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Harry T Orr

Publications and source records attributed to Harry T Orr.

At least 19 recordsLinked to original sources
PubMed2006

ATAXIN-1 interacts with the repressor Capicua in its native complex to cause SCA1 neuropathology.

Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein, in this case, ATAXIN-1 (ATXN1). A key question in the field is whether neurotoxicity is mediated by aberrant, novel interactions with the expanded protein or whether its wild-type functions are augmented to a deleterious degree. We examined soluble protein complexes from mouse cerebellum and found that the majority of wild-type and expanded ATXN1 assembles into large stable complexes containing the transcriptional repressor Capicua. ATXN1 directly binds Capicua and modulates Capicua repressor activity in Drosophila and mammalian cells, and its loss decreases the steady-state level of Capicua. Interestingly, the S776A mutation, which abrogates the neurotoxicity of expanded ATXN1, substantially reduces the association of mutant ATXN1 with Capicua in vivo. These data provide insight into the function of ATXN1 and suggest that SCA1 neuropathology depends on native, not novel, protein interactions.

Amino Acid Sequence↗
PubMed2006

RORalpha-mediated Purkinje cell development determines disease severity in adult SCA1 mice.

Spinocerebellar ataxia type 1 (SCA1) is one of nine inherited, typically adult onset, polyglutamine neurodegenerative diseases. To examine whether development impacts SCA1, we used a conditional transgenic mouse model of SCA1 to delay the postnatal expression of mutant ATXN1 until after completion of cerebellar development. Delayed postnatal expression of mutant ATXN1 led to a substantial reduction in severity of disease in adults in comparison with early postnatal gene expression. This was linked to a destabilization of RORalpha, a transcription factor critical for cerebellar development. In SCA1 mice, there was a depletion of RORalpha and a reduction in expression of genes controlled by RORalpha. Partial loss of RORalpha enhanced mutant ATXN1 pathogenicity. Additionally, evidence points to the existence of a complex containing ATXN1, RORalpha, and the RORalpha coactivator Tip60. These studies indicate RORalpha and Tip60 have a role in SCA1 and suggest a mechanism by which compromising cerebellar development contributes to severity of neurodegeneration in an adult.

Animals↗
PubMed2006

Targeted deletion of a single Sca8 ataxia locus allele in mice causes abnormal gait, progressive loss of motor coordination, and Purkinje cell dendritic deficits.

Spinocerebellar ataxia type 8 (SCA8) patients typically have a slowly progressive, adult-onset ataxia. SCA8 is dominantly inherited and is caused by large CTG repeat expansions in the untranslated antisense RNA of the Kelch-like 1 gene (KLHL1), but the molecular mechanism through which this expansion leads to disease is still unknown. To more fully characterize the underlying molecular mechanisms involved in SCA8, we developed a mouse model in which Klhl1 is deleted in either all tissues or is deleted specifically in Purkinje cells only. We found that mice that are either homozygous or heterozygous for the Klhl1 deletion have significant gait abnormalities at an early age and develop a significant loss of motor coordination by 24 weeks of age. This loss progresses more rapidly in homozygous knock-outs. Mice with Klhl1 specifically deleted in only Purkinje cells had a loss of motor coordination that was almost identical to the total-tissue deletion mice. Finally, we found significant Purkinje cell dendritic deficits, as measured by the thickness of the molecular layer, in all mice in which Klhl1 was deleted (both total and Purkinje cell-specific deletions) and an intermediate reduction in molecular layer thickness in mice with reduced levels of Klhl1 expression (heterozygous deletions). The results from this mouse model show that even a partial loss of Klhl1 function leads to degeneration of Purkinje cell function and indicates that loss of KLHL1 activity is likely to play a significant part in the underlying pathophysiology of SCA8.

Alleles↗
PubMed2006

Polyglutamine neurodegenerative diseases and regulation of transcription: assembling the puzzle.

The polyglutamine disorders are a class of nine neuro-degenerative disorders that are inherited gain-of-function diseases caused by expansion of a translated CAG repeat. Even though the disease-causing proteins are widely expressed, specific collections of neurons are more susceptible in each disease, resulting in characteristic patterns of pathology and clinical symptoms. One hypothesis poses that altered protein function is fundamental to pathogenesis, with protein context of the expanded polyglutamine having key roles in disease-specific processes. This review will focus on the role of the disease-causing polyglutamine proteins in gene transcription and the extent to which the mutant proteins induce disruption of transcription.

Animals↗
PubMed2005

The AXH domain of Ataxin-1 mediates neurodegeneration through its interaction with Gfi-1/Senseless proteins.

