Peripheral androgen receptor gene suppression rescues disease in mouse models of spinal and bulbar muscular atrophy

Summary

Spinal and bulbar muscular atrophy (SBMA) is caused by the polyglutamine androgen receptor (polyQ AR), a protein expressed by both lower motor neurons and skeletal muscle. Although viewed as a motor neuronopathy, data from patients and mouse models suggest that muscle contributes to disease pathogenesis. Here we tested this hypothesis using AR113Q knock-in and human BAC transgenic mice that express the full-length polyQ AR and display androgen-dependent weakness, muscle atrophy and early death. We developed antisense oligonucleotides that suppressed AR gene expression in the periphery, but not CNS, following subcutaneous administration. Suppression of polyQ AR in the periphery rescued deficits in muscle weight, fiber size and grip strength, reversed changes in muscle gene expression, and extended lifespan of mutant males. We conclude that polyQ AR expression in the periphery is an important contributor to pathology in SBMA mice and that peripheral administration of therapeutics should be explored for SBMA patients.

Introduction

Spinal and bulbar muscular atrophy (SBMA) is one of nine untreatable diseases caused by CAG/glutamine tract expansions. In SBMA, a polyglutamine (polyQ) tract near the amino terminus of the androgen receptor (AR) leads to hormone-dependent protein unfolding, and to the loss of lower motor neurons in the brainstem and spinal cord of affected males (Lieberman and Fischbeck, 2000). Clinical onset occurs in adolescence to adulthood and is characterized initially by muscle cramps and elevated serum creatine kinase (Katsuno et al., 2006b; Sperfeld et al., 2002). These myopathic features commonly precede muscle weakness, which inevitably develops as the disease progresses and is most severe in the proximal limb and bulbar muscles. As with all of the polyglutamine disorders, the mechanisms that lead to selective neuronal dysfunction and degeneration are poorly understood, and disease-modifying therapies are currently unavailable.

Several general principles have emerged from the study of SBMA model systems that guide our understanding of disease pathogenesis. Binding of testosterone or dihydrotestosterone to the polyQ AR promotes ligand-dependent unfolding and nuclear translocation of the mutant protein (Katsuno et al., 2002; Takeyama et al., 2002). These steps are required for pathogenesis and underlie the occurrence of disease only in men. The mutation leads to a partial loss of transactivation function (Chamberlain et al., 1994; Irvine et al., 2000; Kazemi-Esfarjani et al., 1995; Lieberman et al., 2002; Mhatre et al., 1993), and while this may contribute to features of androgen insensitivity, neuromuscular degeneration is mediated by a toxic gain of function conferred by protein unfolding. In SBMA, as in other CAG/polyQ disorders, the mutant protein disrupts multiple downstream pathways, and toxicity likely results from the cumulative effects of altering a diverse array of cellular processes including transcription, RNA splicing, axonal transport and mitochondrial function (Katsuno et al., 2006a; Kemp et al., 2011; McCampbell et al., 2000; Morfini et al., 2006; Ranganathan et al., 2009; Szebenyi et al., 2003; Yu et al., 2009). The existence of divergent mechanisms of toxicity suggests that potential treatments targeting a single downstream pathway are likely to be incomplete or unsuccessful. In contrast, efforts to target the polyQ AR as the proximal mediator of toxicity by harnessing cellular machinery to promote its degradation hold promise for therapeutic intervention. As the Hsp90-based chaperone machinery controls proteostasis of the AR (Morishima et al., 2008; Thomas et al., 2004; Thomas et al., 2006; Wang et al., 2010), genetic and pharmacological approaches to promote Hsp70-dependent ubiquitination have been shown to facilitate degradation of the mutant protein (Wang et al., 2013).

