamtanteil der Neurone nur geringfügig erhöhte und die übrigen Zellen unbeeinflusst blieben. Zusätzlich wurden ein erhöhter intrazellulärer DA-Gehalt und eine gesteigerte Aufnahme von [3H]DA um 20 % nachgewiesen. Die [3H]DA-Aufnahme und morphologische Untersuchungen zeigten funktionale und reife DA Neurone, es wurde aber auch die Theorie der Neuentstehung durch mögliche Differenzierungsprozesse untersucht. Interessanterweise wurde die Genexpression von einem breiten Spektrum neurotropher Faktoren (Shh, Wnt1, Wnt5a) und Transkriptionsfaktoren (En1, Nurr1, Pitx3), die für die Differenzierung und Entwicklung DA Neurone entscheidend sind, durch die Behandlung mit 9-Me-BC hochreguliert. Zusätzlich erhöhte sich die Expression der DA Markergene Aldh1a1, Dat und Th. Dabei war die Hochregulierung der Genexpression bei allen Faktoren bis auf Shh und Wnt1 von der Anwesenheit des BC abhängig. Ein weiterer Aspekt, der auf eine Differenzierung hindeuten könnte, war die verringerte Anzahl mitotischer BrdU-positiver Zellen. Das Erscheinen DA Neurone könnte also auf Differenzierung und Entwicklung von undifferenzierten Zellen oder Vorläuferzellen beruhen. Jedoch wäre auch eine Induktion der TH von vorher TH-negativen Zellen denkbar. Eine weitere Erklärung könnte das Vorherrschen eines dynamischen Gleichgewichts von Absterben und Neuentstehung DA Neurone innerhalb der Primärzellkultur sein und der Absterbeprozess durch protektive Effekte von 9-Me-BC unterbunden wurde. Zukünftige Experimente sollten zu einer weiteren Aufklärung, der diesem Phänomen zu Grunde liegendenen Mechanismen beitragen. Auch durch die Behandlung mit dem Harman 1,9-DiMe-BC erhöhte sich die Anzahl der DA Neurone konzentrationsabhängig, jedoch erwies sich im Vergleich zu 9-Me-BC nur eine Konzentration von 50 µM als signifikant. Innerhalb dieser Arbeit wurden auf Genexpressionsebene mit Hilfe von Microarrays und qRT-PCR mögliche neuroprotektive Effekte des Dopaminagonisten Lisurid im gleichen Zellkulturmodell untersucht. Lisurid gehört zur Substanzklasse der Ergotalkaloide und wird zur Behandlung der Parkinson-Krankheit eingesetzt. Bei Voruntersuchungen in der DA mesencephalen Primärzellkultur wies Lisurid eine protektive Wirkung für DA Neurone gegen Glutamattoxizität auf. Durch qRT-PCR konnten nur 50 % der ausgewählten Gene der Microarraydaten validiert werden. Nach 24 h Behandlung mit Lisurid wurde die Genexpression von dem Transportprotein Transthyretin (Ttr) hochreguliert, dessen erhöhte Biosynthese und Sekretion interessanterweise mit einer Verminderung der Aggregation des Amyloid-β-Proteins assoziiert wird. Die Genexpression der Aldoketoreduktase 1c20 (Ark1c20) wurde um 50 % herrunterreguliert. Die Bedeutung dieses Ergebnisses bedarf weiterer Abklärung, da eine gewebspezifische Expression bisher nur für die Leber gefunden wurde. Das Thyroidhormonrezeptorbindende Protein 3 (Thrap3), die Mitogen aktivierte Kinase Kinase Kinase 12 (Map3k12) und der G-Protein gekoppelte Rezeptor 27 (Gpr27) waren in ihrer Genexpression hochreguliert. Ein Einfluss von Lisurid auf Signaltransduktionswege konnte somit nachgewiesen werden. Des Weiteren wurde durch Lisurid die Expression der Transkriptionsfaktoren NeuroD1 und Tcf3 hochreguliert, die in Differenzierungsprozesse involviert sind. NeuroD1 gilt dabei als proneurales Gen und ist somit möglicherweise an Vorgängen der Neuroprotektion beteiligt. Keines der validierten differentiell exprimierten Gene des 24 h Experimentes war nach einem Behandlungszeitraum von 6 h reguliert. Die Änderungen der Genexpression nach Preinkubation mit Lisurid und anschließender Glutamatbehandlung und Behandlung mit Glutamat allein überschnitten sich weitestgehend. Demnach war vor allem die Glutamatbehandlung für die differentielle Genexpression verantwortlich. Eine zusätzliche Neusynthese von radikalfangenden Proteinen durch Preinkubation mit Lisurid konnte auf Genebene nicht gefunden werden. Es ist jedoch nicht auszuschließen, da eine Regulation auf post-transkriptioneller Ebene möglich ist.

