Huntington’s disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in exon 1 of the huntingtin gene, resulting in an expanded polyglutamine (polyQ) repeat in the huntingtin protein. Patients receive symptomatic treatment for motor, emotional, and cognitive impairments; however, there is no treatment to slow the progression of the disease, with death occurring 15-20 years after diagnosis. Mutant huntingtin protein interferes with multiple cellular processes leading to cellular dysfunction and neuronal loss. Due to the complexity of mutant huntingtin toxicity, many approaches to treating each effect are being investigated. Unfortunately, addressing one cause of toxicity might not result in protection from other toxic insults, necessitating a combination of treatments for HD patients. Ideally, single therapy targeting the mutant mRNA or protein could prevent all downstream toxicities caused by mutant huntingtin. In this work, I used animal models to investigate a potential therapeutic target for decreasing mutant huntingtin protein, and I apply bioluminescent imaging to investigate RNA interference to silence mutant huntingtin target sites. The enzyme puromycin sensitive aminopeptidase (PSA) has the unique property of degrading polyQ peptides and been implicated in the degradation of huntingtin. In this study, we looked for an effect of decreased PSA on the pathology and behavior in a mouse model of Huntington’s disease. To achieve this, we crossed HD mice with mice with one functional PSA allele and one inactivated PSA allele. We found that PSA heterozygous HD mice develop a greater number of pathological inclusion bodies, representing an accumulation of mutant huntingtin in neurons. PSA heterozygous HD mice also exhibit worsened performance on the raised-beam test, a test for balance and coordination indicating that the PSA heterozygosity impairs the function of neurons with mutant huntingtin. In order to test whether increasing PSA expression ameliorates the HD phenotype in mice we created an adeno-associated virus (AAV) expressing the human form of PSA (AAV-hPSA). Unexpectedly, testing of AAV-hPSA in non-HD mice resulted in widespread toxicity at high doses. These findings suggest that overexpression of PSA is toxic to neurons in the conditions tested. In the second part of my dissertation work, I designed a model for following the silencing of huntingtin sequences in the brain. Firefly luciferase is a bioluminescent enzyme that is extensively used as a reporter molecule to follow biological processes in vivo using bioluminescent imaging (BLI). I created an AAV expressing the luciferase gene containing huntingtin sequences in the 3'-untranslated region (AAV-Luc-Htt). After co-injection of AAV-Luc-Htt with RNA-silencing molecules (RNAi) into the brain, we followed luciferase activity. Using this method, we tested cholesterol-conjugated siRNA, un-conjugated siRNA, and hairpin RNA targeting both luciferase and huntingtin sequences. Despite being able to detect silencing on isolated days, we were unable to detect sustained silencing, which had been reported in similar studies in tissues other than the brain. We observed an interesting finding that co-injection of cholesterol-conjugated siRNA with AAV-Luc-Htt increased luminescence, findings that were verified in cell culture to be independent of serotype, siRNA sequence, and cell type. That cc-siRNA affects the expression of AAV-Luc-Htt reveals an interesting interaction possibly resulting in increased delivery of AAV into cells or an increase in luciferase expression within the cell. My work presents a method to follow gene silencing of huntingtin targets in the brain, which needs further optimization in order to detect sustained silencing. Finally, in this dissertation I continue the study of bioluminescent imaging in the brain. We use mice that have been injected in the brain with AAV-Luciferase (AAV-Luc) to screen 34 luciferase substrate solutions to identify the greatest light-emitting substrate in the brain. We identify two substrates, CycLuc1 and iPr-amide as substrates with enhanced light-emitting properties compared with D-luciferin, the standard, commercially available substrate. CycLuc1 and iPr-amide were tested in transgenic mice expressing luciferase in dopaminergic neurons. These novel substrates produced luminescence unlike the standard substrate, D-luciferin which was undetectable. This demonstrates that CycLuc1 and iPr-amide improve the sensitivity of BLI in low expression models. We then used CycLuc1 to test silencing of luciferase in the brain using AAV-shRNA (AAV-shLuc). We were unable to detect silencing in treated mice, despite a 50% reduction of luciferase mRNA. The results from this experiment identify luciferase substrates that can be used to image transgenic mice expressing luciferase in dopaminergic neurons. My work contributes new data on the study of PSA as a modifier of Huntington’s disease in a knock-in mouse model of Huntington’s disease. My work also makes contributions to the field of bioluminescent imaging by identifying and testing luciferase substrates in the brain to detect low level of luciferase expression.

