Nuclear localization of a non-caspase truncation product of atrophin-1, with an expanded polyglutamine repeat,increases cellular toxicity

Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder similar to Huntington's disease, with clinical manifestations including chorea, incoordination, ataxia, and dementia. It is caused by an expansion of a CAG trinucleotide repeat encoding polyglutamine in the atrophin-1 gene. Both patients and DRPLA transgenic mice have nuclear accumulation of atrophin-1, especially an approximately 120-kDa fragment, which appears to represent a cleavage product. We now show that this is an N-terminal fragment that does not correspond to the previously described caspase-3 fragment, or any other known caspase cleavage product. The atrophin-1 sequence contains a putative nuclear localization signal in the N terminus of the protein and a putative nuclear export signal in the C terminus. We have tested the hypothesis that endogenous localization signals are functional in atrophin-1, and that nuclear localization and proteolytic cleavage contribute to atrophin-1 cell toxicity. In transient cell transfection experiments using a neuroblastoma cell line, full-length atrophin-1 with 26 (normal) or 65 (expanded) glutamines localized to both nucleus and cytoplasm, with no significant difference in toxicity between the normal and mutant proteins. A construct with 65 glutamine repeats encoding an N-terminal fragment (which removes an NES) of atrophin-1 similar in size to the truncation product in DRPLA patient tissue, showed increased nuclear labeling, and an increase in cellular toxicity, compared with a similar fragment with 26 glutamines. Full-length atrophin-1 with 65 polyglutamine repeats and mutations inactivating the NES also yielded increased nuclear localization and increased toxicity. These data suggest that truncation enhances cellular toxicity of the mutant protein, and that the NES is a relevant region deleted during truncation. Furthermore, mutating the NLS in the truncated protein shifted atrophin-1 more to the cytoplasm and eliminated the increased toxicity, consistent with the idea that nuclear localization enhances toxicity. In none of the experiments were inclusions visible in the nucleus or cytoplasm suggesting that inclusion formation is unrelated to cell death. These data indicate that truncation of atrophin-1 may alter its ability to shuttle between the nucleus and cytoplasm, leading to abnormal nuclear interactions and cell toxicity.     

Cathepsins L and Z are critical in degrading polyglutamine-containing proteins within lysosomes

In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins.     

Analyzing the aggregation of polyglutamine-expansion proteins and its modulation by molecular chaperones

Polyglutamine (polyQ)-expansion proteins cause protein aggregation in the cytosol and nucleus of neuronal cells, leading to neurodegenerative diseases. For example, expansion of the polyQ tract (>40 repeats) in huntingtin (htt) proteins leads to Huntington disease, while polyQ-expanded ataxins cause several types of ataxias. PolyQ-rich inclusions are found in neuronal cells of patients, suggesting that polyQ disease is caused by protein misfolding. However, the mechanisms by which polyQ-expansion proteins exert neuronal toxicity are largely unknown. Here, we review experimental procedures to analyze the roles of molecular chaperones in preventing polyQ aggregation and toxicity as well as to measure the characteristics and dynamics of polyQ aggregation, particularly focusing on cellular models and dynamic imaging of fluorescently-labeled polyQ-expansion proteins and their modulation by chaperones.     

Dynamic imaging by fluorescence correlation spectroscopy identifies diverse populations of polyglutamine oligomers formed in vivo

Protein misfolding and aggregation are exacerbated by aging and diseases of protein conformation including neurodegeneration, metabolic diseases, and cancer. In the cellular environment, aggregates can exist as discrete entities, or heterogeneous complexes of diverse solubility and conformational state. In this study, we have examined the in vivo dynamics of aggregation using imaging methods including fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS), to monitor the diverse biophysical states of expanded polyglutamine (polyQ) proteins expressed in Caenorhabditis elegans. We show that monomers, oligomers and aggregates co-exist at different concentrations in young and aged animals expressing different polyQ-lengths. During aging, when aggregation and toxicity are exacerbated, FCS-based burst analysis and purified single molecule FCS detected a populational shift toward an increase in the frequency of brighter and larger oligomeric species. Regardless of age or polyQ-length, oligomers were maintained in a heterogeneous distribution that spans multiple orders of magnitude in brightness. We employed genetic suppressors that prevent polyQ aggregation and observed a reduction in visible immobile species with the persistence of heterogeneous oligomers, yet our analysis did not detect the appearance of any discrete oligomeric states associated with toxicity. These studies reveal that the reversible transition from monomers to immobile aggregates is not represented by discrete oligomeric states, but rather suggests that the process of aggregation involves a more complex pattern of molecular interactions of diverse intermediate species that can appear in vivo and contribute to aggregate formation and toxicity.     

