Die vorliegende Studie bringt neue Erkenntnisse bezüglich der Rolle und Ver-teilung der Mikroglia und der eingewanderten Monozyten im Verlauf der Alz-heimer Erkrankung in postmortem Gehirnen. Im Gegensatz zu Studien an Tiermodellen konnten wir in unserer Kohorte eine nur sehr geringe Beteili-gung myeloischer Monozyten an der AD Pathologie beobachten, so dass man annehmen kann, dass bei Menschen die Immunantwort des Gehirns haupt-sächlich von den hirneigenen Mikrogliazellen getragen wird. Dies wurde an humanem postmortem Hirngewebe bis zu diesem Zeitpunkt noch nicht unter-sucht. Zudem konnte gezeigt werden, dass die vulnerablen, früh von Tangles und Plaques betroffenen Hirnregionen auch eine frühe Mikrogliareaktion aufwei-sen und insbesondere von proinflammatorischen Zellen besiedelt werden und dass die Reaktion in manchen Regionen im Verlauf zunimmt, während in an-deren eine Abflachung oder sogar Abnahme beobachtet wird. / The present study provides new insights into the role and distribution of microglia and migrated monocytes in the progression of AD in postmortem brains. In contrast to studies using animal models, we observed very little involvement of myeloid monocytes in AD pathology in our cohort, suggesting that in humans the immune response of the brain is mainly carried by the brain's own microglial cells. This has not yet been investigated in human postmortem brain tissue. In addition, it could be shown that the vulnerable brain regions affected early by tangles and plaques also exhibit an early microglial response and are colonized in particular by proinflammatory cells and that the response in some regions increases during the course, while in others a flattening or even decrease is observed.

Parkinson's disease (PD) is a chronic, progressive disorder of the central nervous system that affects approximately 1.5 million Americans. One of the principal pathological features of PD is dopamine deficiency in the substantia nigra of the brain. A key enzyme that has been associated with the neurodegeneration seen in PD is monoamine oxidase-B (MAO-B). Several inhibitors of this enzyme have resulted in neuroprotection in the mouse model of PD. One such compound is 7-nitroindazole (1). This thesis describes the synthesis and MAO activity of several indazolyl and benzimidazolyl prodrugs that are designed to release an enzyme inhibitor in the affected brain area. These studies have provided information regarding the nucleophilic aromatic substitutions of the ambident nucleophiles under consideration. We have also discovered a compound that releases the enzyme inhibitor upon bioactivation by MAO. These results as well as a MPTP mouse study with the aforementioned compound are detailed within. / Master of Science

The human VAMP-associated protein B (hVAPB) has been shown to cause a range of motor neurodegenerative diseases, including amyotrophic lateral sclerosis 8 (ALS8) and spinal muscular atrophy (SMA). However, the molecular mechanisms underlying VAPB-induced neurodegeneration remain elusive. We sought to address this question by identifying VAPB interacting proteins, which may be affected by the disease causative mutations. Using a combination of biochemical and genetic approaches in Drosophila, we confirmed the evolutionarily conserved phosphoinositide phosphatase Sac1 (Suppressor of Actin 1), as a DVAP binding partner and showed that the two proteins colocalise in the endoplasmic reticulum. We also show that DVAP function is required to maintain normal levels of phosphoinositides (PIs) and that downregulation of either Sac1 or DVAP at the larval neuromuscular junction (NMJ) affects a number of synaptic processes, including axonal transport, synaptic growth, microtubule integrity and localisation of several postsynaptic components. We found that double knock down of DVAP and Sac1 induces no further increase in the severity of the mutant phenotypes when compared to either single mutant alone. This, together with the similarity in mutant phenotypes, indicates that the two genes function in a common pathway. In flies carrying the ALS8 mutation (DVAP-P58S), we observed reduced viability, locomotion defects and early death in surviving adults, closely matching the phenotypes of both DVAP and Sac1 downregulation. Additionally, transgenic expression of DVAP-P58S in the motor system elicits synaptic defects similar to those of either Sac1 or DVAP loss-of-function, including an increase in the levels of PtdIns-4-Phosphate (PI4P), the substrate of Sac1. Consistent with these observations, we found that Sac1 is sequestered into DVAP-P58S mediated aggregates and that downregulation of PI4P in neurons rescues the neurodegenerative and the synaptic phenotypes associated with DVAP-P58S transgenic expression. Together our data unveil a previously unknown function for Sac1 in neurodegeneration and synaptic function, as well as provide evidence for a dominant negative mechanism for phosphoinositide-mediated ALS8 pathogenesis. We also highlight a causative role for increased PI4P levels in VAPB-P56S induced neurodegeneration.