A list of genes have been identified to carry mutations causing familial ALS such as SOD1, TARDBP, C9orf72. But for sporadic ALS, which is 90% of all ALS cases, the underlying genetic variants are still largely unknown. There are multiple genome-wide association study (GWAS) for sporadic ALS, but usually a large number nominated SNP can hardly be replicated in larger cohort analysis. Also majority of GWAS SNP lie within noncoding region of genome, imposing a huge challenge to study their biological role in ALS pathology. With the rapid development of next-generation sequencing technology, we are able to sequence exome and whole-genome of a large number of ALS patients to search for novel genetic variants and their potential biological function. Here by analyzing exam data, we discovered two novel or extremely rare missense mutations of DPP6 from a Mestizo Mexican ALS family. We showed the two mutations could exert loss-of-function effect by affecting electrophysiological properties of Potassium channels as well as the membrane localization of DPP6. To our knowledge this is the first report of DPP6 nonsynonymous mutations in familial ALS patients. In addition, by analyzing whole-genome data, we discovered strong linkage disequilibrium between SNP rs12608932, a repeatedly significant ALS GWAS signal, and one polymorphic TGGA tetra-nucleotide tandem repeat, which is further flanked by large TGGA repetitive sequences. We also demonstrated rs12608932 risk allele is associated with reduced UNC13A expression level in human cerebellum and UNC13A knockout could lead to shorter survival in SOD1-G93A ALS mice. Thus the TGGA repeat might be the real underlying genetic variation that confer risk to sporadic ALS.

Canavan’s Disease is a fatal Central Nervous System disorder caused by genetic defects in the enzyme – aspartoacylase and currently has no effective treatment options. We report additional phenotypes in a stringent preclinical aspartoacylase knockout mouse model. Using this model, we developed a gene therapy strategy with intravenous injections of the aspartoacylase gene packaged in recombinant adeno associated viruses (rAAVs). We first investigated the CNS gene transfer abilities of rAAV vectors that can cross the blood-brain-barrier in neonatal and adult mice and subsequently used different rAAV serotypes such as rAAV9, rAAVrh.8 and rAAVrh.10 for gene replacement therapy. A single intravenous injection rescued lethality, extended survival and corrected several disease phenotypes including motor dysfunctions. For the first time we demonstrated the existence of a therapeutic time window in the mouse model. In order to limit off-target effects of viral delivery we employed a synthetic strategy using microRNA mediated posttranscriptional detargeting to restrict rAAV expression in the CNS. We followed up with another approach to limit peripheral tissue distribution. Strikingly, we demonstrate that intracerebroventricular administration of a 50-fold lower vectors dose can rescue lethality and extend survival but not motor functions. We also study the contributions of several peripheral tissues in a primarily CNS disorder and examine several molecular attributes behind pathogenesis of Canavan’s disease using primary neural cell cultures. In summary, this thesis describes the potential of novel rAAV-mediated gene replacement therapy in Canavan’s disease and the use of rAAVs as a tool to tease out its pathological mechanism.

Amyotrophic lateral sclerosis (ALS) is a progressive adult neurodegenerative disease that causes death of both upper and lower motor neurons. Approximately 90 percent of ALS cases are sporadic (SALS), and 10 percent are inherited (FALS). Mutations in the PFN1 gene have been identified as causative for one percent of FALS. PFN1 is a small actin-binding protein that promotes actin polymerization, but how ALS-linked PFN1 mutations affect its cognate functions or acquire gain-of-function toxicity remains largely unknown. To elucidate the contribution of ALS-linked PFN1 mutations to neurodegeneration, we have characterized these mutants in both mammalian cultured cells and Drosophila models. In mammalian neuronal cells, we demonstrate that ALS-linked PFN1 mutants form ubiquitinated aggregates and alter neuronal morphology. We also show that ALS-linked PFN1 mutants have partial loss-of-function effects on actin polymerization in growth cones of mouse primary motor neurons and larval neuromuscular junctions (NMJ) in Drosophila. In Drosophila, we also observe that PFN1 level influences integrity of adult motor neurons, as demonstrated by locomotion, lifespan, and leg NMJ morphology. In sum, the work presented in this dissertation has shed light on PFN1- linked ALS pathogenesis by demonstrating a loss-of-function mechanism. We have also developed a Drosophila PFN1 model that will serve as a valuable tool to further uncover PFN1-associated cellular pathways that mediate motor neuron functions.