Efficient induction of nuclear aggresomes by specific single missense mutations in the DNA-binding domain of a viral AP-1 homolog

Nuclear aggresomes induced by proteins containing an expanded polyglutamine (polyQ) tract are pathologic hallmarks of certain neurodegenerative diseases. Some GFP fusion proteins lacking a polyQ tract may also induce nuclear aggresomes in cultured cells. Here we identify single missense mutations within the basic DNA recognition region of Bam HI Z E B virus replication activator (ZEBRA), an Epstein-Barr virus (EBV)-encoded basic zipper protein without a polyQ tract, that efficiently induced the formation of nuclear aggresomes. Wild-type (WT) ZEBRA was diffusely distributed within the nucleus. Four non-DNA-binding mutants, Z(R179E), Z(R183E), Z(R190E), and Z(K178D) localized to the periphery of large intranuclear spheres, to discrete nuclear aggregates, and to the cytoplasm. Other non-DNA-binding mutants, Z(N182K), Z(N182E), and Z(S186E), did not exhibit this phenotype. The interior of the spheres contained promyelocytic leukemia and HSP70 proteins. ZEBRA mutants directly induced the nuclear aggresome pathway in cells with and without EBV. Specific cellular proteins (SC35 and HDAC6) and viral proteins (WT ZEBRA, Rta, and BMLF1) but not other cellular or viral proteins were recruited to nuclear aggresomes. Co-transfection of WT ZEBRA with aggresome-inducing mutants Z(R183E) and Z(R179E) inhibited late lytic viral protein expression and lytic viral DNA amplification. This is the first reported instance in which nuclear aggresomes are induced by single missense mutations in a viral or cellular protein. We discuss conformational changes in the mutant viral AP-1 proteins that may lead to formation of nuclear aggresomes.     

Evidence for a recruitment and sequestration mechanism in Huntington's disease.

Polyglutamine (polyQ) extension in the coding sequence of mutant huntingtin causes neuronal degeneration associated with the formation of insoluble polyQ aggregates in Huntington's disease. We constructed an array of CAG/CAA triplet repeats, coding for a range of 25-300 glutamine residues, which was used to generate expression constructs with minimal flanking sequence. Normal-length (25 glutamine residues) polyQ did not aggregate when transfected alone. Remarkably, when co-transfected with extended (100-300 glutamine residues) polyQ tracts, normal-length polyQ-containing peptides were trapped in insoluble detergent-resistant aggregates. Aggregates formed in the cytoplasm but were visible in the nucleus only when a strong nuclear localization signal was present. Intermolecular interactions between polyQ tracts mediated the localization of heterogeneous aggregates into the nucleolus by nucleolin protein. Our results suggest that extended polyQ can interact with cellular polyQ-containing proteins, transport them to ectopic cellular locations, and form heterogeneous polyQ aggregates. We provide evidence for a recruitment mechanism for pathogenesis in the polyQ neurodegenerative disorders. In susceptible cells, extended polyQ tracts in huntingtin might interact with and sequester or deplete certain endogenous polyQ-containing cellular proteins.     

VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration

Neuronal cell death, abnormal protein aggregates, and cytoplasmic vacuolization are major pathologies observed in many neurodegenerative disorders such as the polyglutamine (polyQ) diseases, prion disease, Alzheimer disease, and the Lewy body diseases, suggesting common mechanisms underlying neurodegeneration. Here, we have identified VCP/p97, a member of the AAA+ family of ATPase proteins, as a polyQ-interacting protein in vitro and in vivo, and report on its characterization. Endogenous VCP co-localized with expanded polyQ (ex-polyQ) aggregates in cultured cells expressing ex-polyQ, with nuclear inclusions in Huntington disease patient brains, and with Lewy bodies in patient samples. Moreover, the expression of VCP mutants with mutations in the 2nd ATP binding domain created cytoplasmic vacuoles, followed by cell death. Very similar vacuoles were also induced by ex-polyQ expression or proteasome inhibitor treatment. These results suggest that VCP functions not only as a recognition factor for abnormally folded proteins but also as a pathological effector for several neurodegenerative phenotypes. VCP may thus be an ideal molecular target for the treatment of neurodegenerative disorders.     

A Model for Small Heat Shock Protein Inhibition of Polyglutamine Aggregation

Polyglutamine (polyQ) repeat expansions that lead to the formation of amyloid aggregates are linked to several devastating neurodegenerative disorders. While molecular chaperones, including the small heat shock proteins (sHsp), play an important role in protection against protein misfolding, the aberrant protein folding that accompanies these polyQ diseases overwhelms the chaperone network. By generating a model structure to explain the observed suppression of spinocerebellar ataxia 3 (SCA3) by the sHsp αB-crystallin, we have identified key vulnerabilities that provide a possible mechanism to explain this heat shock response. A docking study involving a small bioactive peptide should also aid in the development of new drug targets for the prevention of polyQ-based aggregation.     

RNA-binding protein TLS is a major nuclear aggregate-interacting protein in huntingtin exon 1 with expanded polyglutamine-expressing cells

Formation of intracellular aggregates is the hallmark of polyglutamine (polyQ) diseases. We analyzed the components of purified nuclear polyQ aggregates by mass spectrometry. As a result, we found that the RNA-binding protein translocated in liposarcoma (TLS) was one of the major components of nuclear polyQ aggregate-interacting proteins in a Huntington disease cell model and was also associated with neuronal intranuclear inclusions of R6/2 mice. In vitro study revealed that TLS could directly bind to truncated N-terminal huntingtin (tNhtt) aggregates but could not bind to monomer GST-tNhtt with 18, 42, or 62Q, indicating that the tNhtt protein acquired the ability to sequester TLS after forming aggregates. Thioflavin T assay and electron microscopic study further supported the idea that TLS bound to tNhtt-42Q aggregates at the early stage of tNhtt-42Q amyloid formation. Immunohistochemistry showed that TLS was associated with neuronal intranuclear inclusions of Huntington disease human brain. Because TLS has a variety of functional roles, the sequestration of TLS to polyQ aggregates may play a role in diverse pathological changes in the brains of patients with polyQ diseases.     

多聚谷氨酰胺延伸蛋白募集细胞内转录因子及对基因转录调控的影响

多聚谷氨酰胺（polyglutamine,PolyQ）疾病是由特定基因序列中CAG三核苷酸的不稳定重复扩增所引发的一类神经退行性疾病。至今已发现9种类型的PolyQ疾病,其中多数疾病的致病蛋白质在转录调控中发挥着重要的病理作用。PolyQ蛋白中谷氨酰胺的异常重复延伸会引发蛋白质错误折叠并在细胞中积聚形成包涵体。积聚的蛋白质可通过自身结构域、泛素修饰和RNA等介导的相互作用,有效地募集细胞内的转录因子、泛素接头或受体蛋白,以及分子伴侣等组分到包涵体中。这些组分在细胞中的可溶性比例减少,使得机体内的转录调控系统功能受损,造成转录失调从而诱发疾病。因此,研究异常延伸的PolyQ蛋白对细胞内转录因子及其他组分的募集作用,可在分子水平上解释神经退行性疾病的发病机制,从而为临床应用提供潜在的预防和治疗方法。     

Polyglutamine pathogenesis.

