SIRT1 modulates aggregation and toxicity through deacetylation of the androgen receptor in cell models of SBMA

Abstract

Posttranslational protein modifications can play a major role in disease pathogenesis; phosphorylation, sumoylation, and acetylation modulate the toxicity of a variety of proteotoxic proteins. The androgen receptor (AR) is substantially modified, in response to hormone binding, by phosphorylation, sumoylation, and acetylation; these modifications might thus contribute to DHT-dependent polyglutamine (polyQ)-expanded AR proteotoxicity in spinal and bulbar muscular atrophy (SBMA). SIRT1, a nuclear protein and deacetylase of the AR, is neuroprotective in many neurodegenerative disease models. Our studies reveal that SIRT1 also offers protection against polyQ-expanded AR by deacetylating the AR at lysines 630/632/633. This finding suggested that nuclear AR acetylation plays a role in the aberrant metabolism and toxicity of polyQ-expanded AR. Subsequent studies revealed that the polyQ-expanded AR is hyperacetylated and that pharmacologic reduction of acetylation reduces mutant AR aggregation. Moreover, genetic mutation to inhibit polyQ-expanded AR acetylation of lysines 630/632/633 substantially decreased its aggregation and completely abrogated its toxicity in cell lines and motor neurons. Our studies also reveal one means by which the AR acetylation state likely modifies polyQ-expanded AR metabolism and toxicity, through its effect on DHT-dependent AR stabilization. Overall, our findings reveal a neuroprotective function of SIRT1 that operates through its deacetylation of polyQ-expanded AR and highlight the potential of both SIRT1 and AR acetylation as powerful therapeutic targets in SBMA.

Figure 1.

SIRT1 abrogates mutant AR aggregation and toxicity in a manner dependent upon its deacetylase activity. PC12 cells expressing AR112Q were stably transfected with either SIRT1 or deacetylase-deficient SIRT1(H363Y). A, Stable overexpression of SIRT1 in AR112Q-expressing PC12 cells resulted in a significant reduction in the number of cells containing nuclear inclusions, compared with control, following treatment with 10 nm DHT for 48 h, while deacetylase-deficient SIRT1(H363Y) increased nuclear inclusions. ANOVA with post hoc Tukey test was performed to determine statistical significance. *p < 0.01 B, Western analysis of AR in cells treated in parallel with those in A reveals a substantial decrease in SDS-insoluble AR112Q upon overexpression of SIRT1, and an increase in high molecular weight species upon overexpression of SIRT1(H363Y). C, Cells were treated for 12 d with either 10 nm DHT or EtOH. Two hundred cells were counted from triplicate wells for each treatment and the percentage of Trypan blue-positive cells was graphed. SIRT1 rescued AR112Q-expressing PC12 cells from DHT-dependent death, while SIRT1(H363Y) did not. Also note that SIRT1(H363Y) enhanced DHT-dependent death. ANOVA with post hoc Tukey test was performed to determine statistical significance. *p < 0.05.

Figure 2.

Reduction of SIRT1 in cells expressing polyglutamine-expanded AR increases nuclear inclusions. PC12 cells were treated with siRNA against SIRT1 or nontargeting siRNA. Twenty-four hours later cells were treated with doxycycline to induce AR112Q expression and 10 nm DHT. A, After 48 h, cells were harvested and expression of SIRT1 and AR (AR-H280) was evaluated by Western analysis. Seventy-seven percent knockdown of SIRT1 was achieved. GAPDH was used as a loading control. B, Cells treated as in A were fixed and immunostained for AR (AR-H280), and 300 cells from each of three wells per treatment were counted and the number of cells with AR nuclear inclusions was determined. Reduction of SIRT1 significantly increased the number of cells with nuclear inclusions of AR112Q. Student's t test was performed to determine statistical significance. *p < 0.01.

Figure 3.

SIRT1 protects motor neurons expressing polyglutamine-expanded AR from DHT-dependent death. Dissociated spinal cord cultures were initiated from transgenic mouse embryos expressing AR112Q. SIRT1, deacetylase deficient SIRT1 (SIRT1(H363Y); both tagged with myc-his) or EGFP were overexpressed using AAV infection. Five days after infection, cultures were treated with DHT or EtOH for an additional 7 d and motor neuron number was determined. SIRT1 expression was assayed using an anti-myc antibody. A, Motor neurons from 10 random fields of cells were counted under 20,000× magnification from triplicate wells for each treatment and the average number of motor neurons was determined. SIRT1 protected AR112Q-expressing motor neurons from DHT-dependent death, while SIRT1(H363Y) did not. B, Images represent motor neurons treated with DHT as described in A. NF-H, Unphosphorylated neurofilament-heavy chain. Percentage infectivity of motor neurons with AAV: 92% EGFP (control), 88% SIRT1, and 94% SIRT1(H363Y). ANOVA with post hoc Tukey test was performed to determine statistical significance. *p < 0.01.

Figure 4.

