Indiana University-Purdue University Indianapolis (IUPUI) / The pathway leading from soluble and monomeric to hyperphosphorylated, insoluble and filamentous tau protein is at the center of many human neurodegenerative diseases, collectively referred to as tauopathies, such as Alzheimer disease (AD). In this report, we discuss the role of neuroimaging, genetics, and biomarkers in better understanding the underlying brain changes in tauopathies. In Chapters 1 and 2, we review current knowledge of tauopathies, the protein tau and FDG PET studies in AD. In Chapter 3, we investigate glucose metabolism using [18F]FDG PET in a family with multiple systems tauopathy with presenile dementia (MSTD), a primary tauopathy cause by a mutation in MAPT. The results from this study suggest that mutation carriers have lower [18F]FDG uptake, which may precede clinical onset. In Chapter 4, we assessed brain glucose metabolism using [18F]Fluorodeoxyglucose (FDG) positron emission tomography (PET) in individuals with Gerstmann–Sträussler–Scheinker Disease (GSS) with the PRNP F198S mutation. The results from this study suggest hypometabolism in the cerebellar and striatal regions, which may be preceded by hypermetabolism. This chapter also evaluated if [11C]Pittsburgh Compound B (PiB) PET is capable of detecting PrP-amyloid in GSS in individuals with the PRNP P102L and F198S mutations. The results from this study suggest that [11C]PiB is not suitable for in vivo assessment of PrP amyloid plaques in GSS. In Chapter 5, we examine a correlation between two peripheral markers of axonal degeneration, plasma tau and neurofilament light (NFL), and MRI. The results from this study suggest that plasma NFL may be a more specific marker for neurodegeneration relative to plasma tau. In Chapter 6, we attempted to create a tau biological network from gene and protein databases and literature search. We identified over 150 genes that are related to tau protein or MAPT that are involved in different biological functions. Overall, the results of this report support the notion that using a combination of techniques may help model progression of tau pathology. Future studies may establish additional markers that may be used in combination with some of these measures as tools for diagnosis and for the evaluation of treatment efficacy in therapeutic trials.

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Amyotrophic Lateral Sclerosis (ALS) is a motor neuron disease characterizeby devastating symptoms, such as muscle weakness, paralysis, and death within 5 years of disease onset. Mutations in human (VAMP)-associated protein B (hVAPB) gene cause ALS8. Interestingly, the Drosophila VAPB (DVAP) is required at the Drosophila larval neuromuscular junction (NMJ), to control bouton morphology. In Pennetta’s lab Drosophila Sac1, a phosphoinositide-4- phosphate (PI4P) phosphatase, was identified during a genome-wide yeast twohybrid screen, as a DVAP binding partner. VAP plays a role in regulating PI4P turnover in yeast and phosphoinositides are implicated in some neurodegenerative processes. In this PhD thesis, Sac1-DVAP interaction is used as the starting point to identify the mechanism that is altered when DVAP function is impaired. Thus, the possibility that the cellular pathways regulated by PI4P are affected in VAPB-mediated neurodegeneration was explored. First, the Sac1-DVAP association was confirmed in vitro by coimmunoprecipitation. Subsequently, we found that these two proteins colocalize in vivo at the ER membranes. Then, immunohistochemical analysis of Drosophila larval NMJ revealed that Sac1 and DVAP are involved in similar pathways. They both have a function in microtubule stabilization in the presynaptic boutons and axonal vesicle transport at the presynaptic compartment. They also seem to contribute to the spectrin-actin cytoskeleton stabilization at the postsynaptic compartment of the NMJ. Lastly, we reported that reduced levels of Sac1 phosphatase cause progressive neurodegeneration. Moreover, Sac1 is trapped into cytosolic aggregates induced by the expression of the ALS8-mutant allele of DVAP, and it does not localize to its original place in the cell. All together these results suggest that in ALS8 hVAPB seems to have a dominant negative effect on Sac1. Sac1 mislocalization could inhibit the dephosphorylation of PI4P. This PhD work further confirms Sac1-DVAP interaction and it suggests a mechanism underlying ALS8 pathogenesis, supporting the idea that altered metabolism of phosphoinositides can cause neurodegeneration.