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by an expanded glutamine tract in human Ataxin-1 (hAtx-1). The expansion stabilizes hAtx-1, leading to its accumulation. To understand how stabilized hAtx-1 induces selective neuronal degeneration, we studied Drosophila Atx-1 (dAtx-1), which has a conserved AXH domain but lacks a polyglutamine tract. Overexpression of hAtx-1 in fruit flies produces phenotypes similar to those of dAtx-1 but different from the polyglutamine peptide alone. We show that the Drosophila and mammalian transcription factors Senseless/Gfi-1 interact with Atx-1's AXH domain. In flies, overexpression of Atx-1 inhibits sensory-organ development by decreasing Senseless protein. Similarly, overexpression of wild-type and glutamine-expanded hAtx-1 reduces Gfi-1 levels in Purkinje cells. Deletion of the AXH domain abolishes the effects of glutamine-expanded hAtx-1 on Senseless/Gfi-1. Interestingly, loss of Gfi-1 mimics SCA1 phenotypes in Purkinje cells. These results indicate that the Atx-1/Gfi-1 interaction contributes to the selective Purkinje cell degeneration in SCA1.

Animals↗
PubMed2005

SUMOylation of the polyglutamine repeat protein, ataxin-1, is dependent on a functional nuclear localization signal.

SUMO (small ubiquitin-like modifier) is a member of the ubiquitin family of proteins. SUMO targets include proteins involved in numerous roles including nuclear transport and transcriptional regulation. The previous finding that mutant ataxin-1[82Q] disrupted promyelocytic leukemia (PML) oncogenic domains prompted us to determine whether ataxin-1 disrupts another component of PML oncogenic domains, Sp100 (100-kDa Speckled protein). Similar to the PML protein, mutant ataxin-1[82Q] redistributed Sp100 to mutant ataxin-1[82Q] nuclear inclusions. Based on the ability of PML and Sp100 to be covalently modified by SUMO, we investigated the ability of ataxin-1 to be SUMOylated. SUMO-1 was found to covalently modify the polyglutamine repeat protein ataxin-1. There was a decrease in ataxin-1 SUMOylation in the presence of the expanded polyglutamine tract, ataxin-1[82Q]. The phospho-mutant, ataxin-1[82Q]-S776A, restored SUMO levels to those of wild-type ataxin-1[30Q]. SUMOylation of ataxin-1 was dependent on a functional nuclear localization signal. Ataxin-1 SUMOylation was mapped to at least five lysine residues. Lys(16), Lys(194) preceding the polyglutamine tract, Lys(610)/Lys(697) in the C-terminal ataxin high mobility group domain, and Lys(746) all contribute to ataxin-1 SUMOylation.

Active Transport, Cell Nucleus↗
PubMed2005

A cell-based screen for modulators of ataxin-1 phosphorylation.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of a glutamine repeat within the SCA1-encoded protein ataxin-1. We have previously shown that serine 776 (S776) of both wild-type and mutant ataxin-1 is phosphorylated in vivo and in vitro. Moreover, preventing phosphorylation of this residue by replacing it with alanine resulted in a mutant protein, which was not pathogenic in spite of its nuclear localization. To further investigate pathways leading to S776 phosphorylation of ataxin-1, we developed a cell-culture based assay to screen for modulators of S776 phosphorylation. In this assay, ataxin-1 expression was monitored by enhanced green fluorescent protein (EGFP) fluorescence in cell lines stably expressing EGFP-ataxin-1 fusion protein. The phospho-S776 ataxin-1 specific antibody (PN1168) was used to assess ataxin-1 S776 phosphorylation. A library of 84 known kinase and phosphatase inhibitors was screened. Analysis of the list of drugs that modified S776 phosphorylation places many of the inhibited kinases into known cell signaling pathways. A pathway associated with calcium signaling resulted in phosphorylation of both wild-type and mutant ataxin-1. Interestingly, inhibitors of the PI3K/Akt pathway predominantly diminished mutant ataxin-1 phosphorylation. These results provide new molecular tools to aid in elucidating the biological role of ataxin-1 phosphorylation and perhaps provide potential leads toward the development of a therapy for SCA1.

Animals↗
PubMed2005

RNA association and nucleocytoplasmic shuttling by ataxin-1.

Spinocerebellar ataxia type 1 (SCA1) is a dominant neurodegenerative disease caused by the expression of mutant ataxin-1 containing an expanded polyglutamine tract. Ataxin-1 is a nuclear protein that localizes to punctate inclusions similar to neuronal nuclear inclusions seen in many polyglutamine expansion disease proteins. We demonstrate that ataxin-1 localization to inclusions and inclusion dynamics within the nucleus are RNA and transcription dependent, but not dependent on the polyglutamine tract. Ataxin-1 nuclear inclusions are distinct from other described nuclear bodies but recruit the mRNA export factor, TAP/NXF1, in a manner that is enhanced by cell heat shock. By FRAP protein dynamic studies in live cells, we found that wild-type, but not mutant, ataxin-1 was capable of nuclear export. These results suggest that the normal role of ataxin-1 may be in RNA processing, perhaps nuclear RNA export. Thus, nuclear retention of mutant ataxin-1 may be an important toxic gain of function in SCA1 disease.