SIRT1 reduces acetylation of polyQ-expanded AR. A, Cells were treated with 10 nm DHT or EtOH for 48 h. Immunoprecipitation (IP) of acetyl-lysine and immunoblotting (IB) for AR (AR-H280) revealed a substantial reduction in AR112Q acetylation when SIRT1, but not SIRT1 (H363Y), was overexpressed. Western analysis revealed equivalent levels of AR between cell lines. B, Relative densitometric values of AR following acetyl-lysine IP and Western analysis of AR in AR112Q-expressing cells and in AR112Q-expressing cells that coexpress either SIRT1 or SIRT1 (H363Y). The level of acetylated AR in AR112Q cells was set as 100. A significant reduction in AR112Q acetylation was observed in the presence of SIRT1. Student's t test was performed to determine statistical significance. *p < 0.05.

Figure 5.

Lysines 630, 632, and 633 within the AR are required for SIRT1 to decrease polyglutamine-expanded AR acetylation. PC12 cells expressing polyQ-expanded AR110Q with an exogenous nuclear localization signal in triplicate (NLSX3) were used for these studies. PC12 cells expressing AR110Q or AR110Q with lysines 630, 632, and 633 mutated to acetylation-null arginine (AR110Q-R3) in the presence and absence of stable overexpression of SIRT1 were treated with 10 nm DHT for 48 h. Acetyl-lysine was immunoprecipitated from protein lysates and AR levels determined by Western analysis (AR-H280). Overexpression of SIRT1 reduced the levels of acetylated AR110Q, while it had no effect on levels of acetylated AR110Q-R3. Note that levels of acetylated AR111Q-R3 were reduced compared with those of AR110Q. Equivalent levels of acetyl-lysine were immunoprecipitated from each sample (data not shown). Western analysis of total protein lysates revealed equivalent levels of AR between cell lines.

Figure 6.

The neuroprotective role of SIRT1 in motor neurons expressing polyQ-expanded AR is dependent upon AR lysines 630, 632, and 633. Dissociated spinal cord cultures were initiated from nontransgenic embryonic mice. AAVs were made to express EGFP, SIRT1-myc-his, AR111Q, or acetylation-mimic AR111Q-Q3 [both forms of AR contain an exogenous nuclear localization signal in triplicate (NLSX3) at the N terminus]. After 5 d, the percentage infectivity of motor neurons with AR, EGFP, and SIRT1 was determined by immunofluorescence. The rest of the cultures were then treated with either EtOH or DHT for an additional 7 d. Immunofluorescence analysis was performed to reveal motor neurons (with antibody SMI32) and AR (AR-318) and Hoechst 33258 was used to stain nuclei. Motor neurons were counted from 10 random fields of cells under 20,000× magnification from triplicate wells for each treatment. The number of viable motor neurons expressing AR is represented in the graph. DHT treatment significantly reduced the number of motor neurons expressing AR111Q/EGFP, as well as those expressing AR111Q-Q3/EGFP. SIRT1 overexpression in motor neurons expressing AR111Q (lysine intact) rescued these motor neurons from DHT-dependent death, while SIRT1 did not protect motor neurons expressing AR111Q-Q3 (acetylation-mimic). ANOVA with post hoc Tukey test was performed to determine statistical significance. *p < 0.001.

Figure 7.

Nuclear polyglutamine-expanded AR is hyperacetylated compared with normal glutamine-length AR. AR10Q and AR112Q-expressing PC12 cells were treated with 10 nm DHT for 48 h. A, Acetylation of AR was evaluated by immunoprecipitation (IP) of acetyl-lysine from cell lysates and subsequent Western analysis. A representative Western blot shows that a larger proportion of AR112Q is acetylated compared with AR10Q, and that acetylated AR112Q is observed in an SDS-insoluble form in the well and stacking layer of the gel. Reprobing of the membrane revealed equivalent acetyl-lysine immunoprecipitated from AR10Q and AR112Q lysates. Western analysis of total lysate reveals equivalent AR levels. GAPDH was used as a loading control for total lysates. B, Nuclear and cytoplasmic fractions of cell lysates were used for immunoprecipitation (IP) of acetyl-lysine, and the immunoprecipitates were electrophoresed by SDS-PAGE and immunoblotted (IB) for AR (ARH280). Nuclear, but not cytoplasmic, AR10Q and AR112Q were immunoprecipitated with acetyl-lysine antibody. The nuclear/cytoplasmic fractions were verified by running a portion of the fractions on an SDS-page gel and probing the membrane for AR, nuclear (HMGB1), and cytoplasmic (GAPDH) markers. Note that the signal in the upper-left portion of the ARH280-probed membrane represents nonspecific background signal. There is only a faint signal of AR10Q in the nuclear fraction at 110 kDa. C, Cytoplasmic fraction; N, nuclear fraction.

Figure 8.