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No description available.

Huntington’s disease (HD) is one of nine polyglutamine diseases and it is caused by a CAG expansion in the HTT gene. HD is an autosomal, dominantly inherited neurodegenerative disease affecting between 2 and 5 individuals per 100,000 worldwide and it is currently untreatable. HD spreads from the striatum to the rest of the brain and causes widespread motor, cognitive, and psychiatric symptoms, including Huntington’s chorea. A fruitful approach to identifying potential therapeutic targets for HD is to modify genes in a model organism in an unbiased manner and screen the effect by testing the model in a functional assay. Drosophila models of HD have emerged as key tools for these large scale genetic screens thanks to their combination of ease of maintenance, and breeding in large numbers and their ability to be tested neurobehaviorally. During the course of HD pathogenesis in mammals, the FL-HTT protein is cleaved by many proteases including caspase-6. This cleavage leads to the co-existence of N-terminal (NT) as well as full-length (FL) forms of mutant HTT in the HD neurons. Drosophila lacks caspase-6 therefore FL-HTT is not naturally cleaved at its target site, this allows us to express either the FL mutant HTT or its cleaved NT fragment independently to characterize their differential pathogenic contribution. This study aims to test the concordance of a sample of 75 NT-HTT modifiers identified through a directed screen by testing them in a FL-HTT model. In doing so, we hope to identify shared modifiers and shared functional genetic networks, which may be particularly central to HD progression and useful areas in which to discover therapeutic targets. Further, this study may help to determine what types of models are necessary for future screens to adequately understand the genetic networks that underpin HD progression. In order to assess the impact of the modifier, flies expressing both the modifier and FL-HTT were tested in a climbing assay that measures motor function taking advantage of the model’s innate negative geotactic behavior. Motor performance is measured as the percentage of flies of each genotype that climb up to a 9 cm threshold in a given time interval. Flies were tested at 6 time points on days 18, 19, 20, 21, 22, and 25 of age in order to observe their level of neurobehavioral function in comparison to a positive control of flies with FL-HTT and no modifier and a negative control of flies without mutant HTT. When NT modifiers were tested in the FL model, there was an enrichment in modifiers relative to what is seen by chance. The NT suppressor sample was significantly enriched in modifier genes that effected motor performance in the FL model. Meanwhile, NT HD enhancers were not enriched with modifiers in the FL model. Some modifiers demonstrated contradictory effects on motor performance depending on the HD model tested. This could be caused by different mechanisms of toxicity inherent to NT versus FL HD or from secondary toxicity as the FL experiment occurred over a longer time period and flies were aged at a higher temperature. There was particular enrichment of modifiers in the calcium signaling and inflammation and cytoskeleton stress response pathways, which are robust functional gene networks identified by previous gene screening. These findings suggest that these shared networks are particularly central to HD progression and both are involved in inhibiting a cell’s ability to cope with stress and promoting excitotoxicity. The aforementioned pathogenic features are associated with impaired autophagy, which many see as the key to HTT clearance and ultimately rescuing neurons from degradation and curing HD. As genetic screening continues in Drosophila, shared networks between models have the potential to reveal new therapeutic targets and broaden our understanding of the mechanisms that lead to HD progression. These lessons will be essential as whole genome unbiased screenings in Drosophila continue and our networks become more robust and interconnected. Despite the enrichment in shared modifiers, our results show not infrequent contradictory effects on motor performance when NT modifiers are tested in the FL model. As such, we suggest that future screens test both FL and NT models independently to best study the causes of HD and to help identify the shared modifiers and networks, which are promising areas to mine for therapeutic targets.

