Mutations in SPTBN2, the gene encoding b-III spectrin, give rise to spinocerebellar ataxia type 5 (SCA5), an autosomal dominant neurodegenerative disease characterized by motor incoordination and cerebellar degeneration. The work reported in this thesis addressed possible mechanisms of disease pathogenesis using genetically modified mice lacking b-III spectrin (b-III-/-) and also investigated the normal function of b-III spectrin through identification of proteins that interact with its amino-terminus. Targeted recombination was successful in eliminating expression of full-length b-III spectrin but b-III spectrin lacking exons 2-6 ( 2-6 b-III spectrin) was found to be present at a low level in b-III-/- spectrin mice. To ascertain whether the novel truncated protein had any obvious gain-of-function or adverse property that would complicate analysis of b-III-/- spectrin mice the aberrant transcript 2-6 b-III spectrin was cloned and a number of in vitro experiments carried out. Protein stability, solubility, cellular localization, and functional assays indicated 2-6 b-III spectrin was less functional than full-length b-III spectrin, confirming the b-III-/- spectrin mouse could be considered a functional knockout. Analysis of b-III-/- spectrin mice revealed that from 18-weeks of age hind limb gait became progressively wider than age-matched wild-type (WT) controls and three behavioural tests (stationary rod, rotarod, and elevated beam) demonstrated a progressive impairment in motor performance and coordination. 3-week old b-III-/- spectrin mice performed worse on the rotating rod than age-matched controls but their performance at 3- and 5-rpm improved with consecutive days of testing. Only at 10-rpm did young b-III-/- spectrin mice fail to improve, whereas 6-month old b-III-/- spectrin mice were unable to stay on the rod even at 3-rpm. The ability to balance on a stationary rod was also worse at 6-months of age and the number of hindlimb slips made by b-III-/- spectrin mice on the elevated beam increased from 12-weeks of age. This progressive motor phenotype mirrors symptoms seen in SCA5 patients. In contrast heterozygous mice (b-III+/-) were shown not to develop an ataxic phenotype or display cerebellar degeneration, even at 2-years of age. Cell culture studies using one mutation (L253P) associated with SCA5 revealed that it interfered with protein trafficking from the Golgi apparatus and had a dominant-negative effect on WT function. Incubation at a lower temperature resulted in L253P b-III spectrin reaching the plasma membrane suggesting an altered protein conformation was responsible for the protein trafficking defect. The intracellular accumulation of proteins at the Golgi did not initiate the unfolded protein response. From this work it was concluded that the b-III-/- spectrin mouse is a new model of cerebellar ataxia and loss of b-III spectrin function underlies SCA5 pathogenesis. The results argued against haploinsufficiency and instead suggested disease-causing mutations have dominantnegative effects on WT function and indicate a deficit of cell membrane proteins could participate in SCA5 pathogenesis. Finally, using a yeast two-hybrid screen the amino terminus of b-III spectrin was found to interact with the carboxy-terminus of prosaposin (a neurotrophic factor) and clathrin light chain. The interactions were confirmed in mammalian cells suggesting neurite outgrowth and movement of membrane vesicles may be normal functions of b-III spectrin.

The expansion of repetitive sequences leads to more than 40 neurological, neurodegenerative and neuromuscular diseases. These diseases are frequently characterized by ongoing DNA repeat instability upon transmission, worsening of disease severity and decreasing age of onset with each successive generation. The mechanism of repeat instability and contribution of repeat instability to disease pathogenesis are unknown. My thesis examines the contribution of cis-elements – sequences around and within repeats as well as surrounding epigenetic environments – to repeat instability, and discusses their possible contribution to repeat diseases. Here I identify the first cis-element regulating repeat instability, a DNA binding site for a trans factor protein, CTCF. Loss of CTCF binding at the spinocerebellar ataxia type 7 disease locus induces somatic and germline instability in an age- and tissue-specific manner in mice. CTCF protects against instability in an epigenetic manner, and may function at other disease loci also possessing CTCF binding sites near the repeat. Given that CTCF flanks many repeat loci and is often situated between a replication origin and disease-associated repeat, I assess the role of CTCF on replication and instability at the myotonic dystrophy repeat locus. Templates with CTCF binding sites reduce overall replication efficiency in primate cells that may be partly due to replication fork stalling. Mutating CTCF binding sites can alter the stability of the repeat depending on the distance from the origin of replication to the repeat. Finally I examine chromatinization of (ATTCT)n repeats from the spinocerebellar ataxia type 10 locus. These repeats induce very strong nucleosome formation, and at physiological conditions form even more strongly on (ATTCT)n repeats with interruptions that are also found in some patients. These data contribute to the understanding of repeat instability in the causation of many diseases, and suggest that the presence of cis-elements at repeat-associated disease loci alter the behaviour of repeats.

