CHIP overexpression reduces mutant androgen receptor protein and ameliorates phenotypes of the spinal and bulbar muscular atrophy transgenic mouse model

Abstract

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine tract within the androgen receptor (AR). The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem and diffuse nuclear accumulation and nuclear inclusions of the mutant AR in the residual motor neurons and certain visceral organs. Many components of the ubiquitin-proteasome and molecular chaperones are also sequestered in the inclusions, suggesting that they may be actively engaged in an attempt to degrade or refold the mutant AR. C terminus of Hsc70 (heat shock cognate protein 70)-interacting protein (CHIP), a U-box type E3 ubiquitin ligase, has been shown to interact with heat shock protein 90 (Hsp90) or Hsp70 and ubiquitylates unfolded proteins trapped by molecular chaperones and degrades them. Here, we demonstrate that transient overexpression of CHIP in a neuronal cell model reduces the monomeric mutant AR more effectively than it does the wild type, suggesting that the mutant AR is more sensitive to CHIP than is the wild type. High expression of CHIP in an SBMA transgenic mouse model also ameliorated motor symptoms and inhibited neuronal nuclear accumulation of the mutant AR. When CHIP was overexpressed in transgenic SBMA mice, mutant AR was also preferentially degraded over wild-type AR. These findings suggest that CHIP overexpression ameliorates SBMA phenotypes in mice by reducing nuclear-localized mutant AR via enhanced mutant AR degradation. Thus, CHIP overexpression would provide a potential therapeutic avenue for SBMA.

Figure 1.

Effect of CHIP on the AR or chaperones in cultured cells. A, Although anti-AR (N20) immunoblotting and densitometry demonstrated a dose-dependent decline of both wild-type (24Q) and mutant (65Q) AR expression in response to CHIP overexpression, the mutant AR decreased more than did the wild type. Mean levels of AR-24Q and AR-65Q expression were relatively compared between CHIP-transfected cells and mock-transfected cells. CHIP overexpression did not increase the expression of Hsp70, Hsp40, and Hsp90. *p < 0.005. B, The decrease in mutant AR in response to CHIP overexpression was much higher than that of the wild type (18.8% vs 38.0%; 6 μg of CHIP). *p < 0.05. C, Pulse-chase analysis of two forms of AR. Data are from one representative experiment for wild-type and mutant AR. D, Pulse-chase assessment of the half-life of wild-type (left) and mutant (right) AR. The percentages of AR-24Q and AR-65Q remaining in the absence (●) and presence (○) of overexpressed CHIP are indicated. Mutant AR was degraded more rapidly than the wild-type AR in the presence of overexpressed CHIP. E, Real-time RT-PCR of wild-type and mutant AR mRNA normalized to GAPDH levels. The wild-type and mutant AR mRNA levels were similar under CHIP overexpression. F, Ubiquitination of AR in control, AR-24Q-, and AR-97Q-transfected cells in the absence (−) or presence (+) of CHIP cotransfection. No ubiquitination was detected in control cells without expression of AR. Although both AR-24Q and AR-97Q were ubiquitinated without coexpression of CHIP, AR-97Q was strongly ubiquitinated. CHIP significantly enhanced the level of mutant AR ubiquitination but only slightly enhanced that of wild-type AR. A, B, D, E, Values represent means ± SEM (n = 5). IP, Immunoprecipitation; Ub, ubiquitin.

Figure 2.

Colocalization of nuclear-localized CHIP with mutant AR. A–L, Anti-CHIP and anti-polyQ immunohistochemistry in spinal cords of 16-week-old AR-97Q mice (A–F) and an SBMA patient (G–L). A–C, Double-immunofluorescence staining for CHIP (A; green), expanded-polyQ (B; red), and overlay of the two signals (C; yellow) in the spinal anterior horn cells. D–F, CHIP (green; D) and mutant AR (red; E) are colocalized in nuclear inclusions (shown in yellow; F) in the spinal anterior horn cell. G–I, Double-immunofluorescence staining in cells of the hypoglossal nucleus of an SBMA patient revealed diffuse nuclear colocalization of CHIP (G) and mutant AR (H, I). J–L, CHIP (green; J) and mutant AR (red; K) were also colocalized in NIs (shown in yellow; L) in the spinal anterior horn cell of SBMA patients.

Figure 3.

Increased CHIP expression in double-transgenic mice. A, Schematic view of the transgene construct. The microinjected fragment was composed of a cytomegalovirus enhancer (E), a chicken β-actin promoter (Pro), full-length human CHIP with a myc tag, and a rabbit β-globin polyadenylation signal sequence (polyA). B, Western blot analysis of total spinal cord and muscle protein lysates from AR-97Q/CHIP^(−/−), AR-97Q/CHIP^(tg/−), and AR-97Q/CHIP^(tg/tg) mice immunolabeled with antibodies against CHIP, Hsp90, Hsp70, and Hsp40. C, Coimmunoprecipitation Western blots for CHIP. Soluble fractions were collected from the spinal cord of AR-97Q/CHIP^(−/−) and AR-97Q/CHIP^(tg/tg) mice. Equal amounts of protein were immunoprecipitated with an antibody to myc and immunoblotted for AR and Hsp70. Coimmunoprecipitation of CHIP and the polyQ-expanded mutant AR or the Hsp70 chaperone was detected. D, Western blot analysis of CHIP expression in total spinal cord and muscle protein lysates from CHIP^(tg/tg) and AR-97Q/CHIP^(tg/tg) mice of the indicated ages, immunolabeled with antibodies against myc. E, F, CHIP immunohistochemistry in spinal anterior horn and skeletal muscle of 16-week-old AR-97Q/CHIP^(tg/tg) mice counterstained with Mayer's hematoxylin. E, CHIP immunoreactivity is localized to the nuclei and cytoplasm, with intense and diffuse staining in the anterior horn cells. F, Skeletal muscle showed diffuse nuclear and cytoplasmic staining. IP, Immunoprecipitation.

