Dentatorubral-pallidoluysian Atrophy Protein Research Articles

Search for target genes of transcriptional regulation by Dentatorubral-pallidoluysian atrophy protein that acts as a transcriptional co-regulator

Search for target genes of transcriptional regulation by Dentatorubral-pallidoluysian atrophy protein that acts as a transcriptional co-regulator

Increased aggregation of polyleucine compared with that of polyglutamine in dentatorubral-pallidoluysian atrophy protein

Polyglutamine (polyQ) diseases result from expansion of CAG trinucleotide repeats in their responsible genes. Although gene products with polyQ expansions undergo conformational changes to aggregate in neurons, the relationship between inclusions and neurotoxicity remains unclear. Dentatorubral-pallidoluysian atrophy (DRPLA) is a polyQ disease, and DRPLA protein, also known as atrophin-1 (ATN1), carries an expanded polyQ tract. To investigate how an expanded polyQ tract influences ATN1 aggregation and localization, we compared the aggregation of ATN1 with a polyQ tract to that of ATN1 with a polyleucine (polyL) tract. In COS-7 cells, polyL-ATN1 triggered more aggregation than polyQ-ATN1 of similar repeat sizes. Immunocytochemical and biochemical studies revealed that replacement of the polyQ tract with polyL alters ATN1 localization, leading to retention of polyL-ATN1 in the cytoplasm. Despite this change in localization, polyL-ATN1 and polyQ-ATN1 demonstrate comparable repeat length dependent toxicity. These results suggest that expanded polyQ repeats in ATN1 may contribute to neurodegeneration via alterations in both protein aggregation and intracellular localization.

DRPLA transgenic mouse substrains carrying single copy of full-length mutant human DRPLA gene with variable sizes of expanded CAG repeats exhibit CAG repeat length- and age-dependent changes in behavioral abnormalities and gene expression profiles

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant progressive neurodegenerative disorder with intellectual deterioration and various motor deficits including ataxia, choreoathetosis, and myoclonus, caused by an abnormal expansion of CAG repeats in the DRPLA gene. Longer expanded CAG repeats contribute to an earlier age of onset, faster progression, and more severe neurological symptoms in DRPLA patients. In this study, we have established DRPLA transgenic mouse lines (sublines) harboring a single copy of the full-length mutant human DRPLA gene carrying various lengths of expanded CAG repeats (Q76, Q96, Q113, and Q129), which have clearly shown motor deficits and memory disturbance whose severity increases with the length of expanded CAG repeats and age, and successfully replicated the CAG repeat length- and age-dependent features of DRPLA patients. Neuronal intranuclear accumulation of the mutant DRPLA protein has been suggested to cause transcriptional dysregulation, leading to alteration in gene expression and neuronal dysfunction. In this study, we have conducted a comprehensive analysis of gene expression profiles in the cerebrum and cerebellum of transgenic mouse lines at 4, 8, and 12weeks using multiple microarray platforms, and demonstrated that both the number and expression levels of the altered genes are highly dependent on CAG repeat length and age in both brain regions. Specific groups of genes and their function categories were identified by further agglomerative cluster analysis and gene functional annotation analysis. Calcium signaling and neuropeptide signaling, among others, were implicated in the pathophysiology of DRPLA. Our study provides unprecedented CAG-repeat-length-dependent mouse models of DRPLA, which are highly valuable not only for elucidating the CAG-repeat-length-dependent pathophysiology of DRPLA but also for developing therapeutic strategies for DRPLA.