In vivo proton magnetic resonance spectroscopy (1H MRS) is the only method available to measure small-molecule metabolites in living human tissue, including the brain, without ionizing radiation or invasive medical procedures. Despite its attendant potential for supporting clinical diagnostics in a range of neurological and psychiatric conditions, the metabolite concentration estimates produced by 1H-MRS experiments, and therefore their sensitivity and specificity to any particular biological phenomenon under study, are readily distorted by a number of confounds. These include but are not limited to static and radiofrequency field characteristics, signal relaxation dynamics, macromolecule and lipid contributions to the spectral baseline, spectral fitting artifacts, and other uncontrolled idiosyncrasies of 1H-MRS data acquisition, processing, and quantification. Using 1H-MRS data obtained via 3-Tesla and 7-Tesla magnetic resonance (MR) scanners from healthy controls, individuals with progressive and relapsing-remitting multiple sclerosis (MS), and individuals with post-traumatic stress disorder (PTSD) and/or major depressive disorder (MDD), this work therefore aims to build and apply a framework for quantifying and thereby reducing such confounds introduced to 1H-MRS estimates of in vivo metabolite concentrations at the steps of data processing and quantification, with an ultimate aim to maximizing the potential of 1H MRS for supporting sensitive and specific clinical diagnosis of neurological or psychiatric disease. The steps examined include spectral quantification by linear combination modeling (Chapter 2), absolute quantification by internal concentration referencing (Chapter 3), and cross-sectional statistical analysis of results (Chapters 4 and 5). Chapter 2 designs and implements a graphical user interface (GUI)-supported validation pipeline for measuring how data quality, spectral baseline, and baseline model affect the precision and accuracy of 1H-MR spectral quantification by linear combination modeling. This validation pipeline is then used to show that spectral data quality indices signal to noise ratio (SNR) and full width at half maximum (FWHM) interact with spectral baseline to influence not only the precision but also the accuracy of resultant metabolite concentration estimates, with fit residuals poorly indicative of true fit error and spectral baselines modeled as regularized cubic splines not significantly outperformed by those employing simulated macromolecules. A novel method for extending the commonly used spectral quantification precision estimate Cramér-Rao Lower Bound (CRLB) to incorporate considerations of continuous and piecewise polynomial baseline shapes is therefore presented, tested, and similarly integrated into a GUI-supported toolkit to improve the correspondence between estimated CRLB and metabolite fit error variability when this now empirically justified approach to spectral baseline modeling is used. In Chapter 3, age- and disease-associated differences in transverse (T2) water signal relaxation measured at 7 Tesla in the prefrontal cortex of individuals with progressive (N=21) relative to relapsing-remitting (N=26) or no (N=25) multiple sclerosis are shown to influence absolute quantification of metabolite concentrations by internal referencing to water. In Chapter 4, these findings from Chapters 2 and 3 are used to justify an evidence-based 1H-MR spectral processing and quantification protocol that focuses optimization efforts on baseline modeling approach and references metabolite concentration estimates to internal creatine instead of water. When this protocol is applied to 7-Tesla prefrontal cortex 1H-MR spectra from the aforementioned multiple sclerosis and control cohorts, it supports metabolite concentration estimates that, in the absence of any additional supporting data, inform supervised-learning-enabled identification of progressive multiple sclerosis at nearly 80% held-out validation sensitivity and specificity. Finally, in Chapter 5, the same processing, quantification, and machine-learning pipeline employed in Aim 3 is independently applied to a new set of 7-Tesla prefrontal cortex 1H-MRS raw data from an entirely different cohort of individuals with (N=20) and without (N=18) PTSD and/or comorbid or primary MDD. Here the processing, quantification, and statistics procedures designed using lessons in Chapters 2 and 3 and optimized for classifying multiple sclerosis phenotype in Chapter 4 generalize directly to metabolite-only classification of PTSD and/or MDD with sensitivity and specificity similarly near to or greater than 80%. In both Chapters 4 and 5, supervised learning avoids dimensionally reducing metabolite feature sets in order to pinpoint the specific metabolites most informative for identifying each disease group. Taken together, these findings justify the potential and continued development of 1H MRS, at least as applied in the human brain and especially as supported by multivariate approaches including supervised learning, as an auxiliary or mainstay of clinical diagnostics for neurological or psychiatric disease.