The expansion of repetitive sequences leads to more than 40 neurological, neurodegenerative and neuromuscular diseases. These diseases are frequently characterized by ongoing DNA repeat instability upon transmission, worsening of disease severity and decreasing age of onset with each successive generation. The mechanism of repeat instability and contribution of repeat instability to disease pathogenesis are unknown. My thesis examines the contribution of cis-elements – sequences around and within repeats as well as surrounding epigenetic environments – to repeat instability, and discusses their possible contribution to repeat diseases. Here I identify the first cis-element regulating repeat instability, a DNA binding site for a trans factor protein, CTCF. Loss of CTCF binding at the spinocerebellar ataxia type 7 disease locus induces somatic and germline instability in an age- and tissue-specific manner in mice. CTCF protects against instability in an epigenetic manner, and may function at other disease loci also possessing CTCF binding sites near the repeat. Given that CTCF flanks many repeat loci and is often situated between a replication origin and disease-associated repeat, I assess the role of CTCF on replication and instability at the myotonic dystrophy repeat locus. Templates with CTCF binding sites reduce overall replication efficiency in primate cells that may be partly due to replication fork stalling. Mutating CTCF binding sites can alter the stability of the repeat depending on the distance from the origin of replication to the repeat. Finally I examine chromatinization of (ATTCT)n repeats from the spinocerebellar ataxia type 10 locus. These repeats induce very strong nucleosome formation, and at physiological conditions form even more strongly on (ATTCT)n repeats with interruptions that are also found in some patients. These data contribute to the understanding of repeat instability in the causation of many diseases, and suggest that the presence of cis-elements at repeat-associated disease loci alter the behaviour of repeats.

Neurodegenerative disorders are among the most devastating human illnesses. They present a significant source of morbidity and mortality, and given an aging population, an impending public health crisis. Disease-modifying treatments remain sparse, with most current therapies focused on reducing symptom burden. The cellular stress response is intimately linked to energy management and has frequently been posited as playing a central role in neurodegeneration. Using two distinct neurodegenerative diseases as ‘case studies’, aberrant cellular stress and energy management are demonstrated as potential pathways contributing to neurodegeneration. First, the Huntington’s disease protein, huntingtin, is observed to rapidly localize to early endosomes, where it is associated with arrest in early-to-late and early-to-recycling endocytic trafficking. Given the energy-dependent nature of vesicular trafficking, this arrest is postulated to free substantial energy within the cell, which may subsequently be diverted to pathways that are critical for the initiation of longer-duration stress responses, such as the unfolded protein response. In the context of Huntington’s disease, impaired recovery from this stress response is observed, suggesting deficits in intracellular vesicular trafficking and energy regulation exist in disease states. In the second ‘case study’, a novel spinocerebellar ataxia variant is characterized, occurring as a result of point mutations within two genes: ATXN7 and TOP1MT, which encode ataxin-7 and the type I mitochondrial topoisomerase (top1mt), respectively. Ataxin-7 has previously been implicated in spinocerebellar ataxia type 7, which occurs as a result of a polyglutamine expansion in the first exon of the protein. Patient cells are noted to have substantially lower mitochondrial respiratory function in comparison to healthy controls and decreased levels of mitochondrial DNA, and ataxin-7 subcellular localization is observed to be abnormal. This suggests that there is important interplay between the mitochondria and proteins implicated in neurodegeneration and provides further support for aberrant cellular bioenergetics as a unifying pathway to neurodegeneration. In the concluding chapters, the nuclear localization signal of ataxin-7 is characterized, and there is analysis comparing conical ‘atraumatic’ lumbar puncture needles with bevel-tipped ‘conventional’ needles. Atraumatic needles are noted to be associated with significantly less patient complications and require fewer return visits to hospital. Moreover, atraumatic needles are demonstrated to have similar rates of success and failure when controlling for important variables like clinician specialty, dispelling common misconceptions surrounding their ease-of-use. As lumbar puncture is ubiquitous within the clinical neurosciences and is important for diagnosis, monitoring, and treatment of disease, as well as clinical trials, this work has far-reaching implications for patient care and future research. / Thesis / Doctor of Philosophy (PhD)