Lower motor neurons of the peripheral nervous system are responsible for innervating skeletal muscle and controlling all voluntary movements of the body. Degeneration of motor neurons underlies conditions such as amyotrophic lateral sclerosis and spinal muscular atrophy. The identification of genetic factors that influence the form and function of the peripheral nervous system in vivo will be important for our understanding of the neuromuscular system in health and disease. Here, I have studied the effects of three different genes and their respective protein products on the peripheral nervous system: yellow fluorescent protein (YFP), apolipoprotein E (apoE) and Ercc1 (excision repair cross-complementing group 1). YFP has been used as a reporter protein in many fields of research, including as a powerful tool for visualising neurons in mice. It is used under the assumption that it is biologically inert. However, my findings have revealed that YFP expressed in neurons in mice is not inert: it induces a cell stress response at both the RNA and the protein level and alters the time course of dying-back neuropathy. ApoE is a lipid transport protein with three distinct isoforms in humans (apoE2, apoE3 and apoE4), which are known to differentially affect risk and outcome in a number of central nervous system disorders. However, the effects of different apoE isoforms on the peripheral nervous system have yet to be established. I have shown that apoE4 delays peripheral nerve regeneration and subsequent neuromuscular junction reinnervation compared to apoE3, in the absence of any effects on normal form or function, degeneration or developmental plasticity. Ercc1 protein is involved in several DNA repair systems. Ercc1Δ/- mice have reduced levels of functional Ercc1 protein, which leads to a reduced life span and motor abnormalities, potentially due to a build of up DNA damage. Here I have shown that Ercc1Δ/- mice also have increased abnormalities at the neuromuscular junction (an early pathological target in neurodegeneration) with age. These findings contribute significantly to our understanding of the influence of specific genes on the form and function of the peripheral nervous system in health and disease.

Neurodegenerative diseases are a diverse group of progressive diseases that include Alzheimer’s disease (AD) and prion disease. Although these diseases differ in etiology, they share a number of similarities at the molecular level. For instance, microRNA (miRNA), small RNA molecules that post-translationally regulate gene expression, are often differentially regulated during disease. A previous study identified key miRNA that are dysregulated during prion disease in the hippocampus. Of these miRNA, miR-16-5p is of particular interest, as it has also been found to be dysregulated in AD. The objective of this thesis is to characterize the role of miR-16-5p within hippocampal neurons in order to understand its function during neurodegeneration. It is hypothesized that hippocampal miR-16-5p, given its induction in hippocampal neurons during preclinical disease, plays a role in regulating the dendritic remodeling and synaptic pruning that is the earliest pathological feature of neuronal degeneration in prion disease. To address this hypothesis, primary hippocampal neurons were dissected from embryonic day 18 mice and treated with a lentiviral vector at maturity. This vector either encoded miR-16 or miRZIP-16, causing overexpression or knockdown of miR-16, respectively. Immunoprecipitation of the miRNA-16 enriched RISC complex was then performed, and the co-immunoprecipitated target mRNA was subjected to a whole genome microarray. Analysis of microarray data in Ingenuity Pathway Analysis pinpointed 181 genes involved in neuronal morphology and neurological disease targeted by miR-16. In particular, the MAPK/ERK pathway was targeted at TrkB, MEK1 and c-Raf. This is of interest, as we know that this pathway is disrupted in other neurodegenerative diseases and is directly implicated in neuronal morphology. Subsequent morphological analysis revealed that overexpression of miR-16 in neuronal cells decreased neurite length and branching, consistent with the downregulation of components of the MAPK/ERK pathway. In conclusion, miR-16 targets many mRNA transcripts within the hippocampus that are important members of pathways involved in neuronal development and neurodegeneration, including the MAPK/ERK pathway. / February 2017

