Caspase activation during apoptotic cell death induced by expanded polyglutamine in N2a cells

Wang, Guanghui; Mitsui, Kentaro; Kotliarova, Svetlana; Yamashita, Akira; Nagao, Yoshiro; Tokuhiro, Shinya; Iwatsubo, Takeshi; Kanazawa, Ichiro; Nukina, Nobuyuki

doi:10.1097/00001756-199908200-00001

Cited by 85 publications

(82 citation statements)

References 4 publications

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“…2 D-F). GFP-Htt(Q150) expression in differentiated, postmitotic mouse neuro2a cells (35) was induced for 3 days in the presence of siRNA, followed by withdrawal of the inducer in the continued presence of siRNA. By day 4, the vast majority of cellular Htt was in IBs, which were twice as abundant in cells exposed to autophagin siRNA, compared with cells treated with control (lacZ) siRNA, suggesting that the autophagy pathway was actively eliminating GFPHtt(Q150) aggregates during the induction period (Fig.…”

Section: Resultsmentioning

confidence: 99%

Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation

Iwata

Christianson

Bucci

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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305

227

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CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous aggregates in mouse models of polyglutamine disease, but the cellular mechanism underlying this phenomenon is unknown. Here, we show that autophagy is essential for the elimination of aggregated forms of mutant huntingtin and ataxin-1 from the cytoplasmic but not nuclear compartments. Human orthologs of yeast autophagy genes, molecular determinants of autophagic vacuole formation, are recruited to cytoplasmic but not nuclear inclusion bodies in vitro and in vivo. These data indicate that autophagy is a critical component of the cellular clearance of toxic protein aggregates and may help to explain why protein aggregates are more toxic when directed to the nucleus.autophagy ͉ huntingtin ͉ Huntington's disease P olyglutamine (polyQ) expansion disorders, such as Huntington's disease (HD) and spinocerebellar ataxia type 1, are caused by the production of mutant proteins, huntingtin (Htt) and ataxin-1 (Atx1), respectively, that adopt cytotoxic, nonnative, oligomeric, or aggregation-prone conformations because of the presence of long homopolymeric tracts of glutamine (1). The length of polyQ tracts in normal humans is polymorphic but always below a threshold of 35-40. PolyQ repeats above this threshold are invariably associated with disease, with a strong inverse correlation between repeat length and age-of-onset of neurological disease (2). These findings, together with studies on Htt fragments expressed in cell culture (3) or polyQ peptides in vitro (4), support a model in which glutamine repeats above the threshold adopt a nonnative conformation that is highly prone to self-associate into high-molecular-weight, stable aggregates. These aggregates accumulate in nuclear or cytoplasmic inclusion bodies (IBs) that are invariably associated with end-stage neurodegenerative disease in patients and animal models (5, 6). Although IBs are probably not directly responsible for cellular toxicity (5) and may even contribute to cytoprotection (7,8), the toxicity of mutant polyQ-expanded proteins appears to depend strongly on the cellular compartment in which they accumulate (9, 10). Experimental redirection of Atx1, normally a nuclear protein, to the cytoplasm, drastically reduces toxicity and IB formation in a mouse model of spinocerebellar ataxia type 1 (11). Conversely, the addition of a nuclear localization sequence to the N-terminal polyQ-containing fragment of Htt, which normally partitions between the nucleus and cytoplasm, directs it to the nucleus and increases its toxicity (12, 13). These experiments have been interpreted to indicate that the primary target of polyQ toxicity is in the nucleus. However, it also is possible that both the nucleus and cytoplasm contain targets that are equally vulnerable to these toxic proteins, but that the cytoplasm may be better endowed to neutralize or destroy the proteotoxic agent.Although protein aggregates are highly insoluble (14), animal models using condit...

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Section: Resultsmentioning

confidence: 99%

Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation

Iwata

Christianson

Bucci

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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305

227

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“…2E). We then examined cytotoxicity of these different thtt amyloids by a standard MTT assay (30). Although similar aggregation levels of thttQ60/Q150-GFP were observed by introduction of both 4°C and 37°C conformations, we found that these amyloid conformations led to different toxicities for both thttQ60-GFP and thttQ150-GFP neuro2a cells (Fig.…”

Section: Cytotoxicity Of Different Conformations Of Thtt Amyloids In mentioning

confidence: 86%

“…For mammalian cells, we used stable neuro2a cells that overexpress thtt-GFP fusion protein containing Q16, Q60, or Q150 under a ponasteroneregulatable promoter (30). After extensive attempts, we succeeded in delivering thtt amyloids into the neuro2a cells efficiently by using a lipid-based procedure.…”