Purpose: To describe the experience of managing relapsing-remitting multiple sclerosis among adult women users of injectable disease modifying drugs, including day-to-day management, medication beliefs, and health care provider influence. Rationale/Significance of the study:Approximately 85% of the 400,000 Americans with multiple sclerosis have relapsing-remitting multiple sclerosis (RRMS), characterized by unpredictable relapses and partial or full remissions of neurological symptoms. Untreated, RRMS may progress to permanent, irreversible disability and decreased quality of life. Current guidelines recommend immediate and sustained treatment with injectable disease modifying drugs (DMDs). However, despite pronounced modest benefits, approximately 30%-62% of patients are not undergoing DMD therapy. A small number of quantitative studies have identified factors that predict adherence to injectable DMDs. However, little is known about injectable DMDs from patients’ perspectives. It is important to develop an understanding of the experience of managing RRMS among adult users of injectable DMDs in order for health care providers to provide ongoing education, counseling, and support. Organizing Framework:The framework, Beliefs About Medicines, was used to guide the study. Design: Qualitative descriptive design. Setting: Data were collected from adult women with RRMS who received care from an MS clinic, a neurology practice, and through snowball sampling. Sample: Purposive and theoretical sampling was used to recruit 32 women with RRMS. Maximum variation sampling ensured the appropriate breadth and depth of experiences. Women currently undergoing injectable DMD therapy (n = 25), as well as women who either discontinued (n = 6), or never used (n = 1) injectable DMDs were interviewed. Methods: A qualitative descriptive design was utilized. Verification occurred through trustworthiness of data, including rich, thick description from qualitative interviews; field notes and memoing; and member checks. Simultaneous data collection, analysis, and interpretation facilitated interview revision in order to elicit or expand emerging themes. Content analysis inductively derived themes and patterns within and across categories. Participant quotes substantiated particular themes. Confirmability of the data analysis process was undertaken in consultation with the research advisor. Implications: Findings elucidated adult women’s subjective experiences concerning management of RRMS among users of DMDs, including day-to-day management, medication beliefs, and health care provider influence. Results from this study can be used to educate, counsel, and support women in the management of RRMS.

No description available.

The hypothesis to be tested states that the pathology of Alzheimer's disease (AD) involves elevated levels of oxidative stress, resulting in elevated levels of cellular oxidative defense mechanisms. If the premise is true, than AD pathologically afflicted cells should have a higher demand for glutathione (GSH) as an innate oxidative defense mechanism hence; greater GSH concentrations, increased GSH resynthesis capabilities, and increased levels of cystathionine gamma-lyase (CNase). Alzheimer diseased and age matched control lymphoblast cells, obtained from OHSU's Oregon Brain Aging Study, were cultured, and GSH biochemistry was subsequently evaluated. GSH was depleted by exposing cells to the GSH depleting agent diethylmaleate (DEM) and the resulting GSH concentrations were measured. GSH resynthesis was measured after depleting GSH with DEM, to a level of approximately half base GSH concentration, then removing the depleting agent, resuspending the cells in fresh medium (DEM-free), and subsequently measuring GSH levels. GSH concentrations were measured by HPLC, and all data was normalized to cellular protein concentration. Cellular CNase specific activity levels were measured by adding cytasthionine, the CNase substrate, and then measuring the amount of cysteine produced by means of the DTNB assay. The AD cell lines showed no increase in base levels of GSH as compared to control cell lines. The AD cell lines showed a statistically significant increase in GSH resynthesis capabilities and cystathionine gamma-lyase specific activity levels. These findings add further weight to the AD oxidative stress hypothesis, which is based on the premise that the causative agent of AD pathogenesis is an increase in the level of cellular free radicals produced.