Pharmacologic reduction of polyglutamine-expanded AR reduces nuclear inclusions. AR112Q-expressing PC12 cells were treated in triplicate for 48 h with 10 nm DHT in the presence and absence of 30 μm anacardic acid (inhibitor of CBP/p300 and PCAF). A, Immunoprecipitation of acetyl-lysine and subsequent Western analysis for AR revealed that AA treatment reduced acetylation of AR112Q. Shown here is a representative Western blot. B, AR112Q-expressing cells treated in parallel were fixed and immunostained for AR. Five-hundred cells were counted from each of three wells and the percentage of cells with nuclear inclusions graphed. Anacardic acid reduced the number of cells with nuclear inclusions. Student's t test was performed to determine statistical significance. *p < 0.01.

Figure 9.

Genetic inhibition of mutant AR acetylation ameliorates polyQ-expanded AR aggregation and toxicity. PC12 cells expressing polyQ-expanded AR110Q with an exogenous nuclear localization signal in triplicate (NLSX3) at the N terminus were used for these studies. Cells expressing a single acetylation-null mutation (AR-K630A–110Q), a triple acetylation-null mutant (AR-R3–110Q), or triple acetylation-mimic mutant (AR-T3–110Q or AR-Q3–110Q) were treated with 10 nm DHT or EtOH for 48 h. A, Cells were immunostained and the percentage of cells (300 cells from triplicate wells) containing nuclear inclusions was graphed. A substantial reduction in the number of cells with nuclear inclusions was observed upon expression of acetylation-null (K630A; R3) expanded AR, while an increase (T3) or a similar inclusion load (Q3) was observed upon expression of acetylation-mimic expanded AR. ANOVA with post hoc Tukey test was performed to determine statistical significance. *p < 0.05 B. Cells were treated for 12 d and evaluated for cell death. Two hundred cells were counted from triplicate wells for each treatment. Normal AR110Q- and acetylation-mimic AR-expressing cells died in response to DHT, while acetylation-null AR-expressing cells remained viable. AR35Q-expressing cells did not form inclusions or die in response to DHT. ANOVA with post hoc Tukey test was performed to determine statistical significance. *p < 0.01 C, Western analysis of AR (AR-H280) revealed equivalent levels of AR between all cell lines. GAPDH served as a loading control. A longer development of the AR immunoblot revealed a substantial reduction in SDS-insoluble polyQ-expanded AR in cells expressing acetylation-null AR, while there was an increase in SDS-insoluble high molecular weight species of AR in cells expressing acetylation-mimic AR.

Figure 10.

Acetylation-null polyQ-expanded AR does not induce DHT-dependent death of motor neurons. Dissociated spinal cord cultures were initiated from nontransgenic embryonic mice. AAVs were made to express AR111Q or acetylation-null AR111Q-R3 with an exogenous nuclear localization signal in triplicate (NLSX3) at the N terminus. The cultures were infected with these viruses to achieve an equal number of motor neurons expressing AR. After 5 d, cultures were treated with either EtOH or DHT for an additional 7 d. A, Image represents immunofluorescence of DHT-treated cultures that were noninfected or infected with virus expressing AR111Q or AR111Q-R3. Percentage infectivity of motor neurons with AAV = 78% AR111Q, 82% AR111Q-R3. B, Protein lysates harvested from DHT-treated cultures and analyzed by Western blotting using AR-H280 and GAPDH as a loading control. Similar levels of AR111Q and AR111Q-R3 were observed. C, The number of motor neurons expressing AR was graphed following 7 d of EtOH or DHT treatment. Immunofluorescence analysis was performed to reveal motor neurons (with antibody SMI32) and AR (AR-318); Hoechst 33258 was used to stain nuclei. Motor neurons were counted from 10 random fields of cells under 20,000× magnification from triplicate wells for each treatment. DHT treatment significantly reduced the number of motor neurons expressing AR111Q, while DHT had no effect on the number of viable motor neurons expressing acetylation-null AR111Q-R3. ANOVA was performed with post hoc Tukey test to determine significance. *p < 0.005.

Figure 11.

Acetylation site mutation alters the stabilization of AR by DHT. PC12 cells expressing polyQ-expanded AR110Q with an exogenous nuclear localization signal in triplicate (NLSX3) at the N terminus were used for these studies. Cells expressing lysine-intact AR (AR-110Q), AR with a single acetylation-null mutation (AR-K630A–110Q), a triple acetylation-null mutant AR (AR-R3–110Q), or triple acetylation-mimic AR mutants (AR-T3–110Q or AR-Q3–110Q) were treated with doxycycline (DOX) to express equivalent levels of AR for 48 h, then DHT samples were treated for an additional 36 h with 10 nm DHT in the absence of DOX. Western analysis of AR (AR-H280) reveals reduced DHT-stabilization of acetylation-null mutant AR110Q. Statistical analysis was performed to determine differences between samples with or without DHT for each cell line. Additionally, the percentage increase in AR levels in the presence of DHT was compared between cell lines. Note that the T3 (acetylation-mimic) AR110Q AR is stabilized substantially more than lysine-intact (K3) AR110Q. Student's t test was used to determine statistical significance.