Section: Efficient Introduction Of In Vitro Thtt Amyloids Into Mammalmentioning

confidence: 99%

“…After extensive attempts, we succeeded in delivering thtt amyloids into the neuro2a cells efficiently by using a lipid-based procedure. First, in vitro thttQ42 amyloids were introduced into thttQ150-GFP cells, followed by induction of thttQ150-GFP expression and cell differentiation (30). We then estimated the aggregation status of the endogenous thtt-GFP after 15 h of thtt expression and cell differentiation.…”

Section: Efficient Introduction Of In Vitro Thtt Amyloids Into Mammalmentioning

confidence: 99%

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Distinct conformations of in vitro and in vivo amyloids of huntingtin-exon1 show different cytotoxicity

Nekooki-Machida¹,

Kurosawa

Nukina

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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A hallmark of polyglutamine diseases, including Huntington disease (HD), is the formation of ␤-sheet-rich aggregates, called amyloid, of causative proteins with expanded polyglutamines. However, it has remained unclear whether the polyglutamine amyloid is a direct cause or simply a secondary manifestation of the pathology. Here we show that huntingtin-exon1 (thtt) with expanded polyglutamines remarkably misfolds into distinct amyloid conformations under different temperatures, such as 4°C and 37°C. The 4°C amyloid has loop/turn structures together with mostly ␤-sheets, including exposed polyglutamines, whereas the 37°C amyloid has more extended and buried ␤-sheets. By developing a method to efficiently introduce amyloid into mammalian cells, we found that the formation of the 4°C amyloid led to substantial toxicity, whereas the toxic effects of the 37°C amyloid were very small. Importantly, thtt amyloids in different brain regions of HD mice also had distinct conformations. The thermolabile thtt amyloid with loop/turn structures in the striatum showed higher toxicity, whereas the rigid thtt amyloid with more extended ␤-sheets in the hippocampus and cerebellum had only mild toxic effects. These studies show that the thtt protein with expanded polyglutamines can misfold into distinct amyloid conformations and, depending on the conformations, the amyloids can be either toxic or nontoxic. Thus, the amyloid conformation of thtt may be a critical determinant of cytotoxicity in HD.Huntington disease ͉ polyglutamine misfolding ͉ aggregation

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“…A founder N2a cell line stably transfected with pVgRXR (Zeocinresistant), which constitutively expresses a functional ecdysone receptor, was established as described (12). The founder cells were then transfected with pIND(SP1)-hCRMP-2 to generate double-stable transfectants expressing hCRMP-2.…”

Section: Generation Of Stable N2amentioning

confidence: 99%

Evidence That Collapsin Response Mediator Protein-2 Is Involved in the Dynamics of Microtubules

Ihara

2000

Journal of Biological Chemistry

160

121

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Collapsin response mediator protein-2 (CRMP-2) is a member of the CRMP/TOAD/Ulip/DRP family of cytosolic phosphoproteins involved in neuronal differentiation and axonal guidance. CRMP-2 mediates the intracellular response to collapsin 1/semaphorin 3A, a repulsive extracellular guidance cue for axonal outgrowth. The mutation of UNC-33, a Caenorhabditis elegans homolog of CRMP-2, results in abnormality of microtubules in neurites, but the mechanism of CRMP-2 action remains to be clarified. Here, we report that overexpression of human CRMP-2 in Neuro2a cells, a mouse neuroblastoma cell line, results in blebbing of the cytoplasm. Furthermore, some cells exhibited intranuclear inclusions, which were labeled with antibodies to CRMP-2 and tubulin. CRMP-2 was found to be associated with microtubule bundles in the spindles at the metaphase and in the midbodies at the late telophase in mitotic cells. Thus, it is most likely that failure of complete disassembly of the spindle microtubules during mitosis is responsible for the formation of these intranuclear inclusions. We suggest that CRMP-2 functions by regulating the dynamics of microtubules.

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Caspase activation during apoptotic cell death induced by expanded polyglutamine in N2a cells

Cited by 85 publications

References 4 publications

Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation

Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation

Distinct conformations of in vitro and in vivo amyloids of huntingtin-exon1 show different cytotoxicity

Evidence That Collapsin Response Mediator Protein-2 Is Involved in the Dynamics of Microtubules

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