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• W2061258627 abstract "We present evidence for a specific role of p53 in the mitochondria-associated cellular dysfunction and behavioral abnormalities of Huntington’s disease (HD). Mutant huntingtin (mHtt) with expanded polyglutamine (polyQ) binds to p53 and upregulates levels of nuclear p53 as well as p53 transcriptional activity in neuronal cultures. The augmentation is specific, as it occurs with mHtt but not mutant ataxin-1 with expanded polyQ. p53 levels are also increased in the brains of mHtt transgenic (mHtt-Tg) mice and HD patients. Perturbation of p53 by pifithrin-α, RNA interference, or genetic deletion prevents mitochondrial membrane depolarization and cytotoxicity in HD cells, as well as the decreased respiratory complex IV activity of mHtt-Tg mice. Genetic deletion of p53 suppresses neurodegeneration in mHtt-Tg flies and neurobehavioral abnormalities of mHtt-Tg mice. Our findings suggest that p53 links nuclear and mitochondrial pathologies characteristic of HD. We present evidence for a specific role of p53 in the mitochondria-associated cellular dysfunction and behavioral abnormalities of Huntington’s disease (HD). Mutant huntingtin (mHtt) with expanded polyglutamine (polyQ) binds to p53 and upregulates levels of nuclear p53 as well as p53 transcriptional activity in neuronal cultures. The augmentation is specific, as it occurs with mHtt but not mutant ataxin-1 with expanded polyQ. p53 levels are also increased in the brains of mHtt transgenic (mHtt-Tg) mice and HD patients. Perturbation of p53 by pifithrin-α, RNA interference, or genetic deletion prevents mitochondrial membrane depolarization and cytotoxicity in HD cells, as well as the decreased respiratory complex IV activity of mHtt-Tg mice. Genetic deletion of p53 suppresses neurodegeneration in mHtt-Tg flies and neurobehavioral abnormalities of mHtt-Tg mice. Our findings suggest that p53 links nuclear and mitochondrial pathologies characteristic of HD. Huntington’s disease (HD) is a genetically dominant neurodegenerative disease caused by a mutation in the huntingtin gene leading to an abnormal Huntingtin protein product (Htt) (The Huntington’s Disease Collaborative Research Group, 1993The Huntington’s Disease Collaborative Research GroupA novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes.Cell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (6496) Google Scholar). Mutant Htt protein (mHtt) contains elongated polyglutamines (polyQ) whose length correlates with an earlier age of disease onset (Ross, 2002Ross C.A. Polyglutamine pathogenesis: emergence of unifying mechanisms for Huntington’s disease and related disorders.Neuron. 2002; 35: 819-822Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, Sawa, 2001Sawa A. Mechanisms for neuronal cell death and dysfunction in Huntington’s disease: pathological cross-talk between the nucleus and the mitochondria?.J. Mol. Med. 2001; 79: 375-381Crossref PubMed Scopus (40) Google Scholar, Tobin and Signer, 2000Tobin A.J. Signer E.R. Huntington’s disease: the challenge for cell biologists.Trends Cell Biol. 2000; 10: 531-536Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Expression of mHtt, especially N-terminal fragments, elicits cytotoxicity in cell models (Cooper et al., 1998Cooper J.K. Schilling G. Peters M.F. Herring W.J. Sharp A.H. Kaminsky Z. Masone J. Khan F.A. Delanoy M. Borchelt D.R. et al.Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture.Hum. Mol. Genet. 