[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · May 2005 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Nov 2004 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: A common finding among the expanded polyglutamine disorders is intracellular protein aggregates. Although the precise role of these aggregates in the disease process is unclear, they are generally ubiquitinated, implicating the ubiquitin-proteasome pathway in neuronal degeneration. To investigate the mechanism of aggregate formation, we have developed a cell culture model to express huntingtin designed to have an altered degradation rate through the ubiquitin-dependent N-end rule pathway. We fused the first 171 amino acids of huntingtin, containing either a pathogenic or normal polyglutamine tract, to the enhanced green fluorescent protein (EGFP). The half-life of soluble huntingtin-EGFP was dependent on the degradation signal and the polyglutamine tract length. However, once huntingtin-EGFP with a pathogenic tract had aggregated, the protein was extremely stable. Huntingtin-EGFP with a pathogenic glutamine tract and a shorter half-life displayed a delayed onset of aggregate formation and was more toxic to transfected cells. These data suggest that rapid clearance through the ubiquitin-proteasome pathway slows aggregate formation, yet increases cellular toxicity. Polyglutamine-induced neurotoxicity may therefore be triggered by non-aggregated protein, and aggregate formation itself may be a cellular defense mechanism.
Full-text · Article · Jun 2004 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: Polyglutamine-induced neurodegeneration in transgenic mice carrying the spinocerebellar ataxia type 1 (SCA1) gene is modulated by subcellular distribution of ataxin-1 and by components of the protein folding/degradation machinery. Since phosphorylation is a prominent mechanism by which these processes are regulated, we examined phosphorylation of ataxin-1 and found that serine 776 (S776) was phosphorylated. Residue 776 appeared to affect cellular deposition of ataxin-1[82Q] in that ataxin-1[82Q]-A776 failed to form nuclear inclusions in tissue culture cells. The importance of S776 for polyglutamine-induced pathogenesis was examined by generating ataxin-1[82Q]-A776 transgenic mice. These mice expressed ataxin-1[82Q]-A776 within Purkinje cell nuclei, yet the ability of ataxin-1[82Q]-A776 to induce disease was substantially reduced. These studies demonstrate that polyglutamine tract expansion and localization of ataxin-1 to the nucleus of Purkinje cells are not sufficient to induce disease. We suggest that S776 of ataxin-1 also has a critical role in SCA1 pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Spinocerebellar ataxia type 1 (SCA1) is one of several neurological disorders caused by a CAG repeat expansion. In SCA1, this expansion produces an abnormally long polyglutamine tract in the protein ataxin-1. Mutant polyglutamine proteins accumulate in neurons, inducing neurodegeneration, but the mechanism underlying this accumulation has been unclear. We have discovered that the 14-3-3 protein, a multifunctional regulatory molecule, mediates the neurotoxicity of ataxin-1 by binding to and stabilizing ataxin-1, thereby slowing its normal degradation. The association of ataxin-1 with 14-3-3 is regulated by Akt phosphorylation, and in a Drosophila model of SCA1, both 14-3-3 and Akt modulate neurodegeneration. Our finding that phosphatidylinositol 3-kinase/Akt signaling and 14-3-3 cooperate to modulate the neurotoxicity of ataxin-1 provides insight into SCA1 pathogenesis and identifies potential targets for therapeutic intervention.
[Show abstract][Hide abstract] ABSTRACT: Biochemical signaling pathways are known to have a critical role in neuronal development and function. A growing body of evidence is accumulating to suggest that signaling pathways also underlie neurodegeneration and neurodegenerative disease. One pathway with a prominent role in neurodegenerative disease is the signaling pathway in which the enzyme glycogen synthase kinase 3 (GSK3) is a key component. In vitro and in vivo evidence point to a key role for GSK3 in promoting neurodegeneration and in Alzheimer's disease plaque and neurofibrillary tangle formation. How GSK3 acts in this regard is still open to debate, but it may involve both extracellular and nuclear apoptotic activities.
No preview · Article · Jul 2002 · Current Opinion in Neurobiology
[Show abstract][Hide abstract] ABSTRACT: Three papers published recently in Cell bring the power of human genetics, Drosophila genetics, and genomics to bear on the understanding of fragile X syndrome. They provide further support for the importance of local protein synthesis within a neuron as a determinant of proper synaptogenesis and the development of cognitive abilities.
[Show abstract][Hide abstract] ABSTRACT: Spinocerebellar ataxia type 7 (SCA7) belongs to a group of neurological disorders caused by a CAG repeat expansion in the
coding region of the associated gene. To gain insight into the pathogenesis of SCA7 and possible functions of ataxin-7, we
examined the subcellular localization of ataxin-7 in transfected COS-1 cells using SCA7 cDNA clones with different CAG repeat tract lengths. In addition to a diffuse distribution throughout the nucleus, ataxin-7
associated with the nuclear matrix and the nucleolus. The location of the putative SCA7nuclear localization sequence (NLS) was confirmed by fusing an ataxin-7 fragment with the normally cytoplasmic protein chicken
muscle pyruvate kinase. Mutation of this NLS prevented protein from entering the nucleus. Thus, expanded ataxin-7 may carry
out its pathogenic effects in the nucleus by altering a matrixassociated nuclear structure and/or by disrupting nucleolar
Full-text · Article · Oct 1999 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: Transgenic mice carrying the spinocerebellar ataxia type 1 (SCA1) gene, a polyglutamine neurodegenerative disorder, develop ataxia with ataxin-1 localized to aggregates within cerebellar Purkinje cells nuclei. To examine the importance of nuclear localization and aggregation in pathogenesis, mice expressing ataxin-1 with a mutated NLS were established. These mice did not develop disease, demonstrating that nuclear localization is critical for pathogenesis. In a second series of transgenic mice, ataxin-1 containing a deletion within the self-association region was expressed within Purkinje cells nuclei. These mice developed ataxia and Purkinje cell pathology similar to the original SCA1 mice. However, no evidence of nuclear ataxin-1 aggregates was found. Thus, although nuclear localization of ataxin-1 is necessary, nuclear aggregation of ataxin-1 is not required to initiate pathogenesis in transgenic mice.
[Show abstract][Hide abstract] ABSTRACT: Nucleotide repeat instability is associated with an increasing number of cancers and neurological disorders. The mechanisms
that govern repeat instability in these biological disorders are not well understood. To examine genetic aspects of repeat
instability we have introduced an expanded CAG trinucleotide repeat into transgenic mice. We have detected intergenerational
CAG repeat instability in transgenic mice only when the transgene was maternally transmitted. These intergenerational instabilities
increased in frequency and magnitude as the transgenic mother aged. Furthermore, triplet repeat variations were detected in
unfertilized oocytes and were comparable with those in the offspring. These data show that maternal repeat instability in
the transgenic mice occurs after meiotic DNA replication and prior to oocyte fertilization. Thus, these findings demonstrate
that advanced maternal age is an important factor for instability of nucleotide repeats in mammalian DNA.
Preview · Article · Dec 1997 · Human Molecular Genetics