Wild-type huntingtin protects from apoptosis upstream of caspase-3

Abstract

Expansion of a polyglutamine sequence in the N terminus of huntingtin is the gain-of-function event that causes Huntington's disease. This mutation affects primarily the medium-size spiny neurons of the striatum. Huntingtin is expressed in many neuronal and non-neuronal cell types, implying a more general function for the wild-type protein. Here we report that wild-type huntingtin acts by protecting CNS cells from a variety of apoptotic stimuli, including serum withdrawal, death receptors, and pro-apoptotic Bcl-2 homologs. This protection may take place at the level of caspase-9 activation. The full-length protein also modulates the toxicity of the poly-Q expansion. Cells expressing full-length mutant protein are susceptible to fewer death stimuli than cells expressing truncated mutant huntingtin.

Fig. 1.

Expression of wt and muHtt in stable cell clones.A, Outline of the Htt constructs used and abbreviation of the stable ST14A subclones obtained. B, Expression of wt and muHtt in stable cell clones. Western blot analysis on lysates from clones expressing FLwt (9–25 clone), FLmu (10–6 clone), N548wt (7–12 clone), N548mu (8–2 clone), N63wt (13–5 clone), and N63mu (14–2 clone). The FL and 548aa-truncation were detected with MAB2166 after electrophoresis on a 6 or 10% SDS-PAGE, respectively. The shorter 63aa protein was revealed by an anti-myc antibody after running the samples on a 15% SDS-PAGE. Each blot includes a lane with lysates from parental ST14A cells. Native huntingtin in the cells is detectable at longer exposures. C, Lysates from 7–12 and 8–2 clones were analyzed for protein expression after 5, 10, and 25 passages in vitro. Equal loading was confirmed by reacting the membrane with an anti-actin antibody.

Fig. 2.

Morphology and confluency of parental ST14A (a, b), N548mu (8–2 clone) (c, d), and N548wt (7–12 clone) (e, f) cells in regular passaging conditions (i.e., at 33°C in serum; a, c, e) and after exposure to serum-deprived medium at 39°C for 48 hr (b, d, f). N548wt evokes increased cell survival, whereas N548mu leads to higher cell death in the cultures with respect to parental ST14A cells.

Fig. 3.

Coulter counter (a, b) and MTT (c, d) assays on parental ST14A (open square), N548mu (open circle), and N548wt (open diamond) cells. a andc refer to the normal passaging conditions, i.e., 33°C in serum. b and d indicate the values obtained after exposure at 39°C in SDM. The graphs show the mean and SE of MTT assays (n = 12 independent experiments) and Coulter counter assays (n = 4 independent experiments) performed on three clones expressing N548wt and on two clones bearing the N548mu cDNA. Two clones expressing N63wt (filled diamond) and two expressing FLwt (asterisk) were analyzed by the same assay. All these clones behaved as parental ST14A cells in normal passaging conditions.

Fig. 4.

A, Modification in cell viability and/or mitochondrial activity after 3-NP exposure. Cells were exposed to the indicated doses of 3-NP and analyzed after 48 hr by MTT assay. Parental ST14A cells and representative clones expressing N548wt or N548mu are shown. Protection from 3-NP is also seen in FLwt cells (*p < 0.05 with respect to parental cells). Shown is one of three independent experiments performed on the different clones that gave the same results. B, Hoechst 33258 staining of the supernatant from ST14A cells shows that 3-NP exposure induces apoptotic cell death.

Fig. 5.

A, DNA laddering analysis in ST14A cells, in N548mu cells (8–2 clone), and N548wt cells (7–12 clone) after the temperature and serum-deprived medium shift. Low molecular weight DNA samples were prepared 1, 4, and 20 hr after the shift. As a control, samples from cells in normal growth conditions (33°C) were included (0 time). These data were replicated in another independent experiment. B, Hoechst 33258 staining of parental ST14A (a, b), N548mu (c, d), and N548wt (e, f) cells at 33°C (a, c, e) and 39°C (b, d, f). A higher number of cells with condensed DNA is visible in N548mu cells (d) with respect to controls (parental ST14A, b). No DNA condensation is observed in N548wt cells (f).

Fig. 6.

A, Measurement of caspase-3-like activity. The release of the fluorogenic amc moiety from the caspase-3-specific substrate Ac-DEVD-amc is reported at various times (0, 3, 6, 9, and 12 hr) after the temperature shift. Basal levels of caspase-3 (time 0, white bar) were obtained at 33°C. Data are expressed as nanomoles of cleaved substrate. Shown is one of three experiments performed on different clones that gave similar results. B, Cotransfection of a constitutively active caspase-3 together with the EGFP plasmid in parental ST14A (open bars), N548wtHtt (black bars), and FLwtHtt (gray bars) cells. Cells were transiently cotransfected with the active caspase-3 or a control vector (pLXSP) together with the EGFP-N1 plasmid (Clontech). Cell viability was determined 30 hr after transfection by counting the ratio of fluorescent cells (EGFP-positive) versus total number of cells (*p < 0.01 vs EGFP vector). Data represent the mean of three independent experiments.

Fig. 7.

TUNEL of the stable cell lines expressing Htt after transfection with different death inducers. ST14A (dotted bars), N548wt (7–12 clone, black bars), N548mu (8–11 clone, horizontal striped bars), and FLmu (10–11 clone, diagonal striped bars) cells were transfected with 1 μg of the indicated plasmids (0.4 μg for BIK and BAK) or a control protein. The inset depicts a typical TUNEL readout. A and B show gating on the EGFP-expressing cell population. C shows TUNEL staining of cells transfected with the control protein; D shows TUNEL staining of cells transfected with p55/TNFR. The control protein used is 14-3-3. In ST14A cells p55, BIK, BAK, and caspase-9 induce apoptosis, whereas BAD and caspase-3 show no effect. Apoptosis by all inducers is blocked in cells expressing wtHtt. FLmu cells show similar levels of apoptosis as the parental line for p55, BIK, BAK, and caspase-9 and a marked increase in BAD and caspase-3-induced apoptosis. This increase is also seen in N548mu cells. Shown is the average of three independent experiments. Error bars show the average deviation.