Fourteen and counting: unraveling trinucleotide repeat diseases.

The pathological expansion of unstable trinucleotide repeats currently is known to cause 14 neurological diseases. Over the past several years, researchers have concentrated on the challenging task of identifying the mechanism by which the expanded trinucleotide repeat leads to abnormal cellular function. As a consequence, the trinucleotide repeat field has grown dramatically since the initial discovery of dynamic mutations less than a decade ago. Trinucleotide repeat expansions may prove to cause pathology through a variety of mechanisms including interference with DNA structure, transcription, RNA-protein interaction and altered protein conformations/interactions. The goal of this review is to provide a brief description of the genes harboring expanded repeats, coupled with new insights into the molecular pathways most likely to be disrupted by these expansions. Data from studies of patient material, cell culture and animal models demonstrate the complexity of the pathogenic mechanisms in each of the diseases.

Trinucleotide repeats: mechanisms and pathophysiology.

Within the closing decade of the twentieth century, 14 neurological disorders were shown to result from the expansion of unstable trinucleotide repeats, establishing this once unique mutational mechanism as the basis of an expanding class of diseases. Trinucleotide repeat diseases can be categorized into two subclasses based on the location of the trinucleotide repeats: diseases involving noncoding repeats (untranslated sequences) and diseases involving repeats within coding sequences (exonic). The large body of knowledge accumulating in this fast moving field has provided exciting clues and inspired many unresolved questions about the pathogenesis of diseases caused by expanded trinucleotide repeats. This review summarizes the current understanding of the molecular pathology of each of these diseases, starting with a clinical picture followed by a focused description of the disease genes, the proteins involved, and the studies that have lent insight into their pathophysiology.

Progress in pathogenesis studies of spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited disorder characterized by progressive loss of coordination, motor impairment and the degeneration of cerebellar Purkinje cells, spinocerebellar tracts and brainstem nuclei. Many dominantly inherited neurodegenerative diseases share the mutational basis of SCA1: the expansion of a translated CAG repeat coding for glutamine. Mice lacking ataxin-1 display learning deficits and altered hippocampal synaptic plasticity but none of the abnormalities seen in human SCA1; mice expressing ataxin-1 with an expanded CAG tract (82 glutamine residues), however, develop Purkinje cell pathology and ataxia. These results suggest that mutant ataxin-1 gains a novel function that leads to neuronal degeneration. This novel function might involve aberrant interaction(s) with cell-specific protein(s), which in turn might explain the selective neuronal pathology. Mutant ataxin-1 interacts preferentially with a leucine-rich acidic nuclear protein that is abundantly expressed in cerebellar Purkinje cells and other brain regions affected in SCA1. Immunolocalization studies in affected neurons of patients and SCA1 transgenic mice showed that mutant ataxin-1 localizes to a single, ubiquitin-positive nuclear inclusion (NI) that alters the distribution of the proteasome and certain chaperones. Further analysis of NIs in transfected HeLa cells established that the proteasome and chaperone proteins co-localize with ataxin-1 aggregates. Moreover, overexpression of the chaperone HDJ-2/HSDJ in HeLa cells decreased ataxin-1 aggregation, suggesting that protein misfolding might underlie NI formation. To assess the importance of the nuclear localization of ataxin-1 and its role in SCA1 pathogenesis, two lines of transgenic mice were generated. In the first line, the nuclear localization signal was mutated so that full-length mutant ataxin-1 would remain in the cytoplasm; mice from this line did not develop any ataxia or pathology. This suggests that mutant ataxin-1 is pathogenic only in the nucleus. To assess the role of the aggregates, transgenic mice were generated with mutant ataxin-1 without the self-association domain (SAD) essential for aggregate formation. These mice developed ataxia and Purkinje cell abnormalities similar to those seen in SCA1 transgenic mice carrying full-length mutant ataxin-1, but lacked NIs. The nuclear milieu is thus a critical factor in SCA1 pathogenesis, but large NIs are not needed to initiate pathogenesis. They might instead be downstream of the primary pathogenic steps. Given the accumulated evidence, we propose the following model for SCA1 pathogenesis: expansion of the polyglutamine tract alters the conformation of ataxin-1, causing it to misfold. This in turn leads to aberrant protein interactions. Cell specificity is determined by the cell-specific proteins interacting with ataxin-1. Submicroscopic protein aggregation might occur because of protein misfolding, and those aggregates become detectable as NIs as the disease advances. Proteasome redistribution to the NI might contribute to disease progression by disturbing proteolysis and subsequent vital cellular functions.