Insights into the mechanisms underlying selective neuromuscular degeneration in SBMA have come from the study of mouse models. Previous analysis of AR113Q knock-in mice suggested that pathology arising in skeletal muscle contributes to the disease phenotype (Yu et al., 2006a). In these mice, denervation and myopathy precede spinal cord pathology, consistent with the notion that myopathy is an early disease manifestation (Jordan and Lieberman, 2008). Supporting a role for muscle in pathogenesis are data from transgenic mice that over-express wild type AR only in skeletal muscle and show hormone-dependent myopathy and motor axon loss (Johansen et al., 2009; Monks et al., 2007). That muscle both contributes to the SBMA phenotype and provides a therapeutic target is supported by data showing diminished disease severity in polyQ AR transgenic mice with genetic over-expression of IGF-1 in skeletal muscle (Palazzolo et al., 2009) or with peripheral IGF-1 administration (Rinaldi et al., 2012).

Here we test an alternative strategy to ameliorate toxicity in mouse models of SBMA by suppressing polyQ AR expression using antisense oligonucleotides (ASO). We use these compounds to specifically target polyQ AR expression in the periphery. We demonstrate using two mouse models that peripheral gene suppression of the polyQ AR rescues deficits in muscle weight, fiber size and grip strength, reverses changes in muscle gene expression, and extends lifespan of mutant males. We conclude that polyQ AR expression in the periphery is an important contributor to pathology in SBMA mice and that peripheral administration of therapeutics should be explored for SBMA patients.

Results

Subcutaneous ASO suppresses polyQ AR expression in the periphery but not spinal cord

We sought to define the contribution of peripherally expressed polyQ AR to the phenotype of SBMA mice and to determine whether peripheral tissue is a therapeutic target. To accomplish this, we suppressed AR expression by subcutaneous administration of ASO. Since these compounds do not cross the blood brain barrier (Geary, 2009; Yu et al., 2007), this strategy selectively targeted AR in peripheral tissues such as skeletal muscle. We developed 16-mer chemically modified ASO complementary to human and mouse or human AR transcripts (ASO1 and ASO2 respectively; Supplemental Table 1). These 2′,4′-constrained ethyl (cEt) gapmer ASO show increased stability, tolerability and potency upon in vivo administration (Seth et al., 2009). Initial characterization demonstrated dose-dependent suppression of human and mouse AR mRNAs in cell culture by targeted but not control ASO (Figure 1a). Similarly, subcutaneous administration of targeted but not control ASO led to dose-dependent suppression of AR mRNA and protein expression in skeletal muscle of wild type male mice (Figure 1b, c). Serum testosterone levels of these males exhibited modest variability, and treatment with targeted ASO did not result in a significant alteration (Supplemental Figure 1).

Figure 1. Dose-dependent suppression of AR expression by targeted ASO.

(a) Human umbilical vein endothelial cells (top) and mouse b. END cells (bottom) were electroporated in the presence of AR targeted or control ASO at the indicated concentrations. Relative AR mRNA levels were determined after 16 hr and are reported as mean +/− SD. UTC, untransfected control.

(b, c) Wild type male mice received subcutaneous injections of ASO1, control ASO or saline from six to 14 weeks (n=3/group). Animals were treated with ASO1 or control ASO at 50 mg/kg/week, or with two lower doses of ASO1 (25 mg/kg/day × 3 days, then 12.5 mg/kg/week; or 17.5 mg/kg/day × 3 days, then 10 mg/kg/week). Quadriceps muscle was harvested at 14 weeks for analysis of AR expression. (b) Relative AR mRNA levels (mean +/− SEM). ^*p<0.05, ^***p<0.001 (c) AR protein as detected by immunoprecipitation and western blot.

(d) BAC fxAR121 males (n=4/group) received subcutaneous injections of control ASO (25 mg/kg/week), ASO2 (25, 12.5 or 6.25 mg/kg/week) or saline from seven to 15 weeks. Quadriceps muscle was harvested 48 hrs after the final dose and analyzed for human and mouse AR mRNA levels. Data are mean +/− SD. ^***p<0.001 compared to saline treatment.

