Browsing by Subject "Neurodegeneration"
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Item Defining a Neuroprotective Pathway for the Treatment of Ataxias(2016-08) Leathley, EmilySpinocerebellar Ataxias (SCAs) are a group of genetic diseases characterized by progressive ataxia caused by neurodegeneration of specific cell types, namely Purkinje Cells (PCs) of the cerebellum. Mouse models of SCA Type 1 (SCA1) can be used to study the molecular mechanisms underlying PC degeneration and death. One SCA1 mouse model, ATXN1[30Q]D776, has an initial ataxia but no progressive degeneration or PC death. RNA-seq experiments identified the up-regulation in the cerebellum of the peptide hormone Cholecystokinin (Cck) in these mice. Knocking out Cck or the Cck1 receptor (Cck1R) in ATXN1[30Q]D776 mice confers a progressive disease where PC death occurs by thirty-six weeks of age. Weighted Gene Co-expression Network Analysis (WGCNA) performed on cerebellar RNA-seq data from ATXN1[30Q]D776;Cck-/- mice identified a disease progression-related gene set named the Pink Module that is influenced by Cck. A Cck1R agonist, A71623, was administered via osmotic minipump to ATXN1[30Q]D776;Cck-/- mice and AXTN1[82Q] mice, which are a more faithful representation of human SCA1 PC degeneration. In both mouse models, A71623 protected against progressive ataxia and PC degeneration. These results suggest that manipulation of the Cck-Cck1R pathway may be a therapeutic target for treatment of diseases involving PC degeneration.Item Disrupting Nuclear Localization of Expanded ATXN1 Mitigates SCA1 Phenotypes and Transcriptomic Perturbations Across Brain Regions(2022-02) Handler, HillarySpinocerebellar ataxia type 1 (SCA1) is a dominant neurodegenerative disease caused by ATAXIN1 (ATXN1) trinucleotide repeat expansion and characterized by motor dysfunction, cognitive impairment, and premature death. Degeneration of cerebellar Purkinje cells is a frequent and prominent pathological feature of SCA1. Previous research found that transport of ATXN1 to Purkinje cell nuclei is required for pathology, where mutant ATXN1 alters transcription. In addition to cerebellar degeneration, cell loss and atrophy has been observed in the medulla, cerebral cortex, hippocampus, and striatum of patients with SCA1. These brain regions have not historically been a major focus of SCA1 research. To test the hypothesis that nuclear localization of ATXN1 has a critical role in pathogenesis across diverse regions of the central nervous associated with SCA1, CRISPR-Cas9 was used to develop a knock-in SCA1 mouse model with an amino acid alteration (K772T) in the nuclear localization sequence of the expanded ATXN1 protein. Characterization of these Atxn1175QK772T/2Q mice indicates that nuclear localization of expanded ATXN1 contributes to many SCA1-like phenotypes including motor dysfunction, cognitive deficits, and premature lethality. The K772T mutation reduces nuclear localization, improves extractability, and delays nuclear inclusion formation of expanded ATXN1 throughout the brain. RNA sequencing analyses show that transcriptomic aspects of SCA1 pathogenesis differ between the cerebellum, medulla, cerebral cortex, hippocampus, and striatum. The findings from this project establish that nuclear localization of expanded ATXN1 is a key aspect of SCA1 pathogenesis throughout the brain and suggest that the specific molecular mechanisms of disease progression are largely unique to each of the various brain regions affected.Item Intra-Regional Differences in Cerebellar Vulnerability of Spinocerebellar Ataxia Type 1 Mice(2023-05) Hamel, KatherineIncreasing evidence demonstrates distinct regional differences across the cerebellum, yet whether these differences contribute to selective vulnerability in cerebellar disease remains an open question. Spinocerebellar Ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by an abnormal expansion of polyglutamine (polyQ) repeats in the ATAXIN1 (ATXN1) gene and characterized by cerebellar degeneration. Recent studies in patients with SCA1 indicate that pathogenesis is not uniform across the cerebellum, with posterior vermis and hemispheres exhibiting worse pathology. Mouse models of SCA1 have been critical to increasing our understanding of mutant ATXN1 toxicity, however most studies only assessed cerebellar degeneration from one lobule or with bulk cerebellar tissue.As a consequence, a question that remains unanswered is whether regions of the cerebellum undergo distinct pathological alterations across distinct regions and, if so, what