Article Figures & Data
Figures
- Fig. 1.
Htt is cleaved by calpains into four major cleavage products. A, Treatment of the in vitro-translated N-terminal Htt construct Htt 1955-15 with μ- and m-calpain produced three products. The cleavage is dependent on enzyme concentrations with low levels (L) of μ- and m-calpain (0.1 and 0.3 μm, respectively) producing 67 and 62 kDa fragments and higher levels (H) of μ- and m-calpain (0.3 and 3.0 μm, respectively) producing a single 47 kDa fragment. B, The two larger Htt fragments produced by μ- and m-calpain at lower (L) enzyme concentrations are better visualized with longer exposure times. C, Calpain cleavage ofin vitro-translated Htt (1955-15) is also dependent on Ca2+ concentration and is inhibited by the calpain inhibitor known as calpain inhibitor 1. D, Cleavage ofin vitro-translated Htt 3949-15 with caspase-3, m-calpain, caspase-2, and caspase-6. E, Table of potential calpain cleavage sites. Caspase cleavage sites in Htt are highlighted in red. Caspase-3 cleaves at 513 and 552, caspase-2 cleaves at 552, and caspase-6 cleaves at 586.a, Molecular weight of a fragment on the gel (MWG , in kilodaltons; a, b andc–f values from two separate experiments);b, predicted molecular weight determined from the relative molecular weight of the caspase-3 cleavage product [MWP , in kilodaltons; MWP = MWG × MWC (caspase-3)/MWG(caspase-3)]; c, calculated molecular weight (MWC ) of a fragment based on predicted cleavage site(s) (in kilodaltons); d, P1 amino acid number and sequence for predicted calpain cleavage site(s) and known caspase cleavage sites; e, predicted amino acid sequence recognized by calpains (P2, P1, and P1′) or the amino acid sequence recognized by caspases; f, amino acid sequence of a calpain cleavage site in protein kinase C; g, amino acid sequence of a calpain cleavage site in caspase-12.F, Htt amino acid sequence from 437 to 586. Caspase sites are highlighted in red, and potential calpain sites are highlighted in blue.
- Fig. 2.
The length of the polyglutamine tract modulates calpain cleavage of Htt. A, Cleavage of in vitro-translated Htt 3949-15 with m-calpain produced three distinct cleavage products, whereas the cleavage of Htt 3949-138 produced only a small amount of the largest calpain repeat-dependent cleavage product and barely detectable levels of the two smaller repeat-dependent cleavage products. B, Incubatingin vitro-translated Htt (Htt 3949-15 and Htt 3949-138) with increasing concentrations of m-calpain demonstrated that the 92 kDa cleavage product derived from mutant Htt is more rapidly produced and degraded at lower enzyme concentrations than the corresponding 72 kDa wild-type Htt cleavage product (n = 3).C, In the presence of m-calpain (30 nm), expanded Htt (Htt 3949-138) is preferentially cleaved relative to the normal protein (Htt 3949-15) (n = 3).
- Fig. 3.
According to Western blot analysis, calpain activation and Htt fragmentation are observed in thapsigargin-treated 293T cells overexpressing Htt. A, 293T cells overexpressing full-length Htt15 treated with thapsigargin (Thaps) and probed with calpain (Cp) regulatory subunit antibody. B, 293T cells overexpressing full-length Htt15 and caspase-resistant full-length Htt15 D513A, D552A, D586A treated with thapsigargin (Thaps) and probed with anti-Htt antibody 2166. The controls in B are lysates from 293T cells overexpressing full-length Htt15, which are subsequently treated with m-calpain.C, 293T cells overexpressing normal Htt 3949-15 and expanded Htt 3949-138 treated with thapsigargin (Thaps) and probed with anti-Htt antibody 2166.
- Fig. 4.
According to Western blot analysis, calpain activation and Htt fragmentation are observed in the HD caudate.A, Human HD and control caudate lysates probed with calpain (Cp) regulatory subunit antibody (n = 3). B, Quantification of calpain regulatory subunit expression in the human HD and control caudate (n = 3). C, Quantification of μ-calpain large subunit expression in the HD and control caudate (n = 3). D, Human HD and control caudate lysates probed with anti-Htt antibody 2166 (n = 3). The controls are lysates from 293T cells overexpressing full-length Htt15 and Htt44, which are subsequently treated with m-calpain. The cleavage product labeled with anasterisk represents an Htt44 N-terminal calpain cleavage product equivalent in size to the largest N-terminal cleavage product found in HD lysates.
- Fig. 5.
Increased expression of the calpain regulatory subunit in the HD caudate. Immunohistochemical analysis shows that high levels of calpain are localized to perinuclear vesicles in cells near the lateral ventricle (A, C, E) as well as to medium spiny neurons deeper within the caudate (B, D, F) in HD patients relative to controls (n = 3). In addition, calpain is expressed at high levels in the Lewy bodies of HD patients (C). Arrowheads indicate perinuclear vesicles. Lewy bodies are to the right of the asterisk. In A, C, andE, the lateral ventricle is toward thebottom of the picture. Insets inC and D represent boundaries of the 100× image in E and F, respectively.
- Fig. 6.
Both μ-calpain and m-calpain are upregulated in the HD caudate. Immunohistochemical analysis shows increased expression of the μ- and m-calpain catalytic subunit, as well as Htt, in the caudate from HD patients (A–I). Intense perinuclear vesicular staining of all three proteins is observed in HD caudate tissue surrounding the lateral ventricle (A–I) as well as in cells deeper within the caudate, including medium spiny neurons. Staining with calpain isoform-specific antibodies also indicates that only μ-calpain and Htt proteins (not m-calpain) are associated with Lewy bodies (data not shown). Arrowheads indicate perinuclear vesicles, and intranuclear staining is indicated by an asterisk. In A–I, the lateral ventricle is toward thebottom of the picture. Insets inB, E, and H represent boundaries of the 100× image in C, F, and I, respectively.
Tables
- Table 1.
Human caudate tissue samples used for Western blot analysis and immunohistochemistry
Tissue type Age Sex PMI (hr) Use Control 69 F 20.7 WB 44 F 10.7 WB 63 M 21.5 WB 73 M 12.0 IHC 71 M 11.0 IHC 71 M 24.0 IHC 70 M 17.3 IHC HD (grade 3): 63 F 16.0 WB 32 F 13.4 WB 55 F 24.4 WB 66 M 22.0 IHC 48 M 19.2 IHC 74 F 21.0 IHC PMI, Postmortem interval; WB, Western blot; IHC, immunohistochemistry; F, female; M, male.