We used these compounds to determine the extent to which suppressing peripheral expression of the polyQ AR rescued the phenotype of SBMA mice. This was accomplished using both AR113Q knock-in (Yu et al., 2006a; Yu et al., 2006b) and human BAC fxAR121 transgenic mice (Cortes et. al., 2013). Both of these models express the full-length polyQ AR under the regulation of its endogenous promoter. These mice display a similar androgen-dependent phenotype characterized by weakness, muscle atrophy and early death. In both models, subcutaneous administration of ASO decreased AR expression in skeletal muscle but not spinal cord. To determine the targeting efficacy and specificity in skeletal muscle, BAC fxAR121 transgenic males were treated with the human AR targeted ASO2. Subcutaneous administration of human AR targeted ASO2, but not control ASO, led to dose-dependent suppression of transgene expression in skeletal muscle without affecting expression of the endogenous mouse allele (Figure 1d). While treatment with ASO2 (100 mg/kg, twice weekly, starting at 11 weeks) specifically suppressed transgene expression in skeletal muscle of BAC transgenic mice, the human and mouse cross-reactive ASO1 suppressed both transgenic human AR and endogenous mouse AR mRNA (Figure 2a) demonstrating target selectivity. Quantitative real-time RT-PCR (qPCR) demonstrated >95% reduction of human AR mRNA levels in skeletal muscle of treated males. No significant change in mouse or human AR mRNA levels was detected in brain or spinal cord of treated mice (Figure 2a), indicating that subcutaneous administration selectively targeted peripheral AR expression. The decrease of AR mRNA in muscle was associated with comparable reduction in AR protein immunoreactivity in skeletal muscle nuclei following treatment (Figure 2b).

Figure 2. Subcutaneous ASO administration suppresses AR expression in muscle but not spinal cord.

(a) 11 week old BAC fxAR121 males (n=4/group) received subcutaneous injections of ASO (100 mg/kg, twice weekly) or saline for four weeks. Quadriceps muscle, brain and spinal cord were harvested 48 hrs after the final dose and analyzed for human (left panel) and mouse (right panel) AR mRNA levels. Data are mean +/− SD. ND, not detected. ^***p<0.001 compared to saline treatment.

(b) Wild type (left panel) and BAC fxAR121 males (middle and right panels) received subcutaneous injections of ASO1 (50 mg/kg weekly, starting at 6 weeks) or saline for four weeks. The external urethral sphincter muscle was harvested and stained for AR by immunohistochemistry. Scale bar, 15 μm.

(c, d) AR113Q (n=10/group) and wild type (n=3/group) males received subcutaneous injections of ASO1 (50 mg/kg, twice weekly for four weeks, and then once weekly) or saline, from eight until 26 weeks. Quadriceps muscle and spinal cord were harvested at the indicated ages and analyzed for (c) AR mRNA (mean +/− SEM) and (d) protein by immunoprecipitation and western blot. ^***p<0.001; n.s., not significant.

AR113Q knock-in males express a hybrid humanized AR in which most of mouse AR exon 1 has been replaced by human sequence (Yu et al., 2006b). Therefore, we used ASO1, a human and mouse AR cross-reactive ASO, to treat these mice. Subcutaneous administration of ASO1 (50 mg/kg, twice/week for four weeks, then once/week) or saline was initiated at eight weeks and continued until 26 weeks. Treatment resulted in a significant decrease in AR mRNA levels in quadriceps muscle but not spinal cord (Figure 2c). This decrease in AR expression was long-lived, as partial mRNA reduction was detected in skeletal muscle harvested from mice at 36 weeks of age, 10 weeks after the termination of treatment; by 46 weeks of age (20 weeks post treatment wash-out), AR mRNA levels in muscle approached those of saline treated controls.

Similarly sustained suppression of transcripts by ASO has been reported in skeletal muscle of myotonic dystrophy mice (Wheeler et al., 2012). Knock-down of AR mRNA levels in muscle was associated with ~90% decrease in AR protein levels (Figure 2d). We observed a slow recovery in AR protein levels in muscle after the termination of treatment, with expression remaining ~80% lower than controls at 10 weeks post-treatment and ~65% lower than controls at 20 weeks post-treatment (age 46 weeks). In contrast, AR mRNA and protein expression in spinal cord were not significantly altered by peripheral ASO delivery.