Immunotherapy for Alzheimer\'s disease has emerged as a promising approach for clearing pathological tau protein conformers. To explore this kind of treatment we tested an active immunization with pseudo-phosphorylated tau fragments in P301L tangle model mice that develop neuronal tau aggregates as observed in frontotemporal dementia and Alzheimer’s disease. We found that an immunization reduces neurodegeneration in α-motor neurons in the spinal cord and slows progression of the tangle-related behavioral phenotype. Performance on behavioral assays correlated with tau pathology at the corresponding spinal cord level. Interestingly, a slowed progression of these tauopathy related characteristics were only seen in mice that received a specific immunization with pseudo-phosphorylated tau fragments, not in animals that received a non-specific activation of the immune system. An immunization witch pseudo-phosphorylated tau fragments may be a valuable therapeutic option in targeting one of the major hallmarks of Alzheimer’s disease and frontotemporal dementia.

ClC-6 und ClC-7 sind Chlorid/Proton Austauscher des späten endozytischen Wegs und lokalisieren entsprechend auf späten Endosomen und Lysosomen. Während die Relevanz von ClC-6 in der humanen Physiologie lange unerkannt war, hat ClC-7 seit lange eine etablierte Rolle als Gen, das, wenn mutiert, zu Neurodegeneration und verschiedenen Arten von Osteopetrose in Menschen führt, eine Pathologie, die von dicken und fragilen Knochen gekennzeichnet wird. Hier berichten wir über eine neue ClC-6Y553C de novo Mutation, die vor Kurzem in 3 unabhängigen Patienten entdeckt wurde, die unter einer früh-entstehenden neurodegenerativen Pathologie litten, die mit generellen Entwicklungsverzögerung, MRI Gehirn-Anomalien, Hypotonie und mangelhaftem Atem verbunden war. Mit-ClC-6Y553C-transfizierten Zellen zeigten ungewӧhnlich erhöhten Strömungen und Unempfindlichkeit an pH, was eine dramatische “gain-of-function” in dem nativen säuren Umfeld von späten Endosomen, wo ClC-6 exprimiert wird, darstellt. Zusätzlich erforschten wir die Relevanz von ClC-7 in der lysosomalen Funktion, um die molekulare Mechanismen besser zu verstehen, die hinter des lysosomalen Speicher und Osteopetrose steht, die in Menschen beobachtet werden, die von ClC-7 Mutationen betroffen sind. Zusammenfassend liefern wir Einblicke in den Konsequenzen von ClC-6 und ClC-7 Mutationen für Zelluläre Homöostase und unterstützen eine wichtige Rolle von ClC-6 und ClC-7 in endolysosomaler Funktion und, wenn mutiert, in humaner neurodegenerativen Pathologie. / ClC-6 and ClC-7 are chloride/proton exchangers of the late endocytic pathway and reside on late endosomes and lysosomes, respectively. While relevance in human physiology for ClC-6 has been long unknown, ClC-7 has an established role as a gene, that, if mutated, causes neurodegeneration and different types of osteopetrosis in humans, a sickness characterized by thick and fragile bones. Here we report a new ClC-6Y553C de novo mutation, recently reported in 3 unrelated patients, affected by an early-onset neurodegenerative disease leading to general developmental delay, MRI brain abnormalities, hypotonia and respiratory insufficiency. Transfected cells revealed abnormally enlarged currents and insensitivity to pH in the Y553C ClC-6 mutant, representing a dramatic gain of function in the native acidic environment of late endosomes, where ClC-6 is expressed. Additionally, we investigated the importance of ClC-7 in lysosomal function to better understand the molecular mechanisms behind the lysosomal storage and osteopetrosis observed in human patients affected by ClC-7 mutations. Collectively, we provide insight into the consequences of ClC-6 and ClC-7 mutations for cellular homeostasis and support a crucial role for both ClC-6 and ClC-7 in endolysosomal function and, if mutated, in human neurodegenerative disease.