Polyglutamine-expanded androgen receptors form aggregates that sequester heat shock proteins, proteasome components and SRC-1, and are suppressed by the HDJ-2 chaperone.

Spinal bulbar muscular atrophy is a neurodegenerative disorder caused by a polyglutamine expansion in the androgen receptor (AR). We show in transiently transfected HeLa cells that an AR containing 48 glutamines (ARQ48) accumulates in a hormone-dependent manner in both cytoplasmic and nuclear aggregates. Electron microscopy reveals both types of aggregates to have a similar ultrastructure. ARQ48 aggregates sequester mitochondria and steroid receptor coactivator 1 and stain positively for NEDD8, Hsp70, Hsp90 and HDJ-2/HSDJ. Co-expression of HDJ-2/HSDJ significantly represses aggregate formation. ARQ48 aggregates also label with antibodies recognizing the PA700 proteasome caps but not 20S core particles. These results suggest that ARQ48 accumulates due to protein misfolding and a breakdown in proteolytic processing. Furthermore, the homeostatic disturbances associated with aggregate formation may affect normal cell function.

Expression and function of the chemokine receptors CXCR1 and CXCR2 in sepsis.

Neutrophils (polymorphonuclear neutrophils; PMN) and a redundant system of chemotactic cytokines (chemokines) have been implicated in the pathogenesis of the acute respiratory distress syndrome in patients with sepsis. PMN express two cell surface receptors for the CXC chemokines, CXCR1 and CXCR2. We investigated the expression and function of these receptors in patients with severe sepsis. Compared with normal donors, CXCR2 surface expression was down-regulated by 50% on PMN from septic patients (p < 0.005), while CXCR1 expression persisted. In vitro migratory responses to the CXCR1 ligand, IL-8, were similar in PMN from septic patients and normal donors. By contrast, the migratory response to the CXCR2 ligands, epithelial cell-derived neutrophil activator (ENA-78) and the growth-related oncogene proteins, was markedly suppressed in PMN from septic patients (p < 0.05). Ab specific for CXCR1 blocked in vitro migration of PMN from septic patients to IL-8 (p < 0.05), but not to FMLP. Thus, functionally significant down-regulation of CXCR2 occurs on PMN in septic patients. We conclude that in a complex milieu of multiple CXC chemokines, CXCR1 functions as the single dominant CXC chemokine receptor in patients with sepsis. These observations offer a potential strategy for attenuating adverse inflammation in sepsis while preserving host defenses mediated by bacteria-derived peptides such as FMLP.

Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice.

Mutant ataxin-1, the expanded polyglutamine protein causing spinocerebellar ataxia type 1 (SCA1), aggregates in ubiquitin-positive nuclear inclusions (NI) that alter proteasome distribution in affected SCA1 patient neurons. Here, we observed that ataxin-1 is degraded by the ubiquitin-proteasome pathway. While ataxin-1 [2Q] and mutant ataxin-1 [92Q] are polyubiquitinated equally well in vitro, the mutant form is three times more resistant to degradation. Inhibiting proteasomal degradation promotes ataxin-1 aggregation in transfected cells. And in mice, Purkinje cells that express mutant ataxin-1 but not a ubiquitin-protein ligase have significantly fewer NIs. Nonetheless, the Purkinje cell pathology is markedly worse than that of SCA1 mice. Taken together, NIs are not necessary to induce neurodegeneration, but impaired proteasomal degradation of mutant ataxin-1 may contribute to SCA1 pathogenesis.