1998; 7: 783-790Crossref PubMed Scopus (301) Google Scholar, Hackam et al., 1998Hackam A.S. Singaraja R. Wellington C.L. Metzler M. McCutcheon K. Zhang T. Kalchman M. Hayden M.R. The influence of huntingtin protein size on nuclear localization and cellular toxicity.J. Cell Biol. 1998; 141: 1097-1105Crossref PubMed Scopus (277) Google Scholar, Wellington et al., 2000Wellington C.L. Singaraja R. Ellerby L. Savill J. Roy S. Leavitt B. Cattaneo E. Hackam A. Sharp A. Thornberry N. et al.Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells.J. Biol. Chem. 2000; 275: 19831-19838Crossref PubMed Scopus (282) Google Scholar) as well as fly models (Jackson et al., 1998Jackson G.R. Salecker I. Dong X. Yao X. Arnheim N. Faber P.W. MacDonald M.E. Zipursky S.L. Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons.Neuron. 1998; 21: 633-642Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, Marsh et al., 2000Marsh J.L. Walker H. Theisen H. Zhu Y.Z. Fielder T. Purcell J. Thompson L.M. Expanded polyglutamine peptides alone are intrinsically cytotoxic and cause neurodegeneration in Drosophila.Hum. Mol. Genet. 2000; 9: 13-25Crossref PubMed Scopus (202) Google Scholar) and elicits behavioral abnormalities in mice (Carter et al., 1999Carter R.J. Lione L.A. Humby T. Mangiarini L. Mahal A. Bates G.P. Dunnett S.B. Morton A.J. Characterization of progressive motor deficits in mice transgenic for the human Huntington’s disease mutation.J. Neurosci. 1999; 19: 3248-3257Crossref PubMed Google Scholar, Hodgson et al., 1999Hodgson J.G. Agopyan N. Gutekunst C.A. Leavitt B.R. LePiane F. Singaraja R. Smith D.J. Bissada N. McCutcheon K. Nasir J. et al.A YAC mouse model for Huntington’s disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration.Neuron. 1999; 23: 181-192Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar, Reddy et al., 1998Reddy P.H. Williams M. Charles V. Garrett L. Pike-Buchanan L. Whetsell Jr., W.O. Miller G. Tagle D.A. Behavioural abnormalities and selective neuronal loss in HD transgenic mice expressing mutated full-length HD cDNA.Nat. Genet. 1998; 20: 198-202Crossref PubMed Scopus (345) Google Scholar, Schilling et al., 1999Schilling G. Becher M.W. Sharp A.H. Jinnah H.A. Duan K. Kotzuk J.A. Slunt H.H. Ratovitski T. Cooper J.K. Jenkins N.A. et al.Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin.Hum. Mol. Genet. 1999; 8: 397-407Crossref PubMed Google Scholar), suggesting that the pathophysiology of HD involves a toxic gain of function. The pathophysiology of HD has been linked to mitochondrial dysfunction (Grunewald and Beal, 1999Grunewald T. Beal M.F. Bioenergetics in Huntington’s disease.Ann. N Y Acad. Sci. 1999; 893: 203-213Crossref PubMed Scopus (92) Google Scholar, Sawa, 2001Sawa A. Mechanisms for neuronal cell death and dysfunction in Huntington’s disease: pathological cross-talk between the nucleus and the mitochondria?.J. Mol. Med. 2001; 79: 375-381Crossref PubMed Scopus (40) Google Scholar, Schapira, 1997Schapira A.H. Mitochondrial function in Huntington’s disease: clues for pathogenesis and prospects for treatment.Ann. Neurol. 1997; 41: 141-142Crossref PubMed Scopus (24) Google Scholar). Mitochondrial enzyme activities of the respiratory chain complexes II/III and IV are impaired specifically in the caudate and putamen of HD patient brains (Browne et al., 1997Browne S.E. Bowling A.C. MacGarvey U. Baik M.J. Berger S.C. Muqit M.M. Bird E.D. Beal M.F. Oxidative damage and metabolic dysfunction in Huntington’s disease: selective vulnerability of the basal ganglia.Ann. Neurol. 1997; 41: 646-653Crossref PubMed Scopus (692) Google Scholar, Tabrizi et al., 1999Tabrizi S.J. Cleeter M.W. Xuereb J. Taanman J.W. Cooper J.M. Schapira A.H. Biochemical abnormalities and excitotoxicity in Huntington’s disease brain.Ann. Neurol. 