Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

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.

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references. Also available online through Digital Dissertations.

Master of Science in Medicine / Saccadic and vestibular-evoked eye movements are similar in that their three-dimensional kinematic properties show eye position-dependence. When the line of sight is directed towards an eccentric target, the eye velocity axis tilts in a manner that depends on the instantaneous position of the eye in the head, with the magnitude of tilt also depending on whether the eye movement is saccadic or vestibular-evoked. The mechanism responsible for producing eye velocity axis tilting phenomena is not well understood. Some authorities have suggested that muscle pulleys in the orbit are critical for implementing eye velocity axis tilting, while others have suggested that the cerebellum plays an important role. In the current study, three-dimensional eye and head rotation data were acquired, using the magnetic search coil technique, to confirm the presence of eye position-dependent eye velocity axis tilting during saccadic eye movements. Both normal humans and humans with cerebellar atrophy were studied. While the humans with cerebellar atrophy were noted to have abnormalities in the two-dimensional metrics and consistency of their saccadic eye movements, the eye position-dependent eye velocity axis tilts were similar to those observed in the normal subjects. A mathematical model of the human saccadic and vestibular systems was utilized to investigate the means by which these eye position-dependent properties may arise for both types of eye movement. The predictions of the saccadic model were compared with the saccadic data obtained in the current study, while the predictions of the vestibular model were compared with vestibular-evoked eye movement data obtained in a previous study. The results from the model simulations suggest that the muscle pulleys are responsible for bringing about eye position-dependent eye velocity axis tilting for both saccadic and vestibular-evoked eye movements, and that these phenomena are not centrally programmed.

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.

Master of Science in Medicine / Saccadic and vestibular-evoked eye movements are similar in that their three-dimensional kinematic properties show eye position-dependence. When the line of sight is directed towards an eccentric target, the eye velocity axis tilts in a manner that depends on the instantaneous position of the eye in the head, with the magnitude of tilt also depending on whether the eye movement is saccadic or vestibular-evoked. The mechanism responsible for producing eye velocity axis tilting phenomena is not well understood. Some authorities have suggested that muscle pulleys in the orbit are critical for implementing eye velocity axis tilting, while others have suggested that the cerebellum plays an important role. In the current study, three-dimensional eye and head rotation data were acquired, using the magnetic search coil technique, to confirm the presence of eye position-dependent eye velocity axis tilting during saccadic eye movements. Both normal humans and humans with cerebellar atrophy were studied. While the humans with cerebellar atrophy were noted to have abnormalities in the two-dimensional metrics and consistency of their saccadic eye movements, the eye position-dependent eye velocity axis tilts were similar to those observed in the normal subjects. A mathematical model of the human saccadic and vestibular systems was utilized to investigate the means by which these eye position-dependent properties may arise for both types of eye movement. The predictions of the saccadic model were compared with the saccadic data obtained in the current study, while the predictions of the vestibular model were compared with vestibular-evoked eye movement data obtained in a previous study. The results from the model simulations suggest that the muscle pulleys are responsible for bringing about eye position-dependent eye velocity axis tilting for both saccadic and vestibular-evoked eye movements, and that these phenomena are not centrally programmed.

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)