Figure 4.

Effects of human CHIP overexpression on the behavioral phenotypes in male AR-97Q mice. A–D, Rotarod task (A; n = 22), cage activity (B; n = 22), body weight (C; n = 26), survival rate (D; n = 26) of the AR-97Q/CHIP^(−/−) (○), AR-97Q/CHIP^(tg/−) (□), and AR-97Q/CHIP^(tg/tg) mice (▵). AR-97Q mice overexpressing human CHIP remained longer on the rotarod and showed higher cage activity than the AR-97Q/CHIP^(−/−). The AR-97Q/CHIP^(−/−) lost weight earlier than the other two double transgenics. D, A Kaplan-Meier plot shows the prolonged survival of AR-97Q/CHIP^(tg/−) and AR-97Q/CHIP^(tg/tg) mice compared with the AR-97Q/CHIP^(−/−). The AR-97Q/CHIP^(−/−) mice were significantly different from either of the other two in all parameters tested. Moreover, the AR-97Q/CHIP^(tg/−) mice were worse off than the AR-97Q/CHIP^(tg/tg) in all parameters tested. E, Footprints of representative 16-week-old AR-97Q/CHIP^(−/−), AR-97Q/CHIP^(tg/−), and AR-97Q/CHIP^(tg/tg) mice. Front paws are indicated in red, and hindpaws are indicated in blue. AR-97Q/CHIP^(−/−) mice exhibit motor weakness with dragging of the legs, AR-97Q/CHIP^(tg/tg) mice walk almost normally, and AR-97Q/CHIP^(tg/−) mice walk with somewhat shorter steps. F, The average length of hindpaw steps in 16-week-old AR-97Q/CHIP^(−/−), AR-97Q/CHIP^(tg/−), and AR-97Q/CHIP^(tg/tg) mice. Values are expressed as means ± SEM (n = 6). *p < 0.005.

Figure 5.

CHIP decreases nuclear-localized mutant AR in double-transgenic mice. A–F, PolyQ immunohistochemistry (1C2) in the spinal anterior horn (A–C) and muscle (D–F) of 16-week-old AR-97Q/CHIP^(−/−) and AR-97Q/CHIP double-transgenic mice. AR-97Q/CHIP^(−/−) mice have intense and frequent staining for 1C2 in the nucleus (A, D). B, C, E, F, AR-97Q/CHIP^(tg/−) (B, E) and AR-97Q/CHIP^(tg/tg) (C, F) mice exhibit low levels of 1C2 staining in the nucleus. G, H, Quantitative assessment of diffuse nuclear staining for 1C2 in the spinal ventral horn (G) and muscle (H). Bars represent the density of 1C2-positive cells in the AR-97Q/CHIP^(−/−), AR-97Q/CHIP^(tg/−), and AR-97Q/CHIP^(tg/tg) mice. There are significantly more 1C2-positive cells in AR-97Q/CHIP^(−/−) mice than in AR-97Q/CHIP^(tg/−) mice or AR-97Q/CHIP^(tg/tg) mice in both tissues. Results are expressed as mean ± SEM for six mice. *p < 0.025; **p < 0.005. I, Immunohistochemical staining with GFAP-specific antibody also showed an obvious reduction in reactive astrogliosis in the spinal anterior horn of AR-97Q/CHIP^(tg/tg) mice. J, Hematoxylin and eosin (HE) staining of muscle tissue in AR-97Q/CHIP^(−/−) mice revealed obvious atrophy and small-angulated fibers, which were not seen in AR-97Q/CHIP^(tg/tg) mice. No., Number.

Figure 6.

CHIP decreases mutant AR protein complexes as well as monomeric mutant AR. A, B, Western blot analysis of total tissue homogenates from the spinal cord (A) and muscle (B) of AR-24Q/CHIP^(−/−), AR-24Q/CHIP^(tg/tg), AR-97Q/CHIP^(−/−), and AR-97Q/CHIP^(tg/tg) mice (16-week-old) probed with an AR-specific antibody (H280). The mutant AR complex appears in the stacking gel, and the monomeric mutant AR appears in the separating gel. Values are expressed as mean ± SEM for six mice. *p < 0.001; **p < 0.0001. C, Real-time RT-PCR of wild-type (AR-24Q) and mutant AR (AR-97Q) mRNA in transgenic mouse spinal cord and skeletal muscle in the absence (wt) and presence (CHIP) of CHIP overexpression. Values are expressed as means ± SE (n = 6). D, Filter trap assay of total tissue homogenates from the spinal cord and muscle of AR-97Q/CHIP^(−/−) and AR-97Q/CHIP^(tg/tg) mice (16 weeks of age), in the absence and presence of CHIP overexpression. Homogenates were filtrated and immunolabeled with an antibody against AR (H280). Large aggregated mutant AR complexes were trapped by the cellulose acetate membrane; soluble monomeric mutant AR passed through the cellulose acetate membrane and was trapped by the nitrocellulose membrane. Endogenous α-tubulin was used as a loading control.