Neurodegeneration with Brain Iron Accumulation (NBIA) disorders, such as Phospholipase A2G6-Associated Neurodegeneration (PLAN) and Pantothenate Kinase-Associated Neurodegeneration (PKAN), are a group of rare early-onset, genetic disorders characterized by neurodegeneration and iron accumulation inside of the basal ganglia (BG), which is accompanied by progressive motor symptoms. In order to address the limitations in available models of NBIA, a B6.C3-Pla2g6m1J/CxRwb mouse model of PLAN was characterized. This model demonstrated key hallmarks of the disease presentation in NBIA, including a severe and early-onset motor deficit, neurodegeneration inside of the substantia nigra (SN) including a loss of dopaminergic function and the formation of abnormal spheroid inclusions as well as iron accumulation. The capture of these hallmarks of NBIA makes this an ideal animal research model for NBIA. Additionally, exploration of candidate systemic biomarkers of NBIA was performed in a case study of a patient with PLAN and in a cohort of 30 patients with PKAN. These investigations demonstrated reductions in transfer and slight, but not significant elevations in soluble transferrin receptor. No significant difference was seen in serum iron parameters. A systemic disease burden including chronic oxidative stress; elevated malondialdehyde, and inflammation; elevated C-reactive protein (CRP), IL-6 and TNFα was noted in both investigations. A number of candidate protein biomarkers including: fibrinogen, transthyretin, zinc alpha-2 glycoprotein and retinol binding protein were also identified. These markers correlated with measures of the severity of iron loading in the globus pallidus (GP); based on R2* magnetic resonance imaging (MRI) and the severity of motor symptoms (Barry-Albright Dystonia Rating Scale) making them potential candidates markers of dysfunction in NBIA. In the patient with PLAN, 37 weeks of therapy with the iron chelator deferiprone (DFP) as well as 20 months of therapy with the antioxidants alpha lipoic acid (ALA) and n-acetylcysteine (NAC) were efficacious in reducing the systemic oxidative and inflammatory disease burden, but it did not significantly alter the progression of the disease. In the antioxidant therapy, this efficacy was primarily due to ALA. When the cohort of patients with PKAN were treated with DFP for 18 months it was highly efficacious in lowering brain iron accumulation in the GP. No significant reduction in the speed of disease progression was seen in DFP treated patients compared to placebo based on initial analysis. Similar to the PLAN patient, DFP also mitigated the systemic disease burden in PKAN patients. In both cases DFP was well tolerated and had minimal impact on serum iron levels, TIBC and transferrin saturation. Collectively these investigations provide valuable insights into disease progression in NBIA. They also provide tools to aid further investigations in NBIA. These are provided in the form of a well-characterized B6.C3-Pla2g6m1J/CxRwb model of PLAN, which robustly captures the disease presentation seen in patients, as well as a panel of systemic blood-based markers of disease burden in NBIA and candidate markers of dysfunction in NBIA. These markers were used to assess two novel therapies in NBIA chelation with DFP and antioxidant therapy with ALA and NAC. / Graduate / 2019-04-19

BACKGROUND: The lysosomal storage disorder, Niemann Pick type C1 (NPC1), presents a variable phenotype including neurovisceral and neurological symptoms. 2-Hydroxypropyl-ss-cyclodextrin (HPssCD)-based therapies are presently the most promising route of intervention. While severe cerebellar dysfunction remains the main disabling feature of NPC1, sensory functions including auditory and olfactory ones are also affected. Morphological and functional anomalies of Npc1 (-/-) mouse retina have also been observed, although the functional integrity of the visual pathway from retina to visual cortex is still unsettled. We have addressed this issue by characterizing the visual evoked potential (VEP) response of Npc1 (-/-) mice and determining if/how HPssCD administration influences the VEPs of both Npc1 (-/-) and Npc1 (+/+) mice. METHODS: VEP elicited by a brief visual stimulus were recorded from the scalp overlying the visual cortex of adult (PN, postnatal days 60, 75, 85 and 100) Npc1 (+/+) and Npc1 (-/-) mice that had received repeated injections of either HPssCD or plain vehicle. The first injection was given at PN4 and was followed by a second one at PN7 and thereafter by weekly injections up to PN49. Cholesterol accumulation and myelin loss were finally assessed by filipin staining and myelin basic protein immunohistochemistry, respectively. RESULTS AND DISCUSSION: We have found that the transmission of visual signals from retina to visual cortex is negatively influenced by the loss of Npc1 function. In fact, the VEP response of Npc1 (-/-) mice displayed a highly significant increase in the latency compared to that of Npc1 (+/+) mice. HPssCD administration fully rescued this defect and counteracted the cholesterol accumulation in retinal ganglion cells and dorsal lateral geniculate nucleus neurons, as well as the myelin loss in optic nerve fibers and axons projecting to the visual cortex observed in of Npc1 (-/-) mice. By contrast, HPssCD administration had no effect on the VEP response of Npc1 (+/+) mice, further strengthening the treatment efficacy. CONCLUSIONS: This study pinpoints the analysis of VEP response as a potentially accurate and non-invasive approach to assess neural activity and visual information processing in NPC1 patients, as well as for monitoring the progression of the disease and assessing the efficacy of potential therapies.