molecular and functional mechanism underlie those differences in mouse models of SCA1. To address this gap in knowledge, I sought to investigate SCA1 pathology across distinct cerebellar regions in a mouse model of SCA1. I hypothesized that SCA1 knock-in mice will exhibit increased vulnerability of the posterior vermis and hemispheres, that these differences in pathology will correlate with underlying distinct gene expression changes, and will result in functional impairment that will be larger in more vulnerable PCs. My results indicate non- uniform degeneration with exacerbated Purkinje cell atrophy and gliosis in the posterior vermis. Importantly, I identified region-enriched genes and pathways of pathogenesis that may contribute to resistance and vulnerability to SCA1 pathogenesis across the cerebellar cortex. I found that calcium activity of Purkinje cells is significantly altered in posterior but not anterior vermis, indicating that worse molecular and pathological changes correlate with functional alterations. Finally, I also found unique genes and pathways altered in the DCN. This work highlights the importance of examining the cerebellum on a regional level in order to better understand disease pathology.Item Investigation of the intranasal delivery method as a means of targeting therapeutic agents to the Injured retina and optic nerve.(2009-09) Alcalá, Sandra R.Ischemic optic neuropathy (ION) is a visually devastating disease process in which there is disruption of arterial blood flow to the optic nerve head. ION, both anterior and posterior, is the most common cause of sudden optic nerve-related vision loss in the developed world. In addition, traumatic optic neuropathy (TON), caused by blunt trauma to the orbit and/or face, can cause tractional, compressive, or ischemic injuries to the optic nerve as well. These types of injuries to the optic nerve can ultimately result in the death of retinal ganglion cells in the retina and, consequently, the functional loss of vision. Currently, there is no effective treatment for these types of injury. There are several issues that stand in the way of adopting treatment modalities for injuries to the optic nerve and retina. Many potential therapeutic drugs are unable to gain access to the affected cells due to the protective blood-retinal and blood-brain barrier. Neurotrophic factors are endogenous large molecular weight neuroprotective proteins that, upon injury, are released in an autocrine and paracrine fashion to reduce apoptotic cellular death. However, these factors possess inherent molecular characteristics that impede their transport through the protective blood-brain barrier. Therefore, the ability to bypass the blood-brain barrier using a non-invasive means would have great clinical potential. This study examined the viability of the intranasal delivery method as a means of targeting therapeutic agents to the injured retina and optic nerve.Item Label-free optical imaging to study brain connectivity and neuropathology(2018-11) Liu, ChaoThe brain is composed of billions of neurons that communicate through an intricate network of axons and dendrites. The difficulty of tracing the 3D neuronal pathways, however, has been a challenge to study the brain connectivity in normal and diseased brains. Polarization-sensitive optical coherence tomography (PS-OCT) provides label-free and depth-resolved contrasts of tissue microstructure. For brain imaging, nerve fiber tracts that are as small as tens of micrometers can be highlighted by polarization-based contrasts due to the birefringent nature of myelin sheath. We applied optical imaging to investigate the anatomical changes associated with neurodegeneration and neuro-oncology. The former includes spinocerebellar ataxia type 1 (SCA1), a fatal inherited genetic disease. The intrinsic optical properties revealed the neuropathology in SCA1 mouse models. To investigate the role of nerve fiber tracts in glioblastoma invasion, we combined PS-OCT with confocal fluorescence microscopy to characterize glioma cell migration behavior in mouse brain slices. Moreover, PS-OCT can be adapted to quantify the inclination angles of nerve fibers and further developed to delineate the complete 3D neuronal pathways. This method and its future advances open up intriguing applications in neurological and psychiatric disorders.Item Mechanisms and consequences of the HSF1 degradation pathway in Huntington's disease(2022-07) Zarate, NicoleHuntington’s disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in exon 1 of the huntingtin gene (HTT). HD is characterized by progressive motor, cognitive, and psychiatric impairments for which there is no cure. Medium spiny neurons (MSNs) of the striatum are preferentially affected by the mutant huntingtin protein (mtHTT) and are subject to neuronal dysfunction and eventual cell death. One driver believed to contribute to MSN dysfunction in HD is excitatory synapse impairment. Deficits in excitatory synapses can range from loss of overall synaptic input to MSNs, loss of synaptic proteins, and transcriptional changes in genes necessary for synaptic stability. However, the mechanisms behind excitatory synapse instability in HD are largely unknown. In this thesis, the role of the stress protective transcription factor, Heat shock factor 1 (HSF1) in excitatory synapse impairment in HD is extensively explored. We show that induction of the tumor suppressor p53 directly binds and regulates expression of Protein Kinase CK2α’, a kinase that was previously demonstrated to phosphorylate HSF1 and lead to its eventual degradation. Reductions in p53 and CK2α’ rescue levels of HSF1 in MSNs, improves expression of HSF1 target genes, and improves levels of T-S synapses in zQ175 HD mice. Further characterization of CK2α’ knockdown in zQ175 HD mice shows that rescued levels of HSF1 parallel improvements in motor behavior, transcriptional deficits associated with synaptic stability, and impairments in synaptic transmission. Given the parallels in improved HSF1 levels and improvements in various synaptic deficits, we next investigated if HSF1 was playing a direct role in excitatory synapse regulation. We demonstrated HSF1 directly regulates the expression of PSD-95 (Dlg4), a protein that participates in stabilizing excitatory synapses, and progressive loss of HSF1 in HD contributes to PSD-95-dependent depletion of excitatory synapse density. Circuit-specific synaptic deficits are reported in HD with an early loss of thalamo-striatal (T-S) synapses followed by changes in cortico-striatal (C-S) synapses at later stages in disease. We showed aged mice lacking one allele of Hsf1(Hsf1(+/-)) presented reduced PSD-95 and T-S synapse levels, indicating HSF1 could play an essential role in the stability of T-S synapses. However, analyses in younger mice showed Hsf1 haploinsufficiency does not significantly influence levels of T-S synapses. Pilot studies using ChIP-seq however indicate genome-wide enrichment of HSF1 binding to genes associated with synapse stability in young mice, other than PSD-95, revealing new potential HSF1 targets that could contribute to synapse dysfunction in HD at different disease stages. Contrarily, acute reduction of HSF1 in the adult striatum using adeno-associated viruses, similar to HSF1 progressive depletion in HD, has negative effects on aspects of striatal function such as MSN protein levels and spatial recognition cognitive behavior. We concluded that HSF1 plays a time-sensitive bimodal role in striatal physiology with an enhanced protective role in the aged brain. Lastly, we demonstrated through in vivo longitudinal analyses of brain neurochemicals, measured by proton magnetic resonance spectroscopy (1H-MRS), that specific neurochemical changes correlate with progressive alterations in T-S and C-S synapses during HD. In addition, CK2α’ knock down and subsequent HSF1 rescue can improve some of these neurochemical alterations. Therefore, these neurochemical measures can be used to monitor circuit dependent synaptic changes. This provides a potential tool for monitoring excitatory synaptic changes in vivo with surrogate biomarkers using MRS. In conclusion, this dissertation provides a holistic view of the mechanisms involved in HSF1 degradation in HD and the pathophysiological roles of this transcription factor in synapse stability. Results from these studies demonstrate how targeting aspects of the HSF1 degradation pathway could be a potential therapeutic avenue in treating HD.Item Mitigating oxidative stress in childhood cerebral adrenoleukodystrophy -an investigation of N-acetylcysteine pharmacology(2014-02) Zhou, JieAdrenoleukodystrophy (ALD) is an X-linked genetic disorder which affects the adrenal glands, peripheral neuronal system, the spinal cord and white matter of central nervous system (CNS). It is a progressive neurology disorder with incidence of 1 in 17,000 newborns. ALD is caused by mutations in the ABCD1 gene, which encodes the peroxisomal membrane transporter for transporting very long chain fatty acids (VLCFAs) into peroxisomes for degradation. As a result, VLCFAs accumulate in