Quantitative comparison of C-X-C chemokines produced by endotoxin-stimulated human alveolar macrophages.

The C-X-C chemokines are a structurally related and functionally redundant family of proteins with neutrophil chemotactic activity. Many of the C-X-C chemokines are produced by endotoxin-stimulated alveolar macrophages (AMs), but knowledge of their relative quantities and their relative contributions to the total chemotactic activity released from these cells is incomplete. Human AMs were stimulated with or without Escherichia coli endotoxin for 2, 4, 8, and 24 h. The mRNA sequences of interleukin (IL)-8, the 78-amino acid epithelial cell-derived neutrophil activator (ENA-78), growth-related protein (GRO) alpha, GRObeta, and GROgamma were cloned by PCR and identified by sequence analysis. The relative mRNA quantities were compared by Northern analysis, and IL-8 was found to predominate. Similarly, IL-8 protein concentrations in the cell supernatants were consistently higher than either the ENA-78 or GRO concentration, and by 24 h, IL-8 concentrations were 10-fold higher than those of the other C-X-C chemokines. Blocking polyclonal antibodies to IL-8 substantially reduced the chemotactic activity in the AM supernatants, whereas antibodies to ENA-78 and GRO had little or no effect. We conclude that IL-8 is the predominant C-X-C chemokine and the dominant neutrophil chemoattractant accumulating in 24-h supernatants of lipopolysaccharide-stimulated human AMs. These studies provide insight into potentially effective strategies of interrupting AM-derived inflammatory signals in the lungs.

Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in ataxin-1. In affected neurons of SCA1 patients and transgenic mice, mutant ataxin-1 accumulates in a single, ubiquitin-positive nuclear inclusion. In this study, we show that these inclusions stain positively for the 20S proteasome and the molecular chaperone HDJ-2/HSDJ. Similarly, HeLa cells transfected with mutant ataxin-1 develop nuclear aggregates which colocalize with the 20S proteasome and endogenous HDJ-2/HSDJ. Overexpression of wild-type HDJ-2/HSDJ in HeLa cells decreases the frequency of ataxin-1 aggregation. These data suggest that protein misfolding is responsible for the nuclear aggregates seen in SCA1, and that overexpression of a DnaJ chaperone promotes the recognition of a misfolded polyglutamine repeat protein, allowing its refolding and/or ubiquitin-dependent degradation.

Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures.

Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative disorders caused by an expansion of a polyglutamine tract. It is characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells. To understand the pathogenesis of SCA1, we examined the subcellular localization of wild-type human ataxin-1 (the protein encoded by the SCA1 gene) and mutant ataxin-1 in the Purkinje cells of transgenic mice. We found that ataxin-1 localizes to the nuclei of cerebellar Purkinje cells. Normal ataxin-1 localizes to several nuclear structures approximately 0.5 microm across, whereas the expanded ataxin-1 localizes to a single approximately 2-microm structure, before the onset of ataxia. Mutant ataxin-1 localizes to a single nuclear structure in affected neurons of SCA1 patients. Similarly, COS-1 cells transfected with wild-type or mutant ataxin-1 show a similar pattern of nuclear localization; with expanded ataxin-1 occurring in larger structures that are fewer in number than those of normal ataxin-1. Colocalization studies show that mutant ataxin-1 causes a specific redistribution of the nuclear matrix-associated domain containing promyelocytic leukaemia protein. Nuclear matrix preparations demonstrate that ataxin-1 associates with the nuclear matrix in Purkinje and COS cells. We therefore propose that a critical aspect of SCA1 pathogenesis involves the disruption of a nuclear matrix-associated domain.