FAT10 Protein Binds to Polyglutamine Proteins and Modulates Their Solubility

Expansion of polyglutamine (pQ) chain by expanded CAG repeat causes dominantly inherited neurodegeneration such as Huntington disease, dentatorubral-pallidoluysian atrophy (DRPLA), and numbers of other spinocerebellar ataxias. Expanded pQ disrupts the stability of the pQ-harboring protein and increases its susceptibility to aggregation. Aggregated pQ protein is recognized by the ubiquitin proteasome system, and the enzyme ubiquitin ligase covalently attaches ubiquitin, which serves as a degradation signal by the proteasome. However, accumulation of the aggregated proteins in the diseased brain suggests insufficient degradation machinery. Ubiquitin has several functionally related proteins that are similarly attached to target proteins through its C terminus glycine residue. They are called ubiquitin-like molecules, and some of them are similarly related to the protein degradation pathway. One of the ubiquitin-like molecules, FAT10, is known to accelerate protein degradation through a ubiquitin-independent manner, but its role in pQ aggregate degradation is completely unknown. Thus we investigated its role in a Huntington disease cellular model and found that FAT10 molecules were covalently attached to huntingtin through their C terminus glycine. FAT10 binds preferably to huntingtin with a short pQ chain and completely aggregated huntingtin was FAT10-negative. In addition, ataxin-1,3 and DRPLA proteins were both positive for FAT10, and aggregation enhancement was observed upon FAT10 knockdown. These findings were similar to those for huntingtin. Our new finding will provide a new role for FAT10 in the pathogenesis of polyglutamine diseases.

Interference with activity-dependent transcriptional activation of BDNF gene depending upon the expanded polyglutamines in neurons

Expanded polyglutamines (polyQ) have been demonstrated to impair the CREB-dependent transcription in established cell lines. Since activity-dependent transcription in neurons, which plays an important role in forming neuronal plasticity, is largely controlled by CREB, it is important to study whether polyQ interferes with the activity-dependent transcriptional activation of genes in neurons. In cultured rat cortical neurons, over-expression of truncated dentatorubral-pallidoluysian atrophy proteins containing expanded polyQ, which form aggregation bodies in nucleus, reduced the calcium (Ca 2+) signal-mediated transcriptional activation of brain-derived neurotrophic factor, c- fos, and pituitary adenylate cyclase-activating polypeptide gene promoters in a dose-dependent manner. The interference with the transcriptional activation was dependent upon the presence of polyQ, the strength of which was increased as the length of polyQ stretches was expanded. Thus, polyQ interferes with the activity-dependent transcription in a polyQ-length-dependent manner, which may correspond to the severity of polyglutamine diseases.

Humanin Attenuates Apoptosis Induced by DRPLA Proteins With Expanded Polyglutamine Stretches

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal-dominant neurodegenerative disorder caused by expansion of CAG repeats in the DRPLA gene, which codes for a polyglutamine (polyQ) stretch. The expanded polyQs are known to form intracellular aggregates and to confer neurotoxic activity. Recent studies have indicated that activation of apoptosis signal-regulating kinase 1 (ASK1) is involved in polyQ-induced apoptosis. Humanin (HN) is an endogenous peptide that inhibits neuronal cell death caused by mutant Alzheimer's disease genes, and this neuroprotective factor has recently been reported to suppress apoptosis by inhibiting activation of ASK1. To test the anti-ASK1 effect of HN on polyQ neurotoxicity, we constructed neuronal PC12 cells expressing expanded polyQs under the control of the Tet-Off system. Using this cell line, we showed that HN suppresses apoptotic cell death induced by expanded polyQs. However, the suppression was incomplete, suggesting that polyQs also stimulate other pathogenic cascades unrelated to ASK1. We further showed that HN suppresses polyQ aggregate formation. This result implied the possibility that aggregation is also related to the polyQ-mediated cascade involving ASK1 activation. Although the details remain uncertain, our results suggest that ASK1 is potentially involved in pathogenesis of DRPLA and that HN might contribute partially to the suppression of neurodegeneration in polyQ diseases.

