Huntington's disease is a progressive neurodegenerative disorder that is associated with a CAG repeat expansion in the gene encoding huntingtin. We found that a 60-kDa protein was increased in Neuro2a cells expressing the N-terminal portion of huntingtin with expanded polyglutamine. We purified this protein, and, using mass spectrometry, identified it as p62, an ubiquitin-associated domain-containing protein. A specific p62 antibody stained the ubiquitylated polyQ inclusions in expanded polyglutamine-expressing cells, as well as in the brain of the huntingtin exon 1 transgenic mice. Furthermore, the level of p62 protein and mRNA was increased in expanded polyglutamine-expressing cells. We also found that p62 formed aggresome-like inclusions when p62 was increased in normal Neuro2a cells by a proteasome inhibitor. Knock-down of p62 does not affect the formation of aggresomes or polyglutamine inclusions, suggesting that p62 is recruited to the aggresome or inclusions secondary to their formation. These results suggest that p62 may play important roles as a responsive protein to a polyglutamine-induced stress rather than as a cross-linker between ubiquitylated proteins. Keywords: aggresome, Huntington's disease, p62, polyglutamine, proteasome inhibitor, RNA interference. Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder with midlife onset that gives rise to progressive, selective neural cell death in the striatum associated with choreic movement and dementia (Vonsattel and DiFiglia 1998). The disease is associated with an unstable expansion of CAG repeats within the coding region of the gene encoding the protein huntingtin. Whereas wildtype chromosomes with a stable CAG repeat possess 6-34 repeats, more than 36 repeats result in an unstable, expanded, disease-associated allele. The mutation in huntingtin produces an expanded stretch of glutamine residues, and the mutant huntingtin aggregates with ubiquitylation, forming neuronal nuclear aggregates or inclusions and dystrophic neuritic inclusions in the HD cortex and striatum. Many observations have suggested that abnormal accumulation of the mutant protein is involved in pathogenesis of various neurodegenerative disorders, including polyglutamine diseases such as HD, by conferring a toxic gain of function (Zoghbi and Orr 2000).How inclusions contribute to altered cell function is not well understood but they could have a variety of effects on the regulation of gene transcription, protein interactions, and protein transport within the nucleus and cytoplasm. Various proteins, for example, ubiquitin, molecular chaperones, and components of the proteasome, co-localize with mutant proteins in the inclusions, which may result in the modification of important cellular functions. Recent studies suggest that cells avoid accumulating potentially toxic aggregates by using molecular chaperones to suppress aggregate formation and by using proteasomes to degrade misfolded proteins. This hypothesis is supported by the fact that chaperone Addres...

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. Huntington disease (HD)2 is a hereditary neurodegenerative disease caused by an expansion of the CAG repeat located in exon 1 of the HD gene (1). Expansion of the polyglutamine (polyQ) stretch in huntingtin (htt), the HD gene product, leads to the formation of intracellular aggregates (2, 3). Previous studies have demonstrated that nuclear accumulation of insoluble polyQ aggregates or formation of neuronal intranuclear inclusions is closely correlated with disease progression (4 -6), and disruption of nuclear physiological processes may account for many of the disease phenotypes in the mouse models generated by expressing mutant N-terminal fragments of htt (7).There are many aggregate-interacting proteins (AIPs), some of which, including the heat shock protein (Hsp) 40, 70, and 90 families, are thought to suppress aggregate formation and cellular toxicity induced by expanded polyQ proteins (8, 9). In addition, functionally important proteins, including transcription factors (10 -12) and members of the ubiquitin-proteosome pathway (13,14), are sequestered in the polyQ aggregates, which could cause their loss of function and result in cellular dysfunction. These studies suggest that the components of AIPs reflect either the cellular defense against polyQ aggregates or the cellular machinery affected by polyQ aggregates. Therefore, identification of AIPs should help elucidate the process of aggregate formation, the cellular response to aggregates, and the mechanisms of cellular dysfunction caused by the polyQ aggregates, but the AIPs are still not fully uncovered.We have previously established a method to identify the components of nuclear polyQ AIPs from Neuro2a cells stably transfected with tNhtt-150Q-EGFP-NLS (HD150Q-NLS cells), which express a cDNA encoding htt exon 1 containing 150 CAG repeats and fused with enhanced green fluorescent pr...

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder. To investigate the mechanism of neurodegeneration induced by mutant huntingtin, we developed a stable neuro2a cell line expressing truncated N-terminal huntingtin (tNhtt) with EGFP using the ecdysone-inducible system. The formation of aggregates and the cell death induced by expression of tNhtt with expanded polyglutamine was repeat length- and dose-dependent. Caspases were activated, and the death substrates of caspases, lamin B and ICAD (an inhibitor of caspase-activated DNase), were cleaved in this cell death process. The cleavage of lamin B was inhibited by caspase inhibitors. These findings suggest that the cell death induced by tNhtt with expanded polyglutamine is mediated by caspases.

Apoptotic cell death of murine leukemia cells induced by E. coli L-asparaginase was studied. Deprivation of L-asparagine from the culture of L5178Y cells by L-asparaginase caused the fragmentation of chromosomal DNA of the leukemia cells within 24 h. Prior to the degradation of DNA, cell cycles of L5178Y cells were found to be arrested in G1 phase, and evidence of the DNA strand breaks was initially observed in G1 phase cells as early as 8 h after the asparaginase treatment. Therefore, apoptosis of leukemia cells induced by L--asparaginase is an event that is associated with the cell cycle arrest in G1 phase.