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

Abstract

The Huntington's disease (HD) mutation is a polyglutamine expansion in the N-terminal region of huntingtin (N-htt). How neurons die in HD is unclear. Mutant N-htt aggregates in neurons in the HD brain; expression of mutant N-htt in vitro causes cell death. Other in vitro studies show that proteolysis by caspase 3 could be important in regulating mutant N-htt function, but there has been no direct evidence for caspase 3-cleaved N-htt fragments in brain. Here, we show that N-htt fragments consistent with the size produced by caspase 3 cleavage in vitro are resident in the cortex, striatum, and cerebellum of normal and adult onset HD brain and are similar in size to the fragments seen after exogenous expression of human huntingtin in mouse clonal striatal neurons. HD brain extracts treated with active caspase 3 had increased levels of N-htt fragments. Compared with the full-length huntingtin, the caspase 3-cleaved N-htt fragments, especially the mutant fragment, preferentially segregated with the membrane fraction. Partial proteolysis of the human caspase 3-cleaved N-htt fragment by calpain occurred in vitro and resulted in smaller N-terminal products; products of similar size appeared when mouse brain protein extracts were treated with calpain. Results support the idea that sequential proteolysis by caspase 3 and calpain may regulate huntingtin function at membranes and produce N-terminal mutant fragments that aggregate and cause cellular dysfunction in HD.

Figure 1

Western blots show caspase 3 cleavage of htt in transfected cells and in mouse brain. (a) X57 cells were transfected with FLAG-htt cDNAs (FH₃₂₂₁) with 18, 46, or 100 glutamines. Blots are from different gels run in parallel and probed with anti-htt antisera ab1 (Left) and 2166 (Right).The N-htt^casp3 fragment varies in size from about 80–100 kDa (arrows) depending on polyglutamine length in htt and is present in cells (C) and in debris (D). The N-htt^casp3 fragment appears as a doublet (left arrow). Proteolytic fragments of the N-htt^casp3 fragment are identified at the level of the arrowheads. The uncleaved protein expressed from FH₃₂₂₁ migrates between 140 and 160 kDa depending on polyglutamine length in htt. Native mouse full-length htt (*) and a small mouse N-htt fragment is seen in cells in blot on left. (b) X57 cells were transfected with FH₃₂₂₁-100 or not transfected (No Tf) and cells (C) and debris (D) were assayed for htt expression with Ab1. In the transfected cells, treatment with Z-VAD-FMK (25 μM) attenuates the level of the N-htt^casp3 fragment (arrow), and there is an increase in the level of a precursor fragment. The N-htt^casp3 fragment is not present in debris (D) from cells treated with Z-VAD-FMK. (c) X57 cells and MCF-7 cells were transfected with FLAG-htt cDNA FH₃₂₂₁-100. The N-htt^casp3 fragment (arrow) appears in cells and debris of X57 cells but is not present in the caspase 3 deficient MCF-7 cells. Proteolytic products of the N-htt^casp3 fragment are identified at arrowheads in the debris of X57 cells. The blot was probed with Ab1. Native mouse htt is at the top. (d) Cleavage of human htt by active caspase 3. Protein extracts from X57 cells transfected with mutant htt cDNA FH₃₂₂₁-100 were treated with active caspase 3 (500 ng) for 1 h at 37°C. There is an increase in the level of the N-htt^casp3 fragment (arrow) after exposure to active caspase 3. The blot was probed with Ab1. In a—d, 10 μg protein were loaded per lane for cell extracts, and equal volumes were loaded per lane for debris.