Master of Science / Department of Biochemistry / Dolores J. Takemoto / Spinocerebellar ataxia type 14 (SCA14) is a rare, autosomal dominant neurodegenerative disease caused by mutations in the gene encoding for protein kinase Cγ (PKCγ). These mutations affect the translocation and activation of the protein and are particularly damaging to the Purkinje cells of the cerebellum. This translocation and activation leads to the down regulation of gap junction activity by direct phosphorylation on the C-terminal tail of connexin proteins. This process is necessary in terminating the propagation of apoptotic signaling and is disrupted by SCA14-type mutations. Gap junctions allow the passive diffusion of small molecules from one adjoining cell to another. Gap junctions function as electrical synapses in neuronal tissue and are formed from connexin proteins. The connexin family of proteins contains approximately 20 members, each of which is expressed in a tissue dependent manner. One of the dominant connexin proteins expressed in Purkinje cells is connexin 57 (Cx57). Here, I have tested if Cx57 is regulated by PKCγ. This thesis shows that activation of PKC and PKCγ caused internalization of Cx57 gap junction plaques in HT-22 cell culture. PKC and PKCγ activation led to the phosphorylation of Cx57 primarily on serine residues. Furthermore, the expression of SCA14-type PKCγ led to increased sensitivity to oxidative stress, resulting decreased cell viability.

Introduction: Spinocerebellar ataxia (SCA) is currently defined as a group of diseases with progressive cerebellar syndrome, which manifests distinctively by ataxic stance and gait. In patients with SCA, deterioration of postural control occurs due to changes of pathophysiological degenerative nervous system. Control of postural stability is significantly influenced by somatosensory afferentation of soft tissue of foot. The work objective is to determine the effect of deep foot massage on postural stability in the group of patients and to compare obtained results with experiments without deep foot massage. Methods: Seven patients of the Clinic Rehabilitation at the Faculty Hospital, Prague Motol (average age 51.9 ± 13.9 years, 5 men, and 2 women) with SCA and clinical signs of cerebellar ataxia were participated in this study. All patients were examined using posturography before and after application of deep foot massage. Measurements were conducted on a firm surface, foam pad with open and closed eyes. Length of COP trajectory, COP area and COP velocity were measured. Results: Significantly lower values of the length and COP velocity were found on the foam pad with closed eyes, when the deep foot massage was applied. Experiments without applications of the deep foot massage did not show any...

The polyglutamine diseases are a clinically heterogeneous group of inherited neurodegenerative disorders caused by expansion of polyglutamine-encoding (CAG)n trinucleotide repeats within the disease genes. It is increasingly clear that the amino acid sequences flanking the polyglutamine expansion in each disease protein, i.e. the specific protein context, contribute to selective neuronal toxicity by influencing the behavior of the disease protein within selectively vulnerable neuronal populations. In the studies described here, I explore the role that protein context plays in the polyglutamine disease, Spinocerebellar ataxia type 3 (SCA3). Toward this end, I utilize biochemical, cell-based, and animal models to gain a broader understanding of the SCA3 disease protein, ataxin-3, and generate tools for further exploration of the molecular properties of ataxin-3 that modulate its toxicity during disease. In Chapter 1, I provide an overview of the recognized polyglutamine diseases, emphasizing the elements of protein context that are distinct among the polyglutamine disease proteins and may contribute to the neuropathological and clinical heterogeneity within this family of diseases. Alternative splicing of the polyglutamine disease gene products adds an additional level of complexity to the tissue-specific protein context of expanded polyglutamine, yet this phenomenon has been underinvestigated. In Chapter 2, I examine the significance of ataxin-3 splice variation. Several minor 5' variants and both known 3' splice variants of ataxin-3, a deubiquitinating enzyme, are expressed at the mRNA level in brain. At the protein level, however, the C-terminal splice isoform with three ubiquitin interacting motifs (3UIM ataxin-3) is the predominant isoform in brain, independent of age or (CAG)n expansion. Although both C-terminal ataxin-3 splice isoforms display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and is more rapidly degraded by the proteasome. These observations demonstrate how alternative splicing of sequences distinct from the polyglutamine-encoding (CAG)n repeat can alter disease-related components of protein context. Knock-in models of polyglutamine diseases utilize pathogenic (CAG)n expansions within the endogenous genomic, transcript, and protein context to recreate key features of individual polyglutamine diseases. In chapter 3, I describe the creation of the first knock-in mouse model of SCA3. Hemizygous knock-in mice transmit the knock-in allele in Mendelian ratios and broadly express both the expanded Atxn3(Q3KQ82) protein and the wildtype murine Atxn3(Q6) protein. In this chapter, I also compare the gene targeting efficiencies and rates of chromosomal instability of a novel C57BL/6J ES cell line (UMB6JD7) and two well established ES cell lines (W4 and Bruce4.G9). Of these, Bruce4.G9 ES cells proved superior based on lower rates of aneuploidy and the production of germline transmitting chimeras. Finally, in Chapter 4 I discuss questions and concepts raised during the course of these studies, and suggest avenues of future research aimed at broadening our understanding of ataxin-3 physiology and of protein context-dependent elements in polyglutamine disease pathogenesis.