Dentatorubral-pallidoluysian atrophy protein is phosphorylated by c-Jun NH2-terminal kinase.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominant-inherited neurodegenerative disease characterized by selective cell loss in particular neuronal pathways. This is caused by expansion of CAG repeats in the coding region of the DRPLA gene, and the extended polyglutamine tract (polyQ) confers a toxic activity. It is valuable to characterize disease gene products for elucidation of the mechanism underlying neuron death at specific anatomical areas of the brain. Here, we define the DRPLA protein as a phosphoprotein, and c-Jun NH(2)-terminal kinase (JNK) is one of the major factors involved in its phosphorylation. Endogenous DRPLA protein was serine-phosphorylated. Phosphorylation was demonstrated in a recombinant JNK activation system in vitro and also in overexpressing cells by transfection after the JNK activation with osmotic pressure. One of the phospho-acceptor sites for JNK appearing in the DRPLA sequence was indeed phosphorylated, which was confirmed by a specific antibody raised against the phosphopeptide. Kinetic studies in the JNK recombinant system showed that expanded polyQ slightly reduced the affinity of JNK to the protein. Thus, the abnormal DRPLA protein seems to be slowly phosphorylated in a certain condition of JNK activation in patients. It may delay a process that is essential in keeping neurons alive.

Expanded polyglutamine stretches form an ‘aggresome’

To understand the pathogenetic mechanisms underlying polyglutamine (polyQ) diseases, we investigated the mechanisms of the formation of aggregate bodies containing expanded polyQ stretches, focusing on dentatorubral-pallidoluysian atrophy (DRPLA). We demonstrated that the expression of a truncated DRPLA protein containing expanded polyQ stretches in COS-7 cells resulted in the formation of perinuclear aggregate bodies that are co-localized with γ-tubulin, a protein marker for the microtubules-organizing center (MTOC). A collapsed vimentin network surrounded these aggregate bodies. Furthermore, disruption of the microtubules (MTs) with nocodazole resulted in the formation of small aggregate bodies that were scattered throughout the cytoplasm. These findings suggest that the truncated DRPLA proteins containing expanded polyQ stretches unfold and form small aggregate bodies in the cell periphery. These aggregates move on MTs to the MTOC, where they remain as distinct ‘aggresomes’.

Distinctive Tissue Distribution and Phosphorylation of IRSp53 Isoforms

An insulin-receptor substrate of 53-kDa protein (IRSp53) is an adapter protein, which interacts with the Rho-family of GTPases and mediates neurite outgrowth. It also binds to DRPLA protein, a product of the gene responsible for a polyglutamine disease, dentatorubral-pallidoluysian atrophy (DRPLA). Isoforms of human IRSp53 have been reported, each with a unique amino acid sequence at the C-terminal end. Here we report the distinctive tissue distribution and phosphorylation of three isoforms (L, S, and T-forms). Western blotting analyses with isoform-specific antibodies demonstrated that the L and S-forms were expressed in the brain, whereas the T-form was not present in any tissues examined, but was found in a cancer cell line. The L and S-forms were phosphorylated upon stimulation with insulin, and the T-form with IGF-I. Since phospho-acceptor sites were localized to the common portion, the difference in phosphorylation seems to be due to the unique C-terminal sequence.

Amino acid sequences flanking polyglutamine stretches influence their potential for aggregate formation.

Expanded polyglutamine stretches have been shown to form aggregates and to be toxic to cells. In this study, we hypothesized that amino acid sequences flanking the polyglutamine stretches influence the aggregate formation potential of these stretches. Green fluorescent protein (GFP) fusion proteins containing glutamine repeats of various lengths and a fixed number of flanking amino acids of ataxin-2, huntingtin, dentatorubral-pallidoluysian atrophy protein (DRPLAP) or ataxin-3 were transiently expressed in COS-7 cells. The aggregate formation potential of ataxin-2 and DRPLAP increased in a CAG-repeat-length-dependent manner, with a threshold between 34 and 36. Truncated ataxin-2-Q56-GFP and truncated huntingtin-Q56-GFP showed a significantly higher aggregate formation potential than truncated DRPLAP-Q56-GFP or truncated ataxin-3-Q56-GFP. These results are in agreement with the clinical observation that ages of disease onset in patients with spinocerebellar ataxia type 2 or Huntington's disease are lower than those in patients with DRPLA or Machado-Joseph disease having expanded CAG repeats of the same length. Furthermore, mutagenesis of the flanking sequence of ataxin-2 markedly reduced its aggregate formation potential. These results indicate that the amino acid sequences flanking the polyglutamine stretches significantly influence their aggregate formation potential.