1999; 45: 25-32Crossref PubMed Scopus (369) Google Scholar) and striatum of mHtt transgenic (mHtt-Tg) mice (Tabrizi et al., 2000Tabrizi S.J. Workman J. Hart P.E. Mangiarini L. Mahal A. Bates G. Cooper J.M. Schapira A.H. Mitochondrial dysfunction and free radical damage in the Huntington R6/2 transgenic mouse.Ann. Neurol. 2000; 47: 80-86Crossref PubMed Scopus (290) Google Scholar). 3-Nitropropionic acid (3-NPA), a selective mitochondrial toxin for the complex II, elicits striatal damage resembling HD (Beal et al., 1993Beal M.F. Brouillet E. Jenkins B.G. Ferrante R.J. Kowall N.W. Miller J.M. Storey E. Srivastava R. Rosen B.R. Hyman B.T. Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid.J. Neurosci. 1993; 13: 4181-4192Crossref PubMed Google Scholar). Mitochondrial disturbances in HD patient brains are not likely secondary to overall neuropathologic alterations, as lymphoblasts from HD patients manifest abnormal mitochondrial membrane depolarization (Panov et al., 2002Panov A.V. Gutekunst C.A. Leavitt B.R. Hayden M.R. Burke J.R. Strittmatter W.J. Greenamyre J.T. Early mitochondrial calcium defects in Huntington’s disease are a direct effect of polyglutamines.Nat. Neurosci. 2002; 5: 731-736Crossref PubMed Scopus (807) Google Scholar, Sawa et al., 1999Sawa A. Wiegand G.W. Cooper J. Margolis R.L. Sharp A.H. Lawler Jr., J.F. Greenamyre J.T. Snyder S.H. Ross C.A. Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization.Nat. Med. 1999; 5: 1194-1198Crossref PubMed Scopus (307) Google Scholar). The mitochondrial defect may be disease specific, as lymphoblasts from the patients with spinocerebellar ataxia type 1 (SCA1), another neurodegenerative condition caused by an expanded polyQ in the disease gene ataxin-1, do not display mitochondrial membrane depolarization (Sawa et al., 1999Sawa A. Wiegand G.W. Cooper J. Margolis R.L. Sharp A.H. Lawler Jr., J.F. Greenamyre J.T. Snyder S.H. Ross C.A. Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization.Nat. Med. 1999; 5: 1194-1198Crossref PubMed Scopus (307) Google Scholar). Nuclear disturbances are also implicated in HD (Hodgson et al., 1999Hodgson J.G. Agopyan N. Gutekunst C.A. Leavitt B.R. LePiane F. Singaraja R. Smith D.J. Bissada N. McCutcheon K. Nasir J. et al.A YAC mouse model for Huntington’s disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration.Neuron. 1999; 23: 181-192Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar, Peters et al., 1999Peters M.F. Nucifora Jr., F.C. Kushi J. Seaman H.C. Cooper J.K. Herring W.J. Dawson V.L. Dawson T.M. Ross C.A. Nuclear targeting of mutant Huntingtin increases toxicity.Mol. Cell. Neurosci. 1999; 14: 121-128Crossref PubMed Scopus (161) Google Scholar, Saudou et al., 1998Saudou F. Finkbeiner S. Devys D. Greenberg M.E. Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions.Cell. 1998; 95: 55-66Abstract Full Text Full Text PDF PubMed Scopus (1331) Google Scholar, Schilling et al., 2004Schilling G. Savonenko A.V. Klevytska A. Morton J.L. Tucker S.M. Poirier M. Gale A. Chan N. Gonzales V. Slunt H.H. et al.Nuclear-targeting of mutant huntingtin fragments produces Huntington’s disease-like phenotypes in transgenic mice.Hum. Mol. Genet. 2004; 13: 1599-1610Crossref PubMed Scopus (79) Google Scholar, Steffan et al., 2000Steffan J.S. Kazantsev A. Spasic-Boskovic O. Greenwald M. Zhu Y.Z. Gohler H. Wanker E.E. Bates G.P. Housman D.E. Thompson L.M. The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription.Proc. Natl. Acad. Sci. USA. 2000; 97: 6763-6768Crossref PubMed Scopus (833) Google Scholar, Wheeler et al., 2000Wheeler V.C. White J.K. Gutekunst C.A. Vrbanac V. Weaver M. Li X.J. Li S.H. Yi H. Vonsattel J.P. Gusella J.F. et al.Long glutamine tracts cause nuclear localization of a novel form of huntingtin in medium spiny striatal neurons in HdhQ92 and HdhQ111 knock-in mice.Hum. Mol. Genet. 