An increasing number of neurodegenerative disorders have been found to be caused by expanding CAG triplet repeats that code for polyglutamine. Huntington's disease (HD) is the most common of these disorders and dentatorubral-pallidoluysian atrophy (DRPLA) is very similar to HD, but is caused by mutation in a different gene, making them good models to study. In this review, we will concentrate on the roles of protein aggregation, nuclear localization and proteolytic processing in disease pathogenesis. In cell model studies of HD, we have found that truncated N-terminal portions of huntingtin (the HD gene product) with expanded repeats form more aggregates than longer or full length huntingtin polypeptides. These shorter fragments are also more prone to aggregate in the nucleus and cause more cell toxicity. Further experiments with huntingtin constructs harbouring exogenous nuclear import and nuclear export signals have implicated the nucleus in direct cell toxicity. We have made mouse models of HD and DRPLA using an N-terminal truncation of huntingtin (N171) and full-length atrophin-1 (the DRPLA gene product), respectively. In both models, diffuse neuronal nuclear staining and nuclear inclusion bodies are observed in animals expressing the expanded glutamine repeat protein, further implicating the nucleus as a primary site of neuronal dysfunction. Neuritic pathology is also observed in the HD mice. In the DRPLA mouse model, we have found that truncated fragments of atrophin-1 containing the glutamine repeat accumulate in the nucleus, suggesting that proteolysis may be critical for disease progression. Taken together, these data lead towards a model whereby proteolytic processing, nuclear localization and protein aggregation all contribute to pathogenesis.     

Identification of anti-prion compounds as efficient inhibitors of polyglutamine protein aggregation in a zebrafish model

Several neurodegenerative diseases, including Huntington disease (HD), are associated with aberrant folding and aggregation of polyglutamine (polyQ) expansion proteins. Here we established the zebrafish, Danio rerio, as a vertebrate HD model permitting the screening for chemical suppressors of polyQ aggregation and toxicity. Upon expression in zebrafish embryos, polyQ-expanded fragments of huntingtin (htt) accumulated in large SDS-insoluble inclusions, reproducing a key feature of HD pathology. Real time monitoring of inclusion formation in the living zebrafish indicated that inclusions grow by rapid incorporation of soluble htt species. Expression of mutant htt increased the frequency of embryos with abnormal morphology and the occurrence of apoptosis. Strikingly, apoptotic cells were largely devoid of visible aggregates, suggesting that soluble oligomeric precursors may instead be responsible for toxicity. As in nonvertebrate polyQ disease models, the molecular chaperones, Hsp40 and Hsp70, suppressed both polyQ aggregation and toxicity. Using the newly established zebrafish model, two compounds of the N'-benzylidene-benzohydrazide class directed against mammalian prion proved to be potent inhibitors of polyQ aggregation, consistent with a common structural mechanism of aggregation for prion and polyQ disease proteins.     

Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila

We tested whether proteins implicated in Huntington's and other polyglutamine (polyQ) expansion diseases can cause axonal transport defects. Reduction of Drosophila huntingtin and expression of proteins containing pathogenic polyQ repeats disrupt axonal transport. Pathogenic polyQ proteins accumulate in axonal and nuclear inclusions, titrate soluble motor proteins, and cause neuronal apoptosis and organismal death. Expression of a cytoplasmic polyQ repeat protein causes adult retinal degeneration, axonal blockages in larval neurons, and larval lethality, but not neuronal apoptosis or nuclear inclusions. A nuclear polyQ repeat protein induces neuronal apoptosis and larval lethality but no axonal blockages. We suggest that pathogenic polyQ proteins cause neuronal dysfunction and organismal death by two non-mutually exclusive mechanisms. One mechanism requires nuclear accumulation and induces apoptosis; the other interferes with axonal transport. Thus, disruption of axonal transport by pathogenic polyQ proteins could contribute to early neuropathology in Huntington's and other polyQ expansion diseases.     