the plasma and tissues of ALD patients. Elevated VLCFAs along with ABCD1 gene mutations are used for the diagnosis of ALD. ALD has various clinical phenotypes. Childhood cerebral adrenoleukodystrophy (CCALD) is the cerebral form of ALD that affects young boys (4~10 years of age), causing progressive, debilitating effects on the CNS leading to death within a few years. The pathophysiology of CCALD is only partially understood, but it is known that VLCFAs accumulate in the plasma, brain and other tissues in CCALD patients, which can cause oxidative stress and downstream neurodegeneration. Recently, oxidative stress, the accumulation of free radicals (reactive molecules), has been shown to cause CNS neurodegeneration and play a major role in CCALD pathophysiology. Currently, the most successful treatment for CCALD is hematopoietic stem cell transplantation (HSCT), which halts disease progression and extends life when CCALD is treated early. But it is much less effective for late-stage CCALD. Based on evidence that oxidative stress plays a role in the disease, the Blood and Marrow transplantation group at University of Minnesota has utilized N-acetylcysteine (NAC) as adjunctive therapy together with HSCT in late-stage CCALD. This combinatorial approach has improved survival rate from 36% to 84% compared to HSCT only in a cohort study (Miller et al., 2011). However, NAC's mechanisms of action are still unclear in CCALD patients. As an FDA-approved drug, NAC is used as an antidote for acetaminophen overdose and as a mucolytic agent to reduce symptoms associated with cystic fibrosis. It has gained renewed attention as a potential therapy for a number of conditions including pulmonary, neurological, psychiatric, and cardiovascular diseases. With a long history of clinical use, several mechanisms including antioxidative and anti-inflammatory activities have been proposed as the basis for its therapeutic effects. However, the exact molecular mechanism by which NAC improves the survival rate of CCALD patients is still unclear. And this missing piece of information, which is the basis for my research work, is required to further optimize the therapy. In my thesis, four research projects were designed and implemented to address the pharmacology of NAC in CCALD related biological models. The first study was to investigate the downstream signaling molecules induced by NAC in the plasma of CCALD patients. Heme oxygenase-1 (HO-1) and ferritin were examined in CCALD patients before and after NAC exposure. Based on the clinical study results that the expression of HO-1 and downstream ferritin were induced by NAC, the second study was further designed in oligodendrocytes, which are CNS glial cells and closely related to demyelination and neurodegneration, to investigate the cytoprotective role of HO-1 induced by NAC. Moreover, we also tried to delineate the role of accumulation of VLCFAs in CCALD and its relationship with oxidative stress and mitochondria. The third study was designed in oligodendrocytes to investigate whether mitochondria and oxidative stress status are affected by pathophysiological concentrations of VLCFAs and if so, whether NAC could be used to reverse this condition. Finally, the fourth pharmacokinetic/pharmacodynamics study was designed and implemented in wild-type mice to address the relationship between NAC concentration and pharmacodynamic endpoints in vivo. This study is also critical to determine the biotransformation of NAC in vivo.The results from my studies indicate HO-1 as the newly discovered downstream mediators for NAC action. Studies also show for the first time that depletion of mitochondrial glutathione (mtGSH) is the pathological cause for CCALD, and that targeting mitochondrial dysfunction can be a potential effective intervention for CCALD patients. In addition, GSH levels, redox ratio, HO-1 and ferritin levels can serve as biomarkers or pharmacodynamic endpoints to evaluate NAC efficacy. In the long term, characterization of NAC mechanisms of action will help to optimize therapy in CCALD patients. In addition, the information generated from my studies on the efficacy of NAC in CCALD is also applicable to other neurodegenerative disorders sharing similar pathologies such as Gaucher's disease, multiple Sclerosis, Alzheimer's disease etc.Item Partial Tip60 loss slows cerebellar degeneration in a Spinocerebellar Ataxia Type 1 (SCA1) mouse model.