Browsing by Subject "Polyglutamine"

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    The role of gene expression and aging in SCA1
    (2013-12) Ingram, Melissa Anne Cornwell
    Spinocerebellar ataxia type 1 is an autosomal dominant disorder caused by a CAG repeat expansion encoding a polyglutamine tract, where patients present with a lack of motor coordination including ataxia. The disease is characterized pathologically by loss of Purkinje cells (PCs) in the cerebellar cortex and neuronal loss in brain stem nuclei and cerebellar dentate nuclei. Besides expansion of the polyglutamine tract, other features of ATXN1 are critical for pathogenesis and severity of disease including phosphorylation of ATXN1 at S776. Mouse models expressing an expanded form of ATXN1, ATXN1[82Q] share initial disease features, such as ataxia, with mice with a normal repeat length with a phosophomimetic aspartic acid substitution, ATXN1[30Q]-D776. However, ATXN1[30Q]-D776 mice do not display late-stage, progressive disease features including PC death. In order to determine molecular pathways leading to the ataxic similarities as well as progressive disease differences in the models, I used RNA-sequencing to examine the cerebellar transcriptome. In addition to identifying candidate genes, Col18a1 and Cck, involved in PC - climbing fiber dynamics, I found splicing correlates with ataxia in the ATXN1[82Q] and ATXN1[30Q]-D776 mice. In order to validate RNA-seq targets and focus on the more biologically relevant splicing candidates, I developed and analyzed RNA-seq from a conditional ATXN1[30Q]-D776 model. In addition, I used this model to examine disease recovery. Interestingly, I found a lack of recovery at older ages without PC death, suggesting older neurons are inherently less able to recover.
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    Unexpected repeat associated proteins
    (2011-02) Gibbens, Brian Ballard
    Spinocerebellar ataxia type 8 (SCA8) is one of a number of dominantly inherited disorders caused by triplet CTG*CAG repeat expansions (2). While investigating the mechanisms of SCA8, Dr. Ranum's lab made the surprising discovery that CAG*CTG expansion constructs express homopolymeric polyglutamine, polyalanine and polyserine expansion proteins without an ATG start codon (3). This repeat associated non-ATG (RAN) translation occurs in transfected cells and lenti-viral transduced cells and brains. Additionally, in vivo mouse and human data demonstrate that RAN-translation across human SCA8 and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins (3). RAN-translation can occur across CAG expansions in a number of different sequence contexts, but the mechanism of this newly discovered phenomenon, which does not follow the previously described rules of translational regulation, is completely unknown. To gain a better understanding of the mechanisms of RAN-translation and their potential role in microsatellite disorders, I chose to test the hypothesis that RNA sequence variations within and outside of the repeat affect the efficiency of RAN-translation. Data described in my thesis support the following conclusions: 1) the efficiency of RAN-translation in different frames can be positively and negatively affected by the nucleotide sequence within and around the repeat tract; 2) non-ATG translation in rabbit reticulocyte lysates (RRLs) is much less permissive than in HEK293T and N2a cells, initiates with methionine, and requires close cognate start codons (e.g. ATT and ATC); 3) RAN-translation in multiple frames can occur in the presence or absence of an ATG-initiated open reading frame; 4) cellular factors found in HEK293T and N2a cells substantially enhance RAN-translation compared to cell free RRLs; 5) RAN-translation is enhanced across repeat motifs which form hairpin structures. These data have led me to propose two models for RAN-translation: the "stalling model" and the "IRES-like model". The stalling model proposes that scanning ribosomes are stalled by repeat-containing hairpins until translation is initiated and that permissive initiation is increased when hairpins or RNA binding proteins interact with the ribosome and its associated translation initiation factors. The IRES-like model proposes that repeat containing hairpins facilitate RAN-translation by mimicking an internal ribosome entry site (IRES) to recruit the ribosome and initiation factors and initiate translation at non-ATG sites.