Ultrastructure of nuclear aggregates formed by expressing an expanded polyglutamine

Intranuclear inclusions have been observed in the brains of patients affected with Huntington's disease (HD). Neuro 2A cells that transiently expressed HD exon 1 bearing 74 glutamine repeats linked to the green fluorescent protein (GFP) and the nuclear localization sequence (NLS) contained aggregates in nuclei. The aggregates were purified by fractionation with centrifugation followed by fluorescence-activated cell sorting (FACS). Heat treatment of the aggregate in an SDS sample buffer caused the dense aggregate cores to disappear and generated a basket-like structure composed of fibrils. Biochemical analysis of the aggregates revealed that the HD exon 1-GFP fusion protein was the major component. The heterogeneous nuclear ribonucleoproteins F and H, histones and ubiquitin were found to be associated with the aggregates. Our observations suggest that the N-terminal fragment of huntingtin may organize the skeletal structure of the aggregates and may disturb normal cellular functions by trapping other proteins within the aggregates.     

Nuclear aggregation of huntingtin is not prevented by deletion of chaperone Hsp104

Polyglutamine expansion causes the disease proteins to aggregate, resulting in stable insoluble aggregates in the nucleus. The in vitro aggregation and cellular toxicity of polyglutamine proteins are reduced by chaperone heat shock proteins (Hsp). In polyglutamine disease animal models, however, polyglutamine inclusions remain in the nucleus despite the suppression of neurodegeneration by Hsp. Studies using yeast genetic approach revealed that the balance of Hsp is important for regulating protein aggregation in the cytoplasm of yeast cells. Here we report that N-terminal fragments of huntingtin with an expanded polyglutamine tract form aggregates only in the cytoplasm of yeast cells and, when tagged with nuclear localization sequences (NLS), are able to aggregate in the nucleus. Deletion of the Hsp104 gene prevents the aggregation of huntingtin in the cytoplasm but is unable to eliminate the aggregation of NLS-tagged huntingtin in the nucleus. The inhibitory effect of Hsp104 deletion on the cytoplasmic aggregation of huntingtin only occurs in viable yeast cells, as aggregates can be formed in Hsp104 deletion cells that have been frozen for 72 h. Fresh cytosolic extracts of the Hsp104 deletion strain inhibit the aggregation of huntingtin in vitro, suggesting that the deletion of Hsp104 may alter the activities of other cytoplasmic factors to inhibit polyglutamine aggregation in the cytoplasm. We propose that the regulatory effects of chaperones may mainly be restricted to the cytoplasm and have much less influence on polyglutamine-containing aggregates in the nucleus.     

Proteolytic processing regulates pathological accumulation in dentatorubral-pallidoluysian atrophy

Dentatorubral-pallidoluysian atrophy is caused by polyglutamine (polyQ) expansion in atrophin-1 (ATN1). Recent studies have shown that nuclear accumulation of ATN1 and cleaved fragments with expanded polyQ is the pathological process underlying neurodegeneration in dentatorubral-pallidoluysian atrophy. However, the mechanism underlying the proteolytic processing of ATN1 remains unclear. In the present study, we examined the proteolytic processing of ATN1 aiming to understand the mechanisms of ATN1 accumulation with polyQ expansion. Using COS-7 and Neuro2a cells that express the ATN1 gene, in which ATN1 was accumulated by increasing the number of polyQs, we identified a novel C-terminal fragment containing a polyQ tract. The mutant C-terminal fragment with expanded polyQ selectively accumulated in the cells, and this was also demonstrated in the brain tissues of patients with dentatorubral-pallidoluysian atrophy. Immunocytochemical and biochemical studies revealed that full-length ATN1 and C-terminal fragments displayed individual localization. The mutant C-terminal fragment was preferentially found in the cytoplasmic membrane/organelle and insoluble fractions. Accordingly, it is assumed that the proteolytic processing of ATN1 regulates the localization of C-terminal fragments. Accumulation of the C-terminal fragment was enhanced by inhibition of caspases in the cytoplasm of COS-7 cells. Collectively, these results suggest that the C-terminal fragment plays a principal role in the pathological accumulation of ATN1 in dentatorubral-pallidoluysian atrophy.