Aberrant phosphorylation of dentatorubral-pallidoluysian atrophy (DRPLA) protein complex in brain tissue

Dentatorubral-pallidoluysian atrophy (DRPLA) is caused by DRPLA protein which carries expansion of a glutamine repeat. Abnormal high-molecular-mass complex formation by DRPLA protein and its pathological ubiquitination comprise the disease processes in the brains of patients with DRPLA. In this study, DRPLA protein complex was isolated and shown to have pathologically stronger bond formation with DRPLA proteins in DRPLA brain tissue compared with control brain tissue. Immunochemical methods and an enzymic dephosphorylation technique were used to demonstrate that DRPLA protein complex is aberrantly phosphorylated in DRPLA brain tissue. Immunohistochemical studies show that both the ubiquitinated cytoplasmic inclusions and the nuclear membrane are aberrantly phosphorylated in DRPLA-affected neurons. This finding suggests that the nuclear membrane is another pathological focus of DRPLA neurodegeneration.

Protein binding of a DRPLA family through arginine-glutamic acid dipeptide repeats is enhanced by extended polyglutamine.

Dentatorubral-pallidoluysian atrophy (DRPLA) is one of the hereditary neurodegenerative disorders caused by expansion of CAG/glutamine repeats. To investigate the normal function of the DRPLA gene and the pathogenic mechanism of neuron death in specific areas of the brain, we isolated and analyzed a gene that shares a notable motif with DRPLA, arginine-glutamic acid (RE) dipeptide repeats. The gene isolated, designated RERE, has an open reading frame of 1566 amino acids, of which the C-terminal portion has 67% homology to DRPLA, whereas the N-terminal portion is distinctive. RERE also contains arginine-aspartic acid (RD) dipeptide repeats and putative nuclear localization signal sequences, but no polyglutamine tracts. RERE is expressed at a low level in most tissues examined. Immunoprecipitation and in vitro binding assays demonstrate that the DRPLA and RERE proteins bind each other, for which one of the RE repeats has a primary role, and extended polyglutamine enhances the binding. With engineered constructs fused with a tag, the RERE protein localized predominantly in the nucleus. Moreover, when RERE is overexpressed, the distribution of endogenous DRPLA protein alters from the diffused to the speckled pattern in the nucleus so as to co-localize with RERE. More RERE protein is recruited into nuclear aggregates of the DRPLA protein with extended polyglutamine than into those of pure polyglutamine. These results reveal a function for the DRPLA protein in the nucleus and the RE repeat in the protein-protein interaction.

Unique Characteristics of Ubiquitin-Bonded Complex Play a Pathological Role in Dentatorubral–Pallidoluysian Atrophy

Abnormal complex formation of dentatorubral–pallidoluysian atrophy (DRPLA) protein and pathological ubiquitination of abnormal complex are two pathological processes involved in DRPLA neurodegeneration. Pathological ubiquitination and solubility in SDS and reducing agent are two unique characteristics of the DRPLA protein complex. Ubiquitination of abnormal DRPLA protein complex in DRPLA brain tissue is heat-resistant and stronger than that in control brain tissue. Pathological ubiquitination of DRPLA protein complex correlates with the onset of symptoms and the size of an expanded glutamine repeat in brain tissue of patients with DRPLA. Pathological ubiquitination plays an important role in DRPLA pathology. DRPLA protein complex is water-insoluble but soluble in SDS and reducing agent, and displays no difference in water insolubility between control and DRPLA brain tissue. Abnormal insoluble complex formation is not developed by a qualitative change in water insolubility of DRPLA protein complex but is developed by a spontaneous accumulation of an abnormally large amount of the DRPLA protein complex.