2000; 9: 503-513Crossref PubMed Scopus (367) Google Scholar). In both HD cell and transgenic animal models, nuclear accumulation of mHtt strongly enhances neurotoxicity. Mutant Htt interacts with nuclear factors such as CREB binding protein (CBP) (Nucifora et al., 2001Nucifora Jr., F.C. Sasaki M. Peters M.F. Huang H. Cooper J.K. Yamada M. Takahashi H. Tsuji S. Troncoso J. Dawson V.L. et al.Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity.Science. 2001; 291: 2423-2428Crossref PubMed Scopus (902) Google Scholar) and Sp1/TAFII130 (Dunah et al., 2002Dunah A.W. Jeong H. Griffin A. Kim Y.M. Standaert D.G. Hersch S.M. Mouradian M.M. Young A.B. Tanese N. Krainc D. Sp1 and TAFII130 transcriptional activity disrupted in early Huntington’s disease.Science. 2002; 296: 2238-2243Crossref PubMed Scopus (559) Google Scholar), whose loss of function may mediate cytotoxicity. The tumor suppressor gene p53 has multiple functions in processes as diverse as angiogenesis and chemotaxis (Vogelstein et al., 2000Vogelstein B. Lane D. Levine A.J. Surfing the p53 network.Nature. 2000; 408: 307-310Crossref PubMed Scopus (5529) Google Scholar). p53 is also expressed in the central nervous system (CNS), where its function is not established (Morrison and Kinoshita, 2000Morrison R.S. Kinoshita Y. The role of p53 in neuronal cell death.Cell Death Differ. 2000; 7: 868-879Crossref PubMed Scopus (116) Google Scholar). While acute excitotoxic insults, such as kainic acid, induce p53 activation and p53 null mice are mildly resistant to neurotoxicity (Morrison et al., 1996Morrison R.S. Wenzel H.J. Kinoshita Y. Robbins C.A. Donehower L.A. Schwartzkroin P.A. Loss of the p53 tumor suppressor gene protects neurons from kainate-induced cell death.J. Neurosci. 1996; 16: 1337-1345Crossref PubMed Google Scholar), it is unclear whether the increase of p53 is primary or secondary to excitotoxicity. Overexpression of p53 in dissociated primary neuron cultures elicits neuronal death (Jordan et al., 1997Jordan J. Galindo M.F. Prehn J.H. Weichselbaum R.R. Beckett M. Ghadge G.D. Roos R.P. Leiden J.M. Miller R.J. p53 expression induces apoptosis in hippocampal pyramidal neuron cultures.J. Neurosci. 1997; 17: 1397-1405Crossref PubMed Google Scholar), but the role of endogenous p53 has not been investigated. Binding of mHtt with p53 has been reported (Steffan et al., 2000Steffan J.S. Kazantsev A. Spasic-Boskovic O. Greenwald M. Zhu Y.Z. Gohler H. Wanker E.E. Bates G.P. Housman D.E. Thompson L.M. The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription.Proc. Natl. Acad. Sci. USA. 2000; 97: 6763-6768Crossref PubMed Scopus (833) Google Scholar), but its pathophysiological implication remains unknown. Immortalized striatal neurons derived from mHtt knockin mouse embryos show elevated levels of p53 protein (Trettel et al., 2000Trettel F. Rigamonti D. Hilditch-Maguire P. Wheeler V.C. Sharp A.H. Persichetti F. Cattaneo E. MacDonald M.E. Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells.Hum. Mol. Genet. 2000; 9: 2799-2809Crossref PubMed Scopus (470) Google Scholar). Genes regulated by p53 are among a large cohort upregulated in cell cultures transfected with mHtt (Sipione et al., 2002Sipione S. Rigamonti D. Valenza M. Zuccato C. Conti L. Pritchard J. Kooperberg C. Olson J.M. Cattaneo E. Early transcriptional profiles in huntingtin-inducible striatal cells by microarray analyses.Hum. Mol. Genet. 