Figure 2

Calpain cleavage of human and mouse htt. (a) Calpain inhibitors ALLN, ALLM, and calpeptin block formation of the proteolytic products of the human N-htt^casp3 fragment. X57 cells were transfected and treated with inhibitors as described in Materials and Methods. Western blots (10 μg protein per lane) were probed with 2166 antibody. Arrows identify the level of the N-htt^casp3 fragment, and arrowheads mark the level of the “C-terminal” portions of the calpain-cleaved fragments that are detected by antibody 2166. (b) Mouse brain homogenates treated with calpain II show increase in the levels of two N-htt fragments at arrowheads migrating at about 65 kDa and 55 kDa. The arrow identifies the level of the caspase-cleaved fragments in mouse htt. Blot was probed with Ab1. Twenty micrograms of protein was loaded per lane. The numbers beside blots in a and b are molecular mass markers.

Figure 3

Western blot analysis of N-terminal htt fragments in control and HD brain. (a) Protein extracts are from controls (C2 and C8), HD patients (A4, A3, A12, J11), and from X57 cells transfected with FLAG-htt cDNA FH₃₂₂₁-46. Wt N-htt (lower arrow on left) is present in control and adult onset HD cases (A4, A3, A12). The larger N-htt fragment (upper arrows) in HD brain migrates at the same size as the N-htt^casp3 fragment produced in X57 cells transfected with a human htt cDNA encoding 46 glutamines. The asterisk identifies the level of the mutant N-htt^casp3 fragment in juvenile patient J11. Blots are from separate gels. Thirty micrograms of protein was loaded per lane for brain and 10 μg protein for the X57 cells. A prominent htt-immunoreactive band is detected with ab1 and runs below the N-htt^casp3 fragments. This fragment is unidentified. (b) Western blot probed with Ab1 shows htt expression in the cortex, striatum, and cerebellum of the same control brain. The N-htt^casp3 fragment (arrow) is present in all regions. Full-length htt and the N-htt^casp3 fragment are higher in the cortex than in the cerebellum. (c) Immunoprecipitation assays by using antisera ab1 (Left) or 2166 (Right) for immunoisolation of N-htt^casp3 fragments from brain extracts. Wt N-htt^casp3 fragment (lower arrow) is present in controls (C2, C18) and in HD patients (A12, J11, A3), and the mutant N-htt^casp3 fragment (upper arrow) is seen in adult onset HD cases (A3 and A12) but not in the juvenile HD cases (J6, J1). The three Western blots were from different experiments. The left blot was probed with 2166 and the middle and right ones with ab1. Full-length htt is at the top of the blots. Fragments are absent when primary antibody (IgG) is omitted from the assay. Twenty-five microliters was loaded per lane. (d) Cleavage of human htt by caspase 3 in homogenates prepared from the brain of adult onset HD patient A4. Protein extracts were treated with active caspase 3 (100–400 ng) for 1 h at 37°C. Wt and mutant N-htt^casp3 fragments of the expected size are produced after treatment (arrows). Proteolysis was markedly attenuated with addition of 5–10 μM Z-VAD-FMK. Actin, another substrate for caspase 3, is also cleaved in the brain extracts in the presence of active caspase 3. Ten micrograms of protein was loaded per lane. Numbers on left are molecular mass markers.

Figure 4

Western blots of htt expression are shown in subcellular fractions prepared from the cortex of a control brain (C2) and an adult onset HD brain (A4). Wt N-htt^casp3 fragment appears in control brain (lower arrow) and wt N-htt^casp3 and mutant N-htt^casp3 fragments (upper arrow) are present in HD brain. N-htt^casp3 fragments are most abundant in the membrane fraction (P2). A smaller product (arrowhead) possibly arising from proteolysis of mutant N-htt^casp3 is detected by 2166 in HD brain. Blots are from separate gels and were probed with ab1 (left) and 2166 (right). Full-length htt (*) appears in all lanes. The stacking gels (brackets) are clear except for the P1 fraction, which was hard to load. Twenty micrograms was loaded per lane. Synaptophysin, a vesicle membrane marker, appears in the P2 membrane fraction and is absent from the S2 fraction. Alpha tubulin was equally present in all fractions (not shown). Numbers beside blots are molecular mass markers.