(2009-07) Gehrking, Kristin MarieSpinocerebellar ataxia type 1 (SCA1) is one of nine dominantly inherited neurodegenerative diseases caused by polyglutamine tract expansion. In SCA1, the expanded polyglutamine tract is in the ataxin-1 (ATXN1) protein. Increased polyglutamine tract length results in earlier disease onset and greater disease severity, which is largely due to cerebellar Purkinje cell degeneration. ATXN1 is part of an in vivo complex with the nuclear receptor (retinoid acid receptor-related orphan receptor alpha [ROR-alpha]) and acetyltransferase (tat-interactive protein 60 kD [Tip60]). ATXN1 and Tip60 interact directly; however, the significance of this interaction is unclear. To test the effect of partial Tip60 loss on SCA1 disease progression, I developed a mutant ATXN1[82Q]/+:Tip60+/- mouse model. Partial Tip60 loss increased ROR-alpha, Rora, and ROR-alpha-mediated gene expression and delayed ATXN1[82]-mediated cerebellar degeneration during midstage disease progression. I also compared ATXN1[82Q]/+ phenotypes between different genetic background strains. Finally in vitro data suggested an ATXN1 polyglutamine length effect on Tip60 acetyltransferase activity. In additional to highlighting genetic background modulation in SCA1 disease, these results suggest a specific temporal role for Tip60 during disease progression and a putative role for Tip60 acetylation in SCA1 disease progression.Item Role of spectrin mutations in spinocerebellar ataxia type five (SCA5)(2009-08) Lorenzo Vila, Damaris NadiaSpinocerebellar ataxia type 5 (SCA5) is a dominant neurodegenerative disorder caused by mutations in the SPBTN2 gene encoding the cytoskeletal protein beta-III spectrin. To get insight into the biology of the disease and the normal function of beta-III spectrin, and to estimate the frequency of SCA5 mutations among ataxia patients, I used a forward human genetic approach to identify novel SPTBN2 mutations. Screening of the SPTBN2 gene in a cohort of families with dominant ataxia of unknown etiology and a large group of ataxia samples identified seventeen novel variants not found in the general population. Putative mutations were identified in the areas comprising the second calponin homology domain, spectrin repeat two to four, and the ninth spectrin repeat of beta-III spectrin. To investigate the downstream effects of the American and German SCA5 mutations in neurons, I established a series of transgenic Drosophila models that express human beta-III-spectrin or fly beta-spectrin proteins containing SCA5 mutations. Through genetic and functional analyses I show that expression of mutant spectrin in the eye causes a progressive neurodegenerative phenotype and expression in larval neurons results in posterior paralysis, reduced synaptic terminal growth, and axonal transport deficits. These phenotypes are genetically enhanced by both dynein and dynactin loss-of-function mutations. I have additionally used the SCA5 fly models to conduct modifier screens and identify genes and biological pathways that may contribute to SCA5 pathogenesis. These studies revealed genetic interactors implicated in a wide range of biological functions including intracellular transport, synapse formation and function, protein homeostasis, and transcription regulation.Item Specific amyloid-beta oligomers in-human cerebrospinal fluid.(2012) Handoko, MaureenAlzheimer’s disease (AD) is a progressive neurodegenerative disease with a long preclinical stage, during which pathological changes and biomarker abnormalities, such as elevations of tau proteins in the cerebrospinal fluid (CSF), occur in the absence of cognitive impairment. Research on the etiology of AD in cell culture and animals have suggested that soluble oligomers of amyloid-β (Aβ), such as Aβ*56, trimers, and dimers, are the synaptotoxic species in AD and are thought to be responsible for initiating the cognitive impairment associated with the early stages of disease. These studies also suggest that tau pathology develops downstream of Aβ and may mediate the detrimental effects of Aβ on learning and memory. However, the whether these Aβ oligomers exist in humans and are associated with disease processes of AD is still unknown. To address this question, we developed a sensitive method to detect Aβ*56, trimers, and dimers in lumbar CSF, enabling the study of these oligomers in clinically characterized living subjects. Biochemical study of CSF from cognitively intact subjects and impaired subjects with clinical diagnosis of AD and mild cognitive impairment (MCI) showed that these oligomers were present in the CSF of both the unimpaired and impaired subjects.Within the unimpaired group, subjects at a higher risk of having