Abnormal Dentatorubral-Pallidoluysian Atrophy (DRPLA) Protein Complex Is Pathologically Ubiquitinated in DRPLA Brains

Dentatorubral-pallidoluysian atrophy (DRPLA) is caused by expansion of a glutamine repeat in DRPLA protein. DRPLA protein undergoes greater complex formation in DRPLA brain tissue, and expanded glutamine repeat enhances complex formation of DRPLA protein. Immunoblots with and without reduction show that the DRPLA protein complex is ubiquitinated only in DRPLA brain tissue. Moreover, immunoblots of regional DRPLA brain tissues reveal that pathological ubiquitination of DRPLA protein complex is found selectively in affected lesions. Double-labeling immunohistochemical studies with antibodies against DRPLA protein and ubiquitin demonstrate that the DRPLA protein is co-localized with ubiquitin in DRPLA neurons and show characteristic neuronal cytoplasmic inclusions with ubiquitinated DRPLA protein complex in the center. Our findings suggest that DRPLA protein undergoes abnormal complex formation with expanded glutamine repeat, and then the complex is pathologically ubiquitinated in DRPLA brain tissue. Pathological ubiquitination of abnormal DRPLA protein complex plays a role in DRPLA pathology.

Adenovirus-mediated expression of mutant DRPLA proteins with expanded polyglutamine stretches in neuronally differentiated PC12 cells. Preferential intranuclear aggregate formation and apoptosis.

To investigate the molecular mechanisms of neurodegeneration caused by expanded CAG repeats in dentatorubral-pallidoluysian atrophy (DRPLA), an autosomal dominant neuro degrees enerative disorder caused by unstable expansion of a CAG trinucleotide repeat in the DRPLA gene on 12p13.31, we established an efficient expression system for truncated and full-length DRPLA proteins with normal or expanded polyglutamine stretches in neuronally differentiated PC12 cells and fibroblasts using an adenovirus expression system. Although aggregate body formation was observed both in neuronally differentiated PC12 cells and in fibroblasts expressing truncated DRPLA proteins with Q82, >97% ( n = 3) of neuronally differentiated PC12 cells showed intra-nuclear inclusions, while only 31 21% ( n = 3) of fibro-blasts had intranuclear inclusions at 3 days after infection. The percentage of apoptotic cells was significantly higher in neuronally differentiated PC12 cells expressing the truncated DRPLA protein with Q82 than in fibroblasts, suggesting the possibility that intranuclear aggregate bodies are formed preferentially in neuronally differentiated PC12 cells and that these cells are more vulnerable than fibroblasts to the toxic effects of expanded polyglutamine stretches in the DRPLA protein. When the full-length DRPLA protein with Q82 was expressed, aggregate bodies were found exclusively in the nuclei of the neuronally differentiated PC12 cells, while they were found in the cytoplasm of fibroblasts. Despite the presence of aggregate bodies, apoptosis was not induced by expression of the full-length DRPLA protein with Q82 in either neuronally differentiated PC12 cells or fibroblasts, suggesting that the presence of intranuclear aggregate bodies is in itself not necessarily toxic to cells.

Dentatorubral-pallidoluysian atrophy protein interacts through a proline-rich region near polyglutamine with the SH3 domain of an insulin receptor tyrosine kinase substrate.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neuro degrees enerative disorder associated with CAG/glutamine repeat expansion. While the DRPLA gene is ubiquitously expressed, neuron death occurs in specific anatomical areas of the brain. This predicts that the DRPLA protein interacts with other proteins and that these interactions may play a role in pathogenesis. Here, we describe a protein that binds to the DRPLA product. One of the clones isolated with a yeast two-hybrid system was identified as a human homolog of the insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53). The gene produced two mRNA forms by differential splicing and encoded 552 and 521 amino acids, respectively. The longer form was mainly expressed in the brain and the shorter one in other tissues. The products were phosphorylated upon stimulation of cultured cells with insulin or insulin-like growth factor 1. Binding of the DRPLA protein to IRSp53 was ascertained by co-immunoprecipitation with antibodies and also by co-localization in perinuclear oval dots in cells expressing engineered constructs. A proline-rich region near the polyglutamine tract of the DRPLA protein and the SH3 domain of IRSp53 were involved in the binding. An extended polyglutamine tract significantly reduced binding ability in yeast cells, but not in in vitro binding assays. The identification of IRSp53 and other proteins detected by the yeast hybrid system predicts that DRPLA functions in a signal transduction pathway coupled with insulin/IGF-1.