Animals↗
PubMed2004

Identification of a novel phosphorylation site in ataxin-1.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease resulting from an expanded CAG repeat in the SCA1 gene that leads to an expanded polyglutamine tract in the gene product. Previous studies have demonstrated that serine at site 776 is phosphorylated [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Studies of ataxin-1 S776 and serine mutated to an alanine, A776, have also shown differential protein-protein interactions and reduced neurodegeneration [H.K. Chen, P. Fernandez-Funez, S.F. Acevedo, Y.C. Lam, M.D. Kaytor, M.H. Fernandez, A. Aitken, E.M. Skoulakis, H.T. Orr, J. Botas, H.Y. Zoghbi, Interaction of Akt_phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1.]. However, mutation of the site serine 776 to an alanine did not abolish all phosphorylation of the protein ataxin-1, suggesting the presence of additional phosphorylation sites [E.S. Emiamian, M.D. Kaytor, L.A. Duvick, T. Zu, S.K. Tousey, H.Y. Zoghbi, H.B. Clark, H.T. Orr, Serine 776 of ataxin-1 is critical for polyglutamine-induced disease in SCA1 transgenic mice, Neuron 38 (2003) 375-387.]. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and mutational analysis demonstrated a novel phosphorylation site at serine 239 of ataxin-1.

Amino Acid Sequence↗
PubMed2004

Recovery from polyglutamine-induced neurodegeneration in conditional SCA1 transgenic mice.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant, polyglutamine-induced neurodegenerative disorder that results in loss of motor coordination caused primarily by a disruption of cerebellar Purkinje cell function. In this study, we developed a conditional SCA1 mouse model to examine whether stopping expression of mutant ataxin-1 alters the disease phenotype. After cessation of SCA1[82Q] transgene expression, mutant ataxin-1, including that in nuclear inclusions, was cleared rapidly from Purkinje cells. At an early stage of disease, Purkinje cell pathology and motor dysfunction were completely reversible. After halting SCA1 expression at later stages of disease, only a partial recovery was seen. Interestingly, restoration of the ability to perform a complex motor task, the accelerating Rotarod, correlated with localization of mGluR1alpha to the Purkinje cell-parallel fiber synapse. These results show that the progression of SCA1 pathogenesis is dependent on the continuous expression of mutant ataxin-1. Of note, even at a late stage of disease, Purkinje cells retain at least some ability to repair the damage caused by mutant ataxin-1.

Animals↗
PubMed2004

Gene profiling links SCA1 pathophysiology to glutamate signaling in Purkinje cells of transgenic mice.

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by the expansion of a polyglutamine repeat within the disease protein, ataxin 1. To elucidate cellular pathways involved in SCA1, we used DNA microarrays to determine the pattern of gene expression in SCA1 transgenic mice at two specific times in the disease process; 5 weeks, a timepoint prior to onset of pathology, and 12 weeks, at the midpoint of the disease progression. Taking advantage of the availability of three SCA1 transgenic mouse lines, each expressing a different form of ataxin-1, we utilized a strategy that resulted in the identification of a limited number of genes with an altered pattern of expression specific to the development of disease. By comparing the pattern of gene expression in the SCA1 ataxic B05-ataxin-1[82Q] transgenic mouse line with those seen in two non-ataxic lines, A02-ataxin-1[30Q] and K772T-[82Q], nine genes were identified whose expression was consistently altered in the cerebellum of B05[82Q] mice at 5 and 12 weeks of age. Interestingly, five of the genes in this group form a biological cohort centered on glutamate signaling pathways in Purkinje cells.

Animals↗
PubMed2004

The effects of the polyglutamine repeat protein ataxin-1 on the UbL-UBA protein A1Up.