Philosophiae Doctor - PhD / The abnormal death of neurons in the central nervous system of individuals suffering from neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease and amyotrophic lateral sclerosis, takes place by an intrinsic cell suicide programme known as apoptosis. This process is triggered by several stimuli, and consists of numerous pathways and cascades which lead to the death of neuronal cells. It is this multifactorial nature of neurodegenerative diseases that has over the years seen many researchers develop compounds that may serve as multi-target directed ligands (MTDLs) which could potentially confer neuroprotection by acting simultaneously on different receptors and target sites implicated in neurodegeneration. This study was aimed at developing MTDLs that may serve as neuroprotective agents by simultaneously (a) inhibiting N-methyl D-aspartate receptors (NMDAR) and blocking L-type voltage gated calcium channels (VGCC) thus regulating the Ca2+ influx mediated excitotoxic process; (b) inhibiting the monoamine oxidase enzymes A and -B (MAO-A/B) thus allowing increase in dopamine levels in the central nervous system and reducing the levels of the highly oxidative products produced by the activity of these enzymes; (c) possessing anti-apoptotic activity to halt the neuronal cell death process. In designing the compounds we focused on the structures of rasagiline and selegiline, two well-known MAO-B inhibitors and proposed neuroprotective agents, and of NGP1-01, a known VGCC blocker and NMDAR antagonist. The first series of compounds (reported in research article 1, Chapter 3), comprised polycyclic propargylamine and acetylene derivatives. Compounds 12, 15 and 16 from this series showed promising VGCC and NMDA receptor channel inhibitory activity ranging from 18 % to 59 % in micromolar concentrations, and compared favourably to the reference compounds. In the MAO-B assay, compound 10 exhibited weak MAO-B inhibition of 73.32 % at 300 μM. The rest of the series showed little to no activity on these target sites, despite showing significant anti-apoptotic activity. This suggested the compounds in this series to be exhibiting their neuroprotective action through some other mechanism(s) unexplored in this study.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Exposure to endocrine-disrupting chemicals as bisphenol A (BPA) during gestation and early postnatal life is known to disrupt normal developmental processes and alter the body’s endocrine system leading to deleterious effects in the developing central nervous system (CNS). BPA is an industrial synthetic chemical commonly used in the production of a range of polymers and consumer products, despite concerns about its safety. There is therefore the need to protect the developing CNS from potential damage through the administration of neuroprotective agents. Most medicinal plants are reported to possess significant protective potential against tissue damage through different mechanisms that prevent cell death, oxidative stress, inflammation, immunodeficiency, etc. In this study, the protective effects of fermented rooibos (Aspalathus linearis) tea against the deleterious effects of BPA were investigated. Rooibos is a herbal beverage indigenous to South Africa with widely acclaimed health benefits often linked to the bioactivity of its polyphenolic compounds, especially aspalathin. The anti-allergic, cardiovascular, antioxidant and neuroprotective effects of this herb have been previously reported hence, the present study aims to investigate if regular consumption of rooibos tea during pregnancy and lactation could protect the developing brain from the deleterious effects of BPA in a Wistar rat model. A total of 40 three-month old adult female pregnant dams, with an average weight of 250g, were divided into four groups (n=10). Group 1 control rats received 9% normal saline ad libitum; group 2 rats received 400μg/kg/day BPA only; group 3 rats received 20% fermented rooibos tea as well as 400μg/kg/day BPA, while group 4 rats received ad libitum 20% fermented rooibos tea only. Offspring rats were housed in the same cages as the dams and only separated after weaning on postnatal day (PND) 21. Neurobehavioural assessment using the open field test was done on postnatal day (PND) 42 after which the final body masses were taken before the rats were decapitated under deep anaesthesia, and the desired CNS parts carefully dissected out and processed for histological, biochemical and immunohistochemical studies. The results obtained showed that there was significant impairment of neurobehavioural activity, decreased cerebral and cerebellar antioxidant enzyme activity, reduced hippocampal CA1 length, significant loss of cerebellar Purkinje cells and significant astrocyte activation demonstrated by increased glial fibrillary acidic protein (GFAP) activity in experimental rats exposed to BPA only. However, co-administration of rooibos tea significantly attenuated the BPA-induced distortions. Taken together, these findings suggest that rooibos could be a potent neuroprotective agent against BPA-induced structural, functional and biochemical alterations in the developing CNS.

Macroautophagy (‘autophagy’ hereafter) mediates the capture of aberrant cytoplasmic material into vesicles called autophagosomes, which then shuttle to lysosomes for degradation. Autophagy is implicated in numerous diseases, largely in a pro-survival role. However, autophagy has also been suggested as a form of programmed cell death (PCD), from cases of dying cells showing autophagosome accumulations. Debate occurs between whether these vesicles drive the lethality, or are instead a failing rescue attempt. This study aimed to provide clarity on this issue. Via the use of chemical and genetic strategies of inducing autophagosome accumulations, we found combining stimulators of autophagosome biogenesis with lysosomal degradation inhibitors gave rise to toxicity. Notably, this effect was dependent on the autophagy machinery and independent of other PCD routes. Research into the underlying mechanisms revealed an energy deficit under these conditions. Since autophagosomes cannot be recycled at lysosomes here, their continued synthesis affords no survival benefits, and instead just serves to deplete cellular energy further. For this reason, we designate this event ‘Futile Autophagosome Synthesis’ (FAS) toxicity. Other contributors to this toxicity include the persistence of harmful agents like Reactive Oxygen Species (ROS). Having established our FAS model, we explored its relevance in both cancer and neurodegeneration. Importantly, we found FAS inducing strategies to be effective in tumour treatment. Also, inhibiting FAS reduced the toxicity seen in neurodegenerative disease. Therefore, not only does this study improve our knowledge of autophagy in PCD, but also indicates it may have important medical implications.