This was an exploratory-descriptive study of fifty children afflicted with myelomeningocele, ages one through six, who were known to the Myelomeningocele Clinic of the Crippled Children’s Division. The study identified the degree of multiple physical, emotional, and environmental stress factors that families must be prepared to cope with. The study identified eleven factors felt to play an important role in family dynamics and how they related to the families response to their child with myelomeningocele. The factors were tested and found to be valid by the use of a pre-test on ten case records. Medical records were then obtained from the Crippled Children's Division for chart review purposes and the appropriate material was recorded. Scores were developed that indicated the degree of stress ranging from minimal involvement to maximum involvement. The study found that a majority of the families in the sample live within commuting distance to needed medical services, have transportation available to them and generally utilize the necessary medical care appropriately. The remainder of the study showed, however, that families could be expected to face a variety of other problems that could only serve to increase family stress. Most of the families had limited financial resources. Over one-half of the families needed special education for their children. A majority of families had no medical insurance. Fifty-eight percent of the families were found to have additional stressful problems to cope with e. g., marital stress, sibling rivalry, additional ill members, etc. Added to this was the information that the child with myelomeningocele was found to be greatly involved in a multiplicity of medical problems at many different levels of functioning. e. g., orthopedic, bowel, neurosurgical, etc., that would be expected to add to the already stressful family dynamics. Among the recommendations developed was a plea for the expansion of the satellite clinic concept, development of parent groups on a geographical basis, development of educational programs for educators and community service personnel, brief orientation programs for parents with the goal of helping them understand and integrate the health care system that they find themselves in.

In vitro models are often used both to characterize and test therapeutics for neurodevelopmental disorders (‘NDDs’). While in vitro models have extraordinary potential to develop therapies for patients, they have historically been confounded by absence of robust phenotypes and/or in vitro phenotypes that fail to translate from laboratory bench to bedside. Within this thesis work, we attempt to address three areas in which in vitro models may be improved – gene selection, model validation and identification of disease-relevant functional assays suited for therapeutic testing. Publicly available databases aggregating identified and annotated disease-causing variants for Mendelian diseases have rapidly expanded over the past two decades. Elucidating mechanisms of disease and developing therapies using in vivo model systems often is both time and cost intensive. Thus, determining which subsets of genes are more likely to generate addressable signals in a dish may lead to more effective drug development. In chapter 1, we identify a set of genes ideally suited for therapeutic inhibition. Specifically, we leverage the aforementioned large genetic databases to identify a set of genes likely to act through a gain-of-function mechanism that are both tolerant to loss-of-function mutations and in the druggable genome. In chapter 2, we aim to characterize the degree of conservation of transcriptomic dysregulation between a human in vitro cortical organoid (‘hCOs’) model, and two mouse models of a severe neurodevelopmental disorder resulting from HNRNPU deficiency. Human model systems may improve upon animal models when human pathogenesis and patient phenotypes are divergent from animal models due to species-specific etiology. However, human model systems often lack the heterogeneity and cell-type specificity and maturity seen in primary fetal samples. Importantly, some mouse models of HNRNPU deficiency have muted phenotypes compared with human patients. We hypothesized that while there are distinctions between humans and mice with HNRNPU deficiency, there will be overlap in transcriptomic dysregulation between human and mouse models. In fact, we find 45-day-old HNRNPU+/- hCOs have consistent transcriptomic dysregulation to embryonic mouse models, but not to perinatal mice. Our findings suggest hCOs are a viable model for characterizing HNRNPU deficiency; however, such models may only be appropriate for elucidating a transcriptomic disease state at a specific developmental time period. Functional assays for neurodevelopmental disorders can aid in understanding whether transcriptomic dysregulation is relevant to patient symptoms, as genomic findings may not always correlate to disease-relevant phenotypes. Further, relevant functional phenotypes can then be utilized for testing potential therapeutics. Importantly, seizures are commonly present in a significant subset of neurodevelopmental disorders and seizure phenotypes have been described as driven by aberrant synchrony in neuronal networks. Using a multielectrode array platform, investigators can use a variety of computational methods to quantify aspects of synchrony in vitro. In chapter 3a, we introduce topological approaches capable of identifying novel synchrony phenotypes in primary neuronal networks from mouse models of neurodevelopmental disorders. Certain mouse models will be confounded by species-specific pathogenesis and/or vastly different developmental timelines and fail to generalize to human patients, motivating the need for functionally active and physiologically relevant human in vitro models. In chapter 3b, we attempt to generate human networks with balanced levels of excitation and inhibition and find confounding lack of functional maturation of inhibitory neuronal subtypes in 90-day-old stem cell-derived neuronal networks. Future work generating in vitro human neuronal networks with functionally mature inhibitory neurons would complement the findings in chapters 1 and 2 and allow for more efficient therapeutic development strategies that may lead to improved patient outcomes.