Inducible PC12 cell model of Huntington’s disease shows toxicity and decreased histone acetylation

Igarashi, Shuichi; Morita, Hokuto; Bennett, Kyla M.; Tanaka, Yuji; Engelender, Simone; Peters, Matthew F.; Cooper, Joseph; Wood, Jonathan; Sawa, Akira; Ross, Christopher A.

doi:10.1097/00001756-200303240-00007

Cited by 70 publications

(44 citation statements)

References 21 publications

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“…Cells were harvested 48 -72 h after transfection. In addition, a stable inducible PC12 cell model expressing full-length Htt (pTet-c-myc-FL148Q) was established as previously described (18). PC12 cells were grown in Dulbecco's modified Eagle's medium with 5% fetal bovine serum, 10% horse serum, 100 g/ml G418, 200 g/ml hygromycin, 200 ng/ml doxycycline, 100 units/ml penicillin, and 100 units/ml streptomycin.…”

Section: Methodsmentioning

confidence: 99%

Huntingtin Phosphorylation Sites Mapped by Mass Spectrometry

Schilling

Gafni

Torcassi

et al. 2006

Journal of Biological Chemistry

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133

114

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Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing fulllength myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt.

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Section: Methodsmentioning

confidence: 99%

Huntingtin Phosphorylation Sites Mapped by Mass Spectrometry

Schilling

Gafni

Torcassi

et al. 2006

Journal of Biological Chemistry

Self Cite

133

114

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“…Cell-based systems include: 1) non-striatal neuronal-like cell lines, particularly PC12, expressing htt with various length polyQ expansions [20]; 2) immortalized striatal-like cell lines with pathogenic htt polyQ expansions [21,22]; and 3) immortalized striatal-like cells derived from polyQ-htt knock-in mice [23]. In vivo models include (Table 1): 1) targeted intrastriatal injections of adenovirus or lentivirus expressing polyQ-htt [24]; and 2) genetically modified mice, using either a cell specific promoter directing expression of polyQ-htt [25], or ROSA26 or BACHD mice combined with cre/lox technology to regulate region-specific expression [15,16,26].…”

Section: What Is Unique About the Msn?mentioning

confidence: 99%

“…2) Repression of transcription secondary to histone hypoacetylation [20,[94][95][96]. 3) Direct decrease in cortical mRNAs, including Lin7b and anterogradely transported BDNF, leading to transneuronal striatal transcriptional dysregulation [60,97,98].…”

Section: Transcriptional Dysregulationmentioning

confidence: 99%

Huntington's Disease and the Striatal Medium Spiny Neuron: Cell-Autonomous and Non-Cell-Autonomous Mechanisms of Disease

Ehrlich

2012

Neurotherapeutics

120

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Huntington's disease is an autosomal dominant disorder caused by a mutation in the gene encoding the protein huntingtin on chromosome 4. The mutation is an expanded CAG repeat in the first exon, encoding a polyglutamine tract. If the polyglutamine tract is >40, penetrance is 100% and death is inevitable. Despite the widespread expression of huntingtin, HD has long been considered primarily as a disease of the striatum. It is characterized by selective vulnerability with dysfunction followed by death of the medium size spiny neuron. Considerable effort is being expended to determine whether striatal damage is cell-autonomous, non-cell-autonomous, requiring cell-cell and region to region communication, or both. We review data supporting both mechanisms. We also attempt to organize the data into common mechanisms that may arise outside the medium, spiny neuron, but ultimately have their greatest impact in the striatum. characterized by selective, or perhaps better described as differential [2], vulnerability with degeneration and death of the medium spiny neuron (MSN). The Gamma-aminobutyric acid (GABA)ergic MSN represents 95% of striatal neurons. They are all output neurons, with extensive intra-striatal connections with other MSNs and striatal interneurons. For the last two decades, increased attention has been paid to the pathology in the cortex (particularly in layers V and VI [3]), which contains the corticostriatal projection neurons.Prior to identification of the HTT gene, the huntingtin protein, and the nature of its mutation, it was assumed that selective neuronal vulnerability in HD would be conferred by the expression pattern of the mutated protein (polyQ-htt). As it is now apparent, htt is expressed throughout the nervous system and periphery, without a preference for, or higher level in, MSNs or cortical projection neurons [4].In the absence of enriched expression of a mutated protein, neuronal subtype vulnerability was assumed to arise from unique protein interactions. For example, in the study of another polyQ disease, spinocerebellar ataxia 7, the interaction between ataxin-7 and the homeobox protein CRX in the retina was reported to specifically lead to pathology [5]. In a followup study, the specific role of CRX was not confirmed [6]. To complicate matters further, the same group recently demonstrated that the interactions between a mutant polyQ protein, Ataxin-1, and 14-3-3epsilon differentially affects disease state in cerebellum and brainstem, both of which are vulnerable regions [7]. The regional distribution of the described huntingtin interacting proteins by and large does not explain MSN vulnerability [8][9][10]. One exception is the htt interacting protein RASD2/Rhes (Ras homologue enriched in striatum), which is striatal-specific. It is a small G-protein that mediates sumoylation of polyQ-htt, and thereby its neurotoxicity.

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“…The mechanisms of Htt induced toxicity are still largely unknown; however, there is mounting evidence that toxic poly(Q)-expanded Htt fragments are formed from fulllength Htt via proteolysis (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Thus, the Htt cleavage pathway and the molecules involved in it may represent potential therapeutic targets for intervention in HD.…”

Section: Huntington Disease (Hd)mentioning

confidence: 99%

Mutant Huntingtin N-terminal Fragments of Specific Size Mediate Aggregation and Toxicity in Neuronal Cells

Ratovitski

Guček

Jiang

et al. 2009

Journal of Biological Chemistry

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124

123

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Huntingtin proteolysis is implicated in Huntington diseasepathogenesis, yet, the nature of huntingtin toxic fragments remains unclear. Huntingtin undergoes proteolysis by calpains and caspases within an N-terminal region between amino acids 460 and 600. We have focused on proteolytic steps producing shorter N-terminal fragments, which we term cp-1 and cp-2 (distinct from previously described cp-A/cp-B). We used HEK293 cells to express the first 511 residues of huntingtin and further define the cp-1 and cp-2 cleavage sites. Based on epitope mapping with huntingtin-specific antibodies, we found that cp-1 cleavage occurs between residues 81 and 129 of huntingtin. Affinity and size exclusion chromatography were used to further purify huntingtin cleavage products and enrich for the cp-1/ cp-2 fragments. Using mass spectrometry, we found that the cp-2 fragment is generated by cleavage of huntingtin at position Arg 167 . This site was confirmed by deletion analysis and specific detection with a custom-generated cp-2 site neo-epitope antibody. Furthermore, alterations of this cleavage site resulted in a decrease in toxicity and an increase in aggregation of huntingtin in neuronal cells. These data suggest that cleavage of huntingtin at residue Arg 167 may mediate mutant huntingtin toxicity in Huntington disease.

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Inducible PC12 cell model of Huntington’s disease shows toxicity and decreased histone acetylation

Cited by 70 publications

References 21 publications

Huntingtin Phosphorylation Sites Mapped by Mass Spectrometry

Huntingtin Phosphorylation Sites Mapped by Mass Spectrometry

Huntington's Disease and the Striatal Medium Spiny Neuron: Cell-Autonomous and Non-Cell-Autonomous Mechanisms of Disease

Mutant Huntingtin N-terminal Fragments of Specific Size Mediate Aggregation and Toxicity in Neuronal Cells

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