The compartmental hypothesis of neurodegeneration proposes that the neurone, long recognized to consist of morphologically and functionally distinct compartments, also houses distinct degeneration mechanisms for the soma, axon and nerve endings. Support for this hypothesis is provided by the phenomenon of the WldS (for Wallerian Degeneration, slow) mouse, a mutant in which axons survive several weeks after transection, rather than degenerating within 24-48 hours as in wild type mice, by virtue of expression of a chimeric Nmnat1/Ube4b protein. In this thesis I used the WldS-mouse to re-examine and extend the theory of compartmental neurodegeneration by focusing specifically on sensory axons and endings; and finally by considering a fourth compartment, the dendrites. The first part of this thesis reports that Ia afferent axons and their annulospiral endings are robustly protected from degeneration in WldS mice. Homozygous or heterozygous WldS mice crossbred with transgenic mice expressing fluorescent protein in neurones were sacrificed at various times after sciatic nerve transection. Fluorescence microscopy of whole mount preparations of lumbrical muscles in these mice revealed excellent preservation of annulospiral endings on muscle spindles for at least 10 days after axotomy. No significant difference was detected in the protection with age or gene copy-number in contrast to the protection of motor nerve terminals, which degenerate rapidly in heterozygote and aged homozygote WldS mice. In an attempt to explain the difference in motor and sensory protection by WldS, examination of three hypotheses was undertaken: a) differences in protein expression, tested by western blot and immunohistochemistry; b) differences in the degree of neuronal branching, tested through examination of g-motor axons and endings which have a degree of branching intermediate to motor and sensory neurons; and c) differences in the activity in the disconnected stumps, through primary culture of the saphenous and phrenic nerve, selected because they comprise largely pure sensory and motor axons respectively. The data suggest that none of these hypotheses provides a sufficient explanation for the difference between sensory and motor protection by WldS. The last part of this thesis attempts to extend the theory of compartmental degeneration. I examine a system for investigation of WldS-mediated protection of dendrites. In preliminary experiments retinal explants from transgenic mice expressing YFP in a subset of retinal ganglion-cell neurones were cultured. The dendritic arbours of these cells were shown to be amenable for repeated visualization and accessible to injury and monitoring of degeneration. Overall the data in this thesis suggest that the level of WldS -mediated protection conferred to an axon or axonal endings varies between different neuronal types. This has implications for the potential applications of WldS research to clinical problems. Specifically, the data imply that sensory neuropathies may benefit more than motor neuropathies from treatments based on the protective effects of WldS. These findings in sensory neurones also challenge some of the assumptions made about WldS- mediated protection of neurones, for example the extent of the age-effect on axonal endings. Further investigation of WldS-mediated protection in the CNS could give renewed impetus to attempts to discover targets for treatment in common neurodegenerative diseases. Finally, a system for investigation of dendritic degeneration has been piloted, suggesting that molecules involved in the degeneration of dendrites or in protection from this degeneration may be amenable to investigation in this system, prospectively extending the compartmental hypothesis of neuronal degeneration.