2002; 11: 1953-1965Crossref PubMed Google Scholar). Although nuclei and mitochondria participate in HD pathology, their relationship has been elusive. Because nuclear p53 regulates many mitochondrial genes and genes for oxidative stress (Sharpless and DePinho, 2002Sharpless N.E. DePinho R.A. p53: good cop/bad cop.Cell. 2002; 110: 9-12Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, Vogelstein et al., 2000Vogelstein B. Lane D. Levine A.J. Surfing the p53 network.Nature. 2000; 408: 307-310Crossref PubMed Scopus (5529) Google Scholar), we hypothesized that p53 might link the nuclear and mitochondrial pathologies in HD. We have investigated p53 in HD cell and transgenic animal models expressing mHtt as well as HD patient brains. We present evidence for a specific role of p53 in the mitochondria-associated cellular dysfunction and behavioral abnormalities of Huntington’s disease. In PC12 cells stably expressing the N-terminal 63 amino acids of Htt with 148 polyQ (N63-148Q) in an inducible manner (Igarashi et al., 2003Igarashi S. Morita H. Bennett K.M. Tanaka Y. Engelender S. Peters M.F. Cooper J.K. Wood J.D. Sawa A. Ross C.A. Inducible PC12 cell model of Huntington’s disease shows toxicity and decreased histone acetylation.Neuroreport. 2003; 14: 565-568Crossref PubMed Scopus (59) Google Scholar), the protein level of p53 is increased more than 10-fold after induction of mHtt, but not wild-type Htt (wtHtt) N63-18Q. The increased p53 occurs selectively in the P1 fractions designated as crude nuclei (Figure 1A ) in 1% SDS-soluble, nonaggregate fractions (Figure 1C), implying that the transactivation-independent role for p53 in the mitochondria is unlikely (Chipuk et al., 2004Chipuk J.E. Kuwana T. Bouchier-Hayes L. Droin N.M. Newmeyer D.D. Schuler M. Green D.R. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis.Science. 2004; 303: 1010-1014Crossref PubMed Scopus (1558) Google Scholar). The increase of p53 protein appears to be posttranslational, as mRNA levels of p53 are increased only 1.4-fold (Figure 1B). In the brains of transgenic mice expressing the N-terminal 171 amino acids of Htt with 82 polyglutamine repeats (N171-82Q) (Schilling et al., 1999Schilling G. Becher M.W. Sharp A.H. Jinnah H.A. Duan K. Kotzuk J.A. Slunt H.H. Ratovitski T. Cooper J.K. Jenkins N.A. et al.Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin.Hum. Mol. Genet. 1999; 8: 397-407Crossref PubMed Google Scholar), p53 is also elevated (Figure 1D). Enhancement of p53 is selective, as kainate, which causes more neuronal death than mHtt, induces a lesser increase in p53. Postmortem brains of human HD patients also manifest substantial p53 increases, with the highest levels in the cases with the most extensive HD pathology (Figure 1E). Only striatum and cerebral cortex show p53 upregulation with no increase in cerebellum, fitting with the regional and cellular selectivity of HD pathology (Sawa, 2001Sawa A. Mechanisms for neuronal cell death and dysfunction in Huntington’s disease: pathological cross-talk between the nucleus and the mitochondria?.J. Mol. Med. 2001; 79: 375-381Crossref PubMed Scopus (40) Google Scholar, Sisodia, 1998Sisodia S.S. Nuclear inclusions in glutamine repeat disorders: are they pernicious, coincidental, or beneficial?.Cell. 1998; 95: 1-4Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, Tobin and Signer, 2000Tobin A.J. Signer E.R. Huntington’s disease: the challenge for cell biologists.Trends Cell Biol. 2000; 10: 531-536Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Confirming