<p>Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease characterized by cerebellar ataxia and visual problems due to a progressive and selective loss of neurons within the cerebellum, brainstem and retina. The disease is caused by the expansion of a CAG repeat in the first coding exon of the SCA7 gene, resulting in an expanded polyglutamine domain in the N-terminal part of ataxin-7, a protein of unknown function.</p><p>To expand our knowledge of the ataxin-7 protein and the mechanism by which mutant ataxin-7 causes disease, we have studied the expression and function of both the normal and the mutated ataxin-7 protein. </p><p>Ataxin-7 expression was examination in brain and non-CNS tissues from SCA7 patients and age-matched controls. Expression was predominantly nuclear in neurons throughout the brain of both healthy and SCA7 individuals. We also observed aggregation of mutant ataxin-7 in the nuclei of neurons. No obvious difference in the expression level of ataxin-7 or the formation of aggregates could be observed between affected and non-affected brain regions in SCA7 patients. Based on these findings, we could conclude that the cell type specific neurodegeneration in SCA7 is not due to differences in expression levels or to the formation of ataxin-7 aggregates.</p><p>To widen our studies on ataxin-7 expression, we isolated and characterized the mouse SCA7 gene homolog. Cloning of the mouse SCA7 gene revealed two SCA7 mRNA isoforms that were highly homologous to their human counterparts. Immunohistochemical analysis also revealed a conserved expression pattern of ataxin-7 in adult mouse brain. In addition, ataxin-7 expression was observed during embryonic development in brain as well as in several non-neuronal tissues such as heart, liver and lung. </p><p>Besides SCA7, eight neurodegenerative disorders are known to be caused by expanded polyglutamine repeats, including SCA 1-3, 6 and 17, DRPLA, SBMA and Huntington’s disease. The polyglutamine disorders have many features in common and a common pathological disease mechanism involving transcriptional dysregulation has been proposed. To investigate the possible involvement of transcriptional dysregulation in SCA7 pathology, we analyzed the effects of both wild-type and expanded ataxin-7 on transcription driven by the co-activator CBP, the Purkinje cell-expressed nuclear receptor RORα1 or a basic TATA promoter. As previously shown for other polyglutamine disease proteins, expansion of the polyglutamine domain in ataxin-7 leads to reduced transcription. Surprisingly, strong repression of CBP-mediated, RORα1-mediated and basal transcription was also observed with wild-type ataxin-7, suggesting that the normal ataxin-7 protein may have a role in transcriptional regulation. </p>