Mutations of the α1A subunit of CaV 2.1 voltage gated calcium (VGCC) channels are responsible for several inherited disorders affecting humans, including familial hemiplegic migraine, episodic ataxia type and spinocerebellar ataxia type. The leaner mouse also carries an autosomal recessive mutation in the α1A subunit of CaV 2.1 VGCCs, which, in the homozygous condition, results in a severe cerebellar atrophy and ataxia. The leaner mutation results in reduced calcium influx through CaV 2.1 VGCCs. To better understand cerebellar neurodegeneration and cerebellar dysfunction we focused our research on elucidating the relationship between mitochondrial function/dysfunction and calcium channel mutations. The aims of this dissertation were: 1) to estimate the extent of neuronal cell death, basal intracellular calcium and mitochondrial (dys)function in cerebellar granule cells (CGC) of adult leaner mice; 2) to analyze the role of the leaner calcium channel mutation on postnatal development of CGCs; and 3) to test whether inducing increased calcium influx by exposing cultured granule cells to potassium chloride can eliminate or reduce the CGC death. By using mechanism independent Fluoro-Jade staining and apoptosis specific TUNEL staining, we demonstrated that leaner CGC death continues into adulthood and the spatial pattern of granule cell death observed during postnatal development also continues into adulthood. The present investigation showed a reduced resting intracellular calcium in CGC from leaner mice as compared to age matched wild type mice, and tottering mice. The tottering mouse is another mutant mouse that carries a mutation in the α1A subunit of CaV 2.1 VGCCs like leaner mouse. However, these mice do not show any neurodegeneration and therefore they were used as a second control. Our results also showed that even though CGC of leaner mice have dysfunctional CaV2.1 channels, there is no change in depolarization induced Ca2+ influx, which suggests a functional compensation for CaV2.1 calcium channels by other VGCCs. Our results showed reduced mitochondrial membrane potential at the time of peak CGC death in leaner mice as compared to wild type CGCs and tottering CGCs. The results of this investigation suggest mitochondrial mediated but reactive oxygen species independent cell death in CGCs of leaner mice.

Prion diseases are a group of fatal neurodegenerative protein-misfolding diseases. Microglia, the resident myeloid cells found within the brain, have been shown to demonstrate a reactive morphology during the disease process with conflicting evidence for both a neurotoxic and neuroprotective role. The studies presented here aimed to investigate the role of microglia activation using transcriptomic and morphological analysis of prion disease in mice. Initially, the host immune response to prion disease was explored using a publically available mouse prion disease dataset. Re-analysis of this dataset was performed using BioLayout Express3D; a novel software tool that supports the visualisation and clustering of correlation networks. Disease-associated genes up-regulated during the later stages of infection were present in two main clusters. The cellular origin of these genes was explored by examining their expression in a dataset comprised of pure populations of cells. This demonstrated that the primary cluster of up-regulated transcripts encompassed genes expressed mainly by microglia and to a lesser extent astrocytes and neurons. The secondary cluster comprised almost exclusively of interferon response genes. The conclusions of these analyses were different from those of the original study that suggested disease-associated genes were primarily neuronal in origin. Mouse models of prion disease were established by infecting a novel line of BALB/cJ inbred mice, expressing EGFP under control of a myeloid specific Csf1r promoter, with the 79A prion strain. Quantification of the morphological changes of EGFP expressing microglia suggested the cells accumulated in the medulla at sites of early misfolded protein deposition with minimal change in their overall appearance. An activated microglia morphology was not observed until protein deposition was extensive. Isolation of EGFP expressing microglia was performed for transcriptome analysis. The vast majority of disease associated genes demonstrated increased expression at the onset of clinical symptoms. The gene list was found to be highly enriched for genes associated with an innate immune response regulated by the NFκB signalling cascade. Also highly enriched were processes associated with protein translation, energy production and stress response. These data suggest a high metabolic load is burdened by proliferating microglia; and as part of a response which is strikingly more pro-inflammatory in nature than has previously been attributed to the microglia phenotype within prion disease. As an active contributor to normal homeostasis, microglia are more than just innate immune surveillance and are now considered an integral component in both the healthy and diseased brain. The ramifications of activation toward the microglia phenotype shown here will have direct and potentially cytotoxic influence on neighbouring microglia and other brain cell types implying microglia as major contributors to the neurotoxic environment found within the CNS during prion disease. Furthermore the identification of genes associated with metabolism offer many intriguing possibilities for manipulating the activity of microglia in pre-clinical therapeutic intervention.

