Tissue-Specific Proteolysis of Huntingtin (htt) in Human Brain: Evidence of Enhanced Levels of N- and C-Terminal htt Fragments in Huntington's Disease Striatum

Mende‐Mueller, Liane; Toneff, Thomas; Hwang, Shin-Rong; Chesselet, Marie‐Françoise; Hook, Vivian

doi:10.1523/jneurosci.21-06-01830.2001

Cited by 139 publications

(108 citation statements)

References 25 publications

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“…Mutant N-htt fragments produced by calpain cleavage might undergo further proteolysis in the cytoplasm. N-htt fragments smaller in size than the calpain-cleaved N-htt fragments were found elevated in HD striatum compared with controls (22).…”

Section: Discussionmentioning

confidence: 76%

Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis

Kim

Sapp

et al. 2001

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

346

250

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A polyglutamine expansion located in the N terminus of huntingtin (N-htt) causes Huntington's disease (HD). How the mutation causes cell death is unknown. Several recent observations implicate altered huntingtin (htt) processing in the pathogenesis of HD. In the HD brain, mutant N-htt fragments aggregate in the nucleus and cytoplasm (1); the expression of mutant N-htt fragments in vitro causes cell death (2, 3). These findings suggest that proteolysis in the N-terminal region of htt may be important in HD pathogenesis. Furthermore, htt cleavage by caspase 3 could contribute to neurodegeneration in HD. Htt can serve as a substrate for caspase activity. Two proximate caspase 3-sensitive sites and one caspase 6-sensitive site are distal to the polyglutamine tract at aspartate residues 513, 552, and 589, respectively, in the wild-type (wt) protein (4-6). Caspase 3-cleaved N-htt products have been observed in vitro after exogenous expression of human htt in HEK 293 cells (4) and clonal striatal neurons (7). Treatment with the broad acting caspase inhibitor Z-VAD-FMK attenuates caspase 3 cleavage of htt and increases cell survival (6, 7). Enhanced immunoreactivity for caspases has been reported in HD striatal neurons compared with control brain, § supporting the involvement of caspase activation in HD pathogenesis. However, there is no evidence for htt proteolysis by caspases in the brain. No non-caspase proteases have been identified that produce a limited proteolysis of htt. Thus, despite the presence of N-htt fragments in adult and juvenile HD brain (1), the proteolytic pathway involved in the production of mutant N-htt fragments in the brain is unknown. That caspase 3 or other proteases cleave the N terminus of mutant htt in the HD brain would provide strong support that N-terminal htt proteolysis is a critical factor in HD pathogenesis.Here, we demonstrate that caspase 3-cleaved N-htt fragments occur in the control and HD brain and are similar in size to fragments produced in vitro after exogenous expression of human wt and mutant htt in mouse clonal striatal cells (7,8). The wt and mutant caspase 3-cleaved N-htt fragments in brain varied in size with polyglutamine length and were preferentially enriched in membrane fractions. Partial proteolysis of the caspase 3-cleaved N-htt fragments by calpain produced smaller Nterminal fragments. We speculate that caspase 3 cleavage regulates the proteolysis of wt and mutant htt in the brain and increases the association of N-terminal htt with membranes. Calpain-induced proteolysis of the caspase 3-cleaved mutant N-htt fragment may lead to the formation of mutant N-htt fragments that can aggregate and form inclusions. Materials and MethodsCell Culture and Transfections. The culturing and transfection of mouse clonal striatal cells (X57 cells) have been described in our recent publications (7,8,9). MCF-7 cells were grown according to the suppliers' recommendations [American Tissue Culture Collection (ATCC)]. DNA was introduced by using Superfect Transfection Reagent (Qiagen, ...

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Section: Discussionmentioning

confidence: 76%

Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis

Kim

Sapp

et al. 2001

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

346

250

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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

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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“…The N-terminal rabbit polyclonal Htt antibody described previously 46 was used for immunoprecipitation. Immunoprecipitation and Western analysis was performed as described above.…”

Section: Cell Culture and Antibodiesmentioning

confidence: 99%

Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease

et al. 2004

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Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

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Tissue-Specific Proteolysis of Huntingtin (htt) in Human Brain: Evidence of Enhanced Levels of N- and C-Terminal htt Fragments in Huntington's Disease Striatum

Cited by 139 publications

References 25 publications

Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis

Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis

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

Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease

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