Hereditary dentatorubral-pallidoluysian atrophy: detection of widespread ubiquitinated neuronal and glial intranuclear inclusions in the brain.

We examined the brains and spinal cords of seven patients with clinicopathologically and genetically confirmed hereditary dentatorubral-pallidoluysian atrophy (DRPLA) using an antibody against ubiquitin, and found small, round immunoreactive intranuclear inclusions in both neurons and glial cells in various brain regions. Ubiquitinated neuronal intranuclear inclusions (uNIIs) were consistently found in the striatum, the pontine nuclei, the inferior olivary complex, the cerebellar cortex and the dentate nucleus. Ubiquitinated glial intranuclear inclusions (uGIIs) were found less frequently than uNIIs. Most of the inclusion-bearing nuclei were of an astrocytic nature. Immunostaining with an antibody against DRPLA protein revealed similar immunoreactive neuronal and glial intranuclear inclusions, but in much smaller in numbers compared with uNIIs and uGIIs. Electron microscopy showed that such inclusions were composed of granular and filamentous structures. These findings strongly suggest that, in DRPLA, the occurrence of uNIIs and uGIIs is directly related to the causative gene abnormality (an expanded CAG repeat encoding polyglutamine), that neurons are affected much more widely than previously recognized and that glial cells are also involved in the disease process.

Different Complex Formations of Dentatorubral-Pallidoluysian Atrophy (DRPLA) Protein in Human and Rat Neurons

Dentatorubral-pallidoluysian atrophy (DRPLA) is caused by expansion of a glutamine repeat in DRPLA protein. Rat DRPLA protein, homologous in sequence to human DRPLA protein, was identified in rat brains. Immunoblots of human control and rat brain tissues with and without reduction show that human DRPLA protein forms more disulfide-bond complexes than rat DRPLA protein. An immunohistochemical study and a subcellular fractionation experiment show that human DRPLA protein produces complexes within the cytoplasm of neurons, whereas rat DRPLA protein rarely does. In spite of the close homology of their amino acid sequences, human and rat DRPLA protein have different protein characters. The differences in these characters suggest structural differences and may be related to these proteins functions.

Expanded Glutamine Repeat Enhances Complex Formation of Dentatorubral-Pallidoluysian Atrophy (DRPLA) Protein in Human Brains

The genetic defect in dentatorubral-pallidoluysian atrophy (DRPLA) is expansion of the CAG repeat. The mutant gene is translated into the protein which carries the expanded glutamine repeat. Immunoblots of human brain tissues with and without reduction show that the DRPLA protein is a disulfide-bond complex and that more of this complex is formed in DRPLA brains than in control brains. This suggests that DRPLA protein undergoes greater complex formation in DRPLA brains and the expanded glutamine repeat may enhance complex formation of untruncated DRPLA protein in DRPLA brains. Immunohistochemical findings show that DRPLA protein is localized in the cytoplasm of the neuron, evidence that it undergoes rare disulfide bonding there.

Intracellular Aggregate Formation of Dentatorubral-Pallidoluysian Atrophy (DRPLA) Protein with the Extended Polyglutamine

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder caused by the abnormal CAG triplet-repeat expansion resulting in an elongated polyglutamine (polyQ) stretch. We have recently showed that the DRPLA protein is cleaved during apoptosis by caspase-3, one of the cysteine protease family members known to be activated during apoptosis. We report here the subcellular localization of the DRPLA protein by fusing the green fluorescent protein as a tag. The full length DRPLA protein is localized predominantly but not exclusively in the nucleus regardless of the length of the polyQ stretch. In contrast, an N-terminal-deleted fragment containing polyQ produced by the proteolytic cleavage with caspase-3 is found both in the nucleus and the cytoplasm. Moreover, the same fragment with the elongated polyQ showed aggregation when overexpressed. Some cells with aggregate formation showed apoptotic phenotype. These findings raise the possibility that the DRPLA protein processed by caspase-3 may lead to aggregation of the protein resulting in the development of neurodegeneration.