the previously reported interaction between exon 1 of Htt (N63-20Q, -51Q, and -83Q) and p53 (Steffan et al., 2000Steffan J.S. Kazantsev A. Spasic-Boskovic O. Greenwald M. Zhu Y.Z. Gohler H. Wanker E.E. Bates G.P. Housman D.E. Thompson L.M. The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription.Proc. Natl. Acad. Sci. USA. 2000; 97: 6763-6768Crossref PubMed Scopus (833) Google Scholar), we observe direct binding of Htt N171 and p53 in vitro (Figure 1F). Furthermore, p53 coimmunoprecipitates with N171 as well as full-length (FL) Htt in transiently transfected 293T cells (Figures 1G and 1H). In both cases, mHtt binds more efficiently to p53 than wtHtt. Coimmunoprecipation (co-IP) of p53 and Htt at endogenous protein levels is evident in stressed HD patient lymphoblasts (Figure 1I). We wondered whether augmented p53 is associated with increased transcriptional activity. Using the p53 binding sequence upstream of a luciferase reporter gene (el-Deiry et al., 1992el-Deiry W.S. Kern S.E. Pietenpol J.A. Kinzler K.W. Vogelstein B. Definition of a consensus binding site for p53.Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1701) Google Scholar), we monitored p53-mediated gene activation (Figures 2A and 2B ). Stable expression of mHtt N63-148Q in PC12 cells elicits a 4-fold increase of p53 transcriptional activation, while in rat primary cortical cultures, transient transfection of mHtt N171-82Q causes an approximately 5-fold amplification of p53 activity. Activation is selective for N171-82Q as well as FL mHtt, since transient expression of ataxin-1 with expanded polyQ fails to influence p53 transcriptional activity (Figure 2C). Vector-based RNA interference (RNAi) to p53 (pSuper-p53) (Brummelkamp et al., 2002Brummelkamp T.R. Bernards R. Agami R. A system for stable expression of short interfering RNAs in mammalian cells.Science. 2002; 296: 550-553Crossref PubMed Scopus (3882) Google Scholar) and a specific p53 inhibitor, pifithrin-α (1 μM) (Duan et al., 2002Duan W. Zhu X. Ladenheim B. Yu Q.S. Guo Z. Oyler J. Cutler R.G. Cadet J.L. Greig N.H. Mattson M.P. p53 inhibitors preserve dopamine neurons and motor function in experimental parkinsonism.Ann. Neurol. 2002; 52: 597-606Crossref PubMed Scopus (183) Google Scholar, Komarov et al., 1999Komarov P.G. Komarova E.A. Kondratov R.V. Christov-Tselkov K. Coon J.S. Chernov M.V. Gudkov A.V. A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy.Science. 1999; 285: 1733-1737Crossref PubMed Scopus (1067) Google Scholar), both block mHtt-induced p53 transactivation (Figures 2D and 2E). We compared protein levels of three p53 targets that directly associate with mitochondria, Apaf-1 (Sang et al., 2005Sang T.K. Li C. Liu W. Rodriguez A. Abrams J.M. Zipursky S.L. Jackson G.R. Inactivation of Drosophila Apaf-1 related killer suppresses formation of polyglutamine aggregates and blocks polyglutamine pathogenesis.Hum. Mol. Genet. 2005; 14: 357-372Crossref PubMed Scopus (52) Google Scholar), Bax (Miyashita and Reed, 1995Miyashita T. Reed J.C. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene.Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (293) Google Scholar), and Puma (Yu et al., 2001Yu J. Zhang L. Hwang P.M. Kinzler K.W. Vogelstein B. PUMA induces the rapid apoptosis of colorectal cancer cells.Mol. Cell. 2001; 7: 673-682Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar), in Htt-transfected PC12 cells. Bax and Puma protein levels are significantly augmented by mHtt, whereas Apaf-1 is only marginally augmented (Figures 2F). Mitochondrial membrane depolarization is enhanced in lymphoblasts of HD but not SCA1 patients treated with low concentrations of cyanide (25 μM) (Sawa et al., 1999Sawa A. Wiegand G.W. Cooper J. Margolis R.L. Sharp A.H. Lawler Jr., J.F. Greenamyre J.T. Snyder S.H. Ross C.A. Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization.Nat. Med. 1999; 5: 1194-1198Crossref PubMed Scopus (307) Google Scholar). As p53 can mediate mitochondrial function and is selectively influenced by mHtt but not by mutant ataxin-1 (Figure 2C), we explored a role for p53 in mHtt-mediated mitochondrial dysfunction. Pretreatment of the cyanide-exposed HD lymphoblasts with 10 μM pifithrin-α blocks depolarization of the mitochondria (Figure 3A). Induction of mHtt N63-148Q in PC12 cells causes pronounced mitochondrial membrane depolarization after 5 days of induction (Figures 3B and 3C ). Treatment with pifithrin-α (1 μM) completely blocks the depolarization, indicating that p53 mediates the mitochondrial abnormality. By contrast, pifithrin-α does not affect the rapid mitochondrial membrane depolarization elicited by carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore and uncoupler of oxidative phosphorylation in mitochondria, demonstrating specificity of pifithrin-α for the p53 pathway. In primary cortical cultures of p53+/+ mice, we observe substantial mitochondrial membrane depolarization associated with transfected mHtt N63-148Q but not wtHtt N63-18Q at a time when nuclei are intact in most neurons (Figures 3D and 3E). Cultures from p53+/− and p53−/− mice (Donehower et al., 1992Donehower L.A. Harvey M. Slagle B.L. McArthur M.J. Montgomery Jr., C.A. Butel J.S. Bradley A. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours.Nature. 1992; 356: 215-221Crossref PubMed Scopus (3904) Google Scholar) are protected from this mitochondrial depolarization. mHtt-Tg mouse models display mitochondrial dysfunction affecting enzymes in the mitochondrial respiratory chain IV (Tabrizi et al., 2000Tabrizi S.J. Workman J. Hart P.E. Mangiarini L. Mahal A. Bates G. Cooper J.M. Schapira A.H. Mitochondrial dysfunction and free radical damage in the Huntington R6/2 transgenic mouse.Ann. Neurol. 2000; 47: 80-86Crossref PubMed Scopus (290) Google Scholar). To evaluate the influence of p53 on such mitochondrial dysfunction in vivo, we injected line 81 N171-82Q mHtt-Tg mice i.p. with pifithrin-α, which efficiently crosses the blood-brain barrier (Duan et al., 2002Duan W. Zhu X. Ladenheim B. Yu Q.S. Guo Z. Oyler J. Cutler R.G. Cadet J.L. Greig N.H. Mattson M.P. p53 inhibitors preserve dopamine neurons and motor function in experimental parkinsonism.Ann. Neurol. 2002; 52: 597-606Crossref PubMed Scopus (183) Google Scholar). The treatment significantly prevents impaired complex IV activity of mHtt-Tg mice (Figure 3F). To address the cellular consequences of mitochondrial dysfunction, we evaluated cytotoxicity following inhibition of p53 with pifithrin-α, RNAi, or genetic deletion. Induction of mHtt N63-148Q (Igarashi et al., 2003Igarashi S. Morita H. Bennett K.M. Tanaka Y. Engelender S. Peters M.F. Cooper J.K. Wood J.D. Sawa A. Ross C.A. Inducible PC12 cell model of Huntington’s disease shows toxicity and decreased histone acetylation.Neuroreport. 