preclinical AD had elevated levels of Aβ oligomers. Furthermore, Aβ*56, dimers, and trimers were positively correlated with total or abnormally phosphorylated tau in the CSF of unimpaired subjects, suggesting that these oligomers are associated with disease processes and may induce tau abnormalities in the preclinical phase of disease. Study of CSF from subjects enrolled in a longitudinal study showed that Aβ*56 was negatively correlated with memory in unimpaired subjects who converted to MCI/AD during follow-up. However, baseline Aβ oligomer levels were not predictive of conversion to MCI/AD from cognitive normality. Further exploratory analysis revealed significant associations between high levels of baseline total tau or low Aβ*56 levels and a faster rate of memory decline. The results of these studies are consistent with the hypothesis that Aβ oligomers may trigger tau abnormalities in preclinical AD, and that Aβ*56 may play a pathological role in preclinical AD but is not sufficient to trigger disease progression. A hypothetical model describing the characteristics of Aβ*56 and tau during the preclinical phase of AD was generated from this data and awaits further evaluation.Item Transcriptional disruptions and functional correlates in a human induced pluripotent stem cell – derived motor neuron model of Spinocerebellar ataxia type 1(2022-08) Sheeler, CarrieIt is hypothesized that degeneration of the motor neurons (MNs) in the brain stem and spinal cord contributes to premature lethality in spinocerebellar ataxia type 1 (SCA1) by affecting the strength of swallowing and respiratory drive. While we can recreate some aspects of MN pathology in mouse models, loss of MNs has only been seen in SCA1 patient populations. This, in addition to other species differences that exist between rodents and humans, accentuates the need for translational human models of disease which can be used to uncover therapeutically relevant cellular and molecular mechanisms underlying dysfunction in vulnerable cell types. To investigate potential mechanisms underlying SCA1 pathology in human MNs, I developed a human induced pluripotent stem cell (iPSC)-derived MN model of SCA1. This is, to our knowledge, the first cell-type specific iPSC-derived model made to study SCA1. Previous work in mouse models has demonstrated both that ATXN1 regulates cellular transcription (Ingram et al 2016, Rousseaux et al 2018, Friedrich et al 2019) and that its entry into the nuclei of cells is important for the longevity of mouse models of SCA1 (Handler et al 2022). Thus, I predicted that the presence of mutant ATXN1 in hiPSC-derived MNs would drive measurable transcriptomic changes in SCA1 samples compared to unaffected controls. Furthermore, transcriptomic disruptions might indicate functional pathways of interest for future studies into the lethality of SCA1. I performed bulk RNA sequencing on MN-enriched cultures to assess underlying transcriptional changes that may be affecting SCA1 human MNs, and subsequently identified disruptions in key cellular processes including extracellular matrix (ECM) regulation, calcium ion binding, and mitotic cell cycle regulation. Proper regulation of ECM is key to many aspects of neuronal development and function, including extension of neurites, proper placement and presentation of receptors and ion channels, cell-cell communication, synapse formation, and intracellular transport. As such, I predicted that broad changes in ECM regulation might result in measurable changes in some of these capabilities in SCA1 MN enriched cultures. Neurite outgrowth was measured in motor neuron progenitors (pMNs) and immature motor neurons (iMNs). I determined that SCA1 samples do not exhibit remarkable disruptions in neurite outgrowth at either timepoint. Assessment of spontaneous calcium activity exhibited a similar degree of physiological maturity between SCA1 and unaffected control MN enriched cultures. Additionally, measurement of miniature excitatory postsynaptic currents (mEPSCs) demonstrated formation of synapses but no functional differences in SCA1 samples compared to controls. However, glutamate evoked calcium activity demonstrated a reduced amplitude of calcium response in SCA1 cultures. This occurred despite any measurable transcriptomic changes in glutamate receptor expression. This indicates potential underlying disruptions in receptor activity and calcium dynamics in SCA1 MN-enriched cultures and provides a potential avenue of interest for future work investigating disruptions in communication in the SCA1 spinal cord.