Angesichts des dramatischen, weltweiten Anstiegs der Prävalenz von Demenzerkrankungen und der aktuellen, unzureichenden Therapieansätze ist die Bereitstellung neuer, wirkungsvoller Behandlungsoptionen von größter Bedeutung. Technologische, pharmakologische und verhaltensbasierte Verfahren des Memory Enhancement könnten zur Lösung dieses Problems beitragen: Hierzu zählt die Stammzelltransplantation, die in mehreren Tierstudien zu einer Verbesserung der Gedächtnisfunktion führte. Zudem wird seit Längerem an einer Impfung gegen die Alzheimer-Krankheit mittels β-Amyloid-Antikörpern geforscht. Ein weiterer therapeutischer Ansatz für die Alzheimer-Krankheit besteht in der optogenetischen Stimulation spezifischer hippocampaler Engramm-Zellen, durch die bei einem Maus-Modell verloren gegangene Erinnerungen wiederhergestellt werden konnten. Unkonventionelle Pharmazeutika wie Erythropoetin führten in Tierstudien und bei Patienten mit neuropsychiatrischen Erkrankungen zu einer Verbesserung der kognitiven Fähigkeiten und des Gedächtnisses. Eine Modifikation der Ernährung und der Einsatz von Pro- und Präbiotika beeinflussen das Gedächtnis über eine Manipulation der Darm-Hirn-Achse. Verhaltensbasierte Maßnahmen wie körperliche Aktivität und der Einsatz von Mnemotechniken stellen effektive Ansätze des Memory Enhancement dar, welche bereits heute von gesunden Individuen implementiert werden können. Für die Anwendung von Augmented Reality (AR) konnten kognitionsfördernde Wirkungen beim Lernen neuroanatomischer Themen und dem Zusammenbau von Objekten nachgewiesen werden. Besonders vielversprechend stellt sich die Entwicklung einer Gedächtnisprothese dar, durch die vergessene Informationen bei Personen mit stattgehabtem Schädel-Hirn-Trauma und apoplektischem Insult reaktiviert werden könnten. Memory Enhancement ist prinzipiell bereits heute bei gesunden und kranken Individuen anwendbar und verspricht wirksame zukünftige Präventions- und Therapieoptionen. Ein realer Einsatz in der klinischen Praxis ist in naher Zukunft jedoch noch nicht zu erwarten. / Due to the worldwide increasing prevalence of dementia and the current, inadequate therapeutic approaches, it is very important to develop new, effective treatment options. Technological, pharmacological and behavior-based methods of memory enhancement could help to solve this problem: This includes stem cell transplantation, which has led to an improvement in memory function in several animal studies. In addition, research into vaccination against Alzheimer's disease using β-amyloid antibodies has been done for several years. Another therapeutic approach for Alzheimer's disease is the optogenetic stimulation of specific hippocampal engram cells, through which lost memories could be restored in a mouse model. Unconventional pharmaceuticals such as erythropoietin have improved cognitive skills and memory in animal studies and in patients with neuropsychiatric disorders. A diet modification and the use of probiotics and prebiotics affect memory by manipulating the gut-brain axis. Behavioral approaches such as physical activity and the use of mnemonics represent effective approaches of memory enhancement that healthy individuals can already implement today. The application of augmented reality (AR) was associated to cognition-promoting effects when learning neuroanatomical topics and assembling objects. The development of a memory prosthesis seems to be particularly promising and could be used to reactivate forgotten information in people with traumatic brain injury and apoplectic insult. In principle, memory enhancement can already be used today in healthy and diseased individuals and promises effective future prevention and therapy options. However, a real use in clinical practice cannot be expected in the near future.