2003; 14: 565-568Crossref PubMed Scopus (59) Google Scholar) or transfection of FL-82Q causes the death of PC12 cells. The tripling of PC12 cell death elicited by mHtt is abolished by pifithrin-α (10 μM) or pSuper-p53 (Figure 4A ). Antisense oligonucleotides against p53 provide about the same degree of protection as pifithrin-α (data not shown). We also employed primary cerebral cortical cultures from wild-type and p53-deleted mice. In p53+/+ cultures, mHtt N171-82Q transfection elicits 2.5 times greater cell death than wtHtt N171-18Q. By contrast, mHtt fails to elicit neurotoxicity in cultures from p53−/− mice. Pronounced, but not complete, protection is also evident in p53+/− cultures (Figures 4B and 4C). We investigated whether p53 is also involved in mHtt-associated aggregate formation (DiFiglia et al., 1997DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2173) Google Scholar), whose role in HD pathogenesis is controversial (Arrasate et al., 2004Arrasate M. Mitra S. Schweitzer E.S. Segal M.R. Finkbeiner S. Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death.Nature. 2004; 431: 805-810Crossref PubMed Scopus (1526) Google Scholar, Sisodia, 1998Sisodia S.S. Nuclear inclusions in glutamine repeat disorders: are they pernicious, coincidental, or beneficial?.Cell. 1998; 95: 1-4Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). We monitored the formation and nuclear targeting of mHtt aggregates in cortical cultures (Figures 4D and 4E). We find no significant difference between p53+/+ and p53−/− neurons in aggregate formation and nuclear localization of mHtt. We observe substantial nuclear fragmentation and condensation in p53+/+ but not in p53−/− preparations of mHtt cells. Thus, in contrast to the critical role of p53 in mitochondrial membrane depolarization and cell death, mHtt-associated nuclear aggregate formation does not appear to be influenced by p53. To explore the role of p53 in mHtt-induced cell death of intact animals, we evaluated the effect of p53 deletion in mHtt-Tg flies (Jackson et al., 1998Jackson G.R. Salecker I. Dong X. Yao X. Arnheim N. Faber P.W. MacDonald M.E. Zipursky S.L. Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons.Neuron. 1998; 21: 633-642Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). The mHtt-Tg flies, which express mHtt N170-120Q under the control of the eye-specific expression GMR promoter (Hay et al., 1994Hay B.A. Wolff T. Rubin G.M. Expression of baculovirus P35 prevents cell death in Drosophila.Development. 1994; 120: 2121-2129Crossref PubMed Google Scholar), manifest prominent cell death of photoreceptor neurons. We crossed mHtt-Tg flies with p53 mutant flies in which the p53 gene was deleted by homologous recombination (Rong et al., 2002Rong Y.S. Titen S.W. Xie H.B. Golic M.M. Bastiani M. Bandyopadhyay P. Olivera B.M. Brodsky M. Rubin G.M. Golic K.G. Targeted mutagenesis by homologous recombination in D. melanogaster.Genes Dev. 2002; 16: 1568-1581Crossref PubMed Scopus (260) Google Scholar) and assessed the effects of deleting p53 in the compound eyes. Wild-type compound eyes contain ∼800 ommatidia, each of which includes seven photoreceptor cells in any given plane of section. The photoreceptor cells contain a microvillar structure referred to as the rhabdomere. Wild-type (wt) and p53 knockout flies (p53) display a normal composition of seven photoreceptor cells in each ommatidium (Figure 5). mHtt-Tg flies (Htt), however, manifest strong age-dependent loss of rhabdomeres and photoreceptor cells, as reported previously (Jackson et al." @default.
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• W2061258627 date "2005-07-01" @default.
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• W2061258627 title "p53 Mediates Cellular Dysfunction and Behavioral Abnormalities in Huntington’s Disease" @default.
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• W2061258627 doi "https://doi.org/10.1016/j.neuron.2005.06.005" @default.
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