Adult neural precursor cells (NPCs) are a heterogeneous population of mitotically active, self-renewing multipotent cells of both adult and developing CNS. They can be expanded in vitro in the presence of mitogens. The B05 transgenic SCA1 mice, expressing human ataxin-1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP-expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks) and significant PC loss (24 weeks). A stereological count demonstrates that mice with significant cell loss exhibit highest survival of grafted NPCs and migration to the vicinity of PCs as compared to wt and younger grafted animals. These animals showed improved motor skills as compared to sham animals. Confocal analysis and profiling shows that many of implanted cells present in the cerebellar cortex have formed gap junctions with host PCs and express connexin43. Grafted cells did not adopt characteristics of PCs, but stereological and morphometric analysis of the cerebellar cortex revealed that grafted animals had more surviving PCs and a better preserved morphology of these cells than the control groups. Perforated patch clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham-treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. In this study, evidence for a neuroprotective effect came, in addition to motor behavior improvement, from stereological and electrophysiological analyses and suggest that timing of stem cell delivery is important to determine its therapeutic effect. In a brain stem cell niche, NSCs reside in a complex cellular and extracellular microenvironment comprising their own progeny, ependymal cells, numerous blood vessels and various extracellular matrix molecules. Recently, it was reported that blood vessel ECs-NSCs crosstalk plays an important role in tissue homeostasis. Bloodstream offers a natural delivery vehicle especially in case of diffuse neurodegenerative diseases which require widespread distribution of exogenous cells. As NSCs are confronted with blood-brain barrier endothelial cells (BBB-ECs) before they can enter into brain parenchyma, we investigated their interaction using primary cultures in an in vitro BBB model. We isolated human fetal neural precursor cells (hfNPCs) from aborted fetal brain tissues and expanded in vitro. We showed that in an in vitro model, human BBB endothelium induces the rapid differentiation of hfNPCs and allows them to cross the endothelial monolayer, with the differentiated progeny remaining in close contact with endothelial cells. These results are not reproduced when using a non-BBB endothelium and are partly dependent on the cytokine MCP1. Our data suggest that, in the presence of attractive signals released by a damaged brain, intravascularly administered NPCs can move across an intact BBB endothelium and differentiate in its vicinity. Overall, our findings have implications for the development of cellular therapies for cerebellar degenerative diseases and understanding of the brain stem cell niche.

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease characterized by cerebellar ataxia and visual problems due to a progressive and selective loss of neurons within the cerebellum, brainstem and retina. The disease is caused by the expansion of a CAG repeat in the first coding exon of the SCA7 gene, resulting in an expanded polyglutamine domain in the N-terminal part of ataxin-7, a protein of unknown function. To expand our knowledge of the ataxin-7 protein and the mechanism by which mutant ataxin-7 causes disease, we have studied the expression and function of both the normal and the mutated ataxin-7 protein. Ataxin-7 expression was examination in brain and non-CNS tissues from SCA7 patients and age-matched controls. Expression was predominantly nuclear in neurons throughout the brain of both healthy and SCA7 individuals. We also observed aggregation of mutant ataxin-7 in the nuclei of neurons. No obvious difference in the expression level of ataxin-7 or the formation of aggregates could be observed between affected and non-affected brain regions in SCA7 patients. Based on these findings, we could conclude that the cell type specific neurodegeneration in SCA7 is not due to differences in expression levels or to the formation of ataxin-7 aggregates. To widen our studies on ataxin-7 expression, we isolated and characterized the mouse SCA7 gene homolog. Cloning of the mouse SCA7 gene revealed two SCA7 mRNA isoforms that were highly homologous to their human counterparts. Immunohistochemical analysis also revealed a conserved expression pattern of ataxin-7 in adult mouse brain. In addition, ataxin-7 expression was observed during embryonic development in brain as well as in several non-neuronal tissues such as heart, liver and lung. Besides SCA7, eight neurodegenerative disorders are known to be caused by expanded polyglutamine repeats, including SCA 1-3, 6 and 17, DRPLA, SBMA and Huntington’s disease. The polyglutamine disorders have many features in common and a common pathological disease mechanism involving transcriptional dysregulation has been proposed. To investigate the possible involvement of transcriptional dysregulation in SCA7 pathology, we analyzed the effects of both wild-type and expanded ataxin-7 on transcription driven by the co-activator CBP, the Purkinje cell-expressed nuclear receptor RORα1 or a basic TATA promoter. As previously shown for other polyglutamine disease proteins, expansion of the polyglutamine domain in ataxin-7 leads to reduced transcription. Surprisingly, strong repression of CBP-mediated, RORα1-mediated and basal transcription was also observed with wild-type ataxin-7, suggesting that the normal ataxin-7 protein may have a role in transcriptional regulation.