Huntington's disease (HD) and Parkinson's disease (PD) are devastating neurodegenerative disorders that are characterized pathologically by degeneration of neurons in the brain and clinically by loss of motor function and cognitive decline in mid to late life. The cause of neuronal degeneration in these diseases is unclear, but both are histologically marked by aggregation of specific proteins in specific brain regions. In HD, fragments of a mutant Huntingtin protein aggregate and cause medium spiny interneurons of the striatum to degenerate. In contrast, PD brains exhibit aggregation of toxic fragments of the alpha synuclein protein throughout the central nervous system and trigger degeneration of dopaminergic neurons in the substantia nigra. Considering the commonalities and differences between these diseases, identifying common biological patterns across HD and PD as well as signatures unique to each may provide significant insight into the molecular mechanisms underlying neurodegeneration as a general process. State-of-the-art high-throughput sequencing technology allows for unbiased, whole genome quantification of RNA molecules within a biological sample that can be used to assess the level of activity, or expression, of thousands of genes simultaneously. In this thesis, I present three studies characterizing the RNA expression profiles of post-mortem HD and PD subjects using high-throughput mRNA sequencing data sets. The first study describes an analysis of differential expression between HD individuals and neurologically normal controls that indicates a widespread increase in immune, neuroinflammatory, and developmental gene expression. The second study expands upon the first study by making methodological improvements and extends the differential expression analysis to include PD subjects, with the goal of comparing and contrasting HD and PD gene expression profiles. This study was designed to identify common mechanisms underlying the neurodegenerative phenotype, transcending those of each unique disease, and has revealed specific biological processes, in particular those related to NFkB inflammation, common to HD and PD. The last study describes a novel methodology for combining mRNA and miRNA expression that seeks to identify associations between mRNA-miRNA modules and continuous clinical variables of interest, including CAG repeat length and clinical age of onset in HD.

The self-assembly of amyloidogenic proteins to form cytotoxic species that give rise to brain deterioration underlies numerous neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Increasing evidence indicates that it is the rare, low-molecular-weight species (oligomers) rather than the more abundant high-molecular-weight fibrils of certain proteins that are the most cytotoxic in several neurodegenerative diseases. However, these species have proven difficult to study using traditional methods due to their transient nature and the heterogeneity of aggregation mixtures. In this thesis, I describe my work to develop advanced methods where I combine single-molecule and ensemble fluorescence techniques with microfluidic strategies to enable the study of protein aggregation, spanning small, transient oligomers to large, insoluble aggregates. In Chapter 1 I give an overview of the biological context and relevance of this work, including the background of neurodegenerative disease, amyloidogenic aggregation and key proteins involved. I then briefly review fluorescence microscopy techniques and the field of microfluidics. In Chapter 2 I describe how complex microfluidics can be integrated with single-molecule confocal techniques to provide a highly sensitive method to continuously probe protein aggregation in vitro. I show, for the first time, that the dilution of aggregating mixtures may be automated, by up to five orders of magnitude, down to the picomolar concentrations suitable for single-molecule measurements. By incorporating this microfluidic dilution device I greatly improve the temporal resolution of the technique and facilitate the observation of more transient species through the ability to rapidly dilute and take fluorescence measurements of samples. In Chapter 3 I overcome the need for in situ labels to monitor amyloidogenic aggregation using single-molecule confocal microscopy. I describe my work to adapt the single-molecule confocal technique to achieve the ultrasensitive detection of individual aggregate species under flow without covalently-attached labels. I have demonstrated the ability of this new method to monitor the aggregation of label-free amyloidogenic proteins using extrinsic labels ex-aggregation, opening the way for biological samples to be probed in a high-throughput manner. In Chapter 4 I describe my work to combine the high precision of confocal microscopy with a microfluidic device developed to directly characterise the sizes and interactions of biomolecules in the continuous phase. By monitoring the spatial and temporal mass transport on the micron scale, the diffusion coefficient, and thus hydrodynamic radius, of species may be determined. The technique delivers much greater sensitivity for size quantification, allowing scarce and other challenging samples to be characterised, and provides significant steps towards accurate sizing for single-molecule aggregation experiments under flow. In Chapter 5 I describe my work to determine the microscopic driving force for the spatial propagation of amyloid-beta. The epifluorescence instrument I built has enabled the proliferation of aggregate species to be monitored over a macro distance on a timescale of minutes. This has greatly improved the scope of the experimental data attained, which will be used in conjunction with Monte Carlo simulations to deliver a model for the propagation of amyloid-beta in vitro. Together this thesis represents my work developing the above novel fluorescence techniques to improve their temporal and size resolution, sensitivity and adaptability to study highly complex and fundamental protein aggregation linked to neurodegenerative disease.