The ataxin-1 interacting ubiquitin-like protein (A1Up) contains an amino-terminal ubiquitin-like (UbL) region, four stress-inducible, heat shock chaperonin-binding motifs (STI1), and an ubiquitin-associated domain (UBA) at the carboxyl terminus of A1Up. Although proteins that have both an UbL and UBA domain are thought to play a crucial role in proteasome-mediated activities, few are characterized, except for hHR23A/B. Similar to other UbL-containing proteins, the UbL of A1Up is essential for the interaction of A1Up with the S5a subunit of the 19S proteasome. Importantly, the interaction with the 19S proteasome was disrupted in the presence of the polyglutamine repeat protein, ataxin-1. The UbL domain of A1Up is ubiquitinated by both Lys(48)-linked and Lys(63)-linked chains. Intact A1Up is stable, suggesting that ubiquitination of A1Up is important for degradation-independent targeting of A1Up to the 19S proteasome. The UBA domain of A1Up binds polyubiquitin chains and has a role in the stability of A1Up and in the subcellular localization of A1Up. When the UBA domain was deleted, the localization of A1Up was entirely cytoplasmic, and it co-localized with the proteasome. Interestingly, the interaction between A1Up and mutant ataxin-1-(82Q) increased the half-life of A1Up, whereas nonpathogenic wild-type ataxin-1-(30Q) or ataxin-1-(82Q)-A776 did not.

Animals↗
PubMed2004

RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia.

The dominant polyglutamine expansion diseases, which include spinocerebellar ataxia type 1 (SCA1) and Huntington disease, are progressive, untreatable, neurodegenerative disorders. In inducible mouse models of SCA1 and Huntington disease, repression of mutant allele expression improves disease phenotypes. Thus, therapies designed to inhibit expression of the mutant gene would be beneficial. Here we evaluate the ability of RNA interference (RNAi) to inhibit polyglutamine-induced neurodegeneration caused by mutant ataxin-1 in a mouse model of SCA1. Upon intracerebellar injection, recombinant adeno-associated virus (AAV) vectors expressing short hairpin RNAs profoundly improved motor coordination, restored cerebellar morphology and resolved characteristic ataxin-1 inclusions in Purkinje cells of SCA1 mice. Our data demonstrate in vivo the potential use of RNAi as therapy for dominant neurodegenerative disease.

Adenoviridae↗
PubMed2004

Overexpression of CREB reduces CRE-mediated transcription: behavioral and cellular analyses in transgenic mice.

The CREB transcription factor mediates neuronal plasticity in many systems, but the relationship between CREB levels and CRE-mediated transcription in individual neurons in vivo is unclear. In FVB/N nontransgenic mice, we observed that Purkinje cells showed low basal levels of Ser(133)-phosphorylated CREB protein yet displayed strong CRE-directed transcription. Transgenic mice overexpressing CREB in Purkinje cells and dentate gyrus granule cells showed a decreased CRE-lacZ signal in the same cells, indicating repression of ATF/CREB family function. Dentate region long-term potentiation was not altered by these changes in CREB expression. CREB transgenic mice demonstrated an inability to perform the rotarod task, without signs of overt ataxia. Our results demonstrate that the level of phosphorylated CREB protein is not a reliable indicator of CRE-mediated function. Furthermore, we conclude that CRE-mediated transcription may be linked to only a subset of cerebellum-mediated motor behaviors and may not be universally required for long-lasting synaptic potentiation.

Activating Transcription Factors↗
PubMed2004

RNA gains a new function: a mediator of neurodegeneration.

Over the past couple of years, it has become evident that some males carrying the fragile X mental retardation (FMR1) premutation, typically viewed to be normal, in fact manifest a neurodegenerative disease with ataxia and tremor. FMR1 premutation alleles uniquely produce FMR1 transcripts with an elongated CGG repeat, leading to the hypothesis that premutant transcripts cause the neurodegenerative disease in carriers. Recently Jin et al. demonstrated, in Drosophila, that FMR1 premutation RNA causes neurodegeneration. These data show RNA can induce neurodegeneration and provide strong evidence that FMR1 RNA mediates the neurodegeneration in human premutation carriers.

Animals↗
PubMed2004

Generation and characterization of LANP/pp32 null mice.

The leucine-rich acidic nuclear protein (LANP) belongs to a family of evolutionarily conserved proteins that are characterized by an amino-terminal domain rich in leucine residues followed by a carboxy-terminal acidic tail. LANP has been implicated in the regulation of a variety of cellular processes including RNA transport, transcription, apoptosis, vesicular trafficking, and intracellular signaling. Abundantly expressed in the developing cerebellum, this protein has also been hypothesized to play a role in cerebellar morphogenesis. LANP has been implicated in disease biology as well, both as a mediator of toxicity in spinocerebellar ataxia type 1 and as a tumor suppressor in cancers of the breast and prostate. To better understand the function of this multifaceted protein, we have generated mice lacking LANP. Surprisingly, these mice are viable and fertile. In addition we could not discern any derangements in any of the major organ systems, including the nervous system, which we have studied in detail. Overall our results point to a functional redundancy of LANP's function, most likely provided by its closely related family members.

Animals↗