Peripheral polyQ AR suppression rescues disease in SBMA mice

Significant amelioration of disease phenotype following AR gene suppression in the periphery was observed in both SBMA mouse models. BAC fxAR121 mice exhibited an age-dependent loss of grip strength and body mass (Figure 3a, b). These deficits were partially rescued in a dose-dependent manner by subcutaneous administration of ASO2 but not control ASO (Figure 3a, b). Higher dose treatment with ASO2 (50 mg/kg/week) starting at an asymptomatic age (six weeks) and continuing for four weeks completely ameliorated the loss of grip strength and body mass in transgenic males (Figure 3c, d). This was associated with rescue of lean body mass as measured by MRI (Figure 3e). BAC fxAR121 males treated with ASO2 showed a dose-dependent extension of lifespan (Figure 4a). Similarly, treatment of AR113Q knock-in males with ASO1 starting at eight weeks resulted in a significant extension of lifespan (Figure 4b), without altering serum testosterone levels (Supplemental Figure 2).

Figure 3. Dose-dependent rescue of grip strength and body mass in BAC fxAR121 males by ASO2.

(a, b) BAC fxAR121 or wild type males (n=10/group) were treated with increasing doses of ASO2 (6.25 - 25 mg/kg/week), control ASO or saline from seven until 15 weeks. Grip strength (a) and body mass (b) are reported as mean +/− SD. Grip strength and body mass of tg mice treated with ASO2 at 12.5 and 25 mg/kg/week are different from saline treated in weeks 15–21, p<0.05. (Tg mice treated with saline show decreased grip strength compared to wt mice in weeks 11–21, p<0.05 by ANOVA, and decreased body mass in weeks 12–21, p<0.05 by ANOVA.)

(c, d) BAC fxAR121 or wild type males (n=7 – 10/group) received subcutaneous injections of ASO2 (50 mg/kg/week, starting at 6 weeks) or saline for four weeks. Age-dependent changes in (c) grip strength and (d) body mass are reported as mean +/− SD. In panel c, ^**p<0.01, ^***p<0.001 compared to saline treated BAC fxAR121 starting at week 13 by ANOVA. In panel d, p<0.05 for ASO2 vs saline treated tg mice from weeks 14–16 by ANOVA.

(e) BAC fxAR121 or wild type males (n=9–10/group) received subcutaneous injections of ASO2 (50 mg/kg/week, starting at 6 weeks) or saline for four weeks. MRI was performed at 16 weeks and used to determine lean body mass. Data are mean +/− SD. ^***p<0.001 compared to saline treated BAC fxAR121 by ANOVA.

Figure 4. Peripheral polyQ AR suppression rescues survival of SBMA mice.

(a) Survival of BAC fxAR121 or wild type males (n=10/group) treated with increasing doses of ASO2 (6.25 - 25 mg/kg/week), control ASO or saline from seven until 15 weeks. The dark blue/black line depicts overlapping survival curves of wild type (saline and ASO2 treated) and transgenic mice treated with ASO2 @ 25 mg/kg/week.

(b) Survival of AR113Q (n=10/group) and wild type (n=3/group) males receiving subcutaneous injections of ASO1 or saline from eight until 26 weeks (as described in figure 1c). The red line depicts overlapping survival of wild type males treated with saline or ASO1. ASO1 treatment significantly extended lifespan of AR113Q males (p=0.016).

To further assess the clinical implications and therapeutic relevance of this strategy, we initiated treatment with ASO2 in a separate cohort of BAC fxAR121 mice at 11 weeks, an age at which these males begin to exhibit diminished grip strength and body mass, as shown in Figure 3. Subcutaneous administration of ASO2 for durations varying from two to eight weeks resulted in a significant increase in survival that reflected the length of treatment (Figure 5a). Furthermore, we detected a significant deficit in grip strength only in those animals treated with ASO for the shortest duration (Figure 5b). These data indicate that therapeutic benefits from peripherally administered ASO are dependent upon total treatment dose and are detected even when initiated at the onset of symptoms.

Figure 5. Phenotype rescue is dependent upon treatment duration and is observed when treatment begins at symptom onset.

BAC fxAR121 or wild type males received subcutaneous injections of ASO2 or saline starting at 11 weeks. Mice were treated (n=6–10/group) for eight weeks with 50 mg/kg/week, or for two, four or eight weeks with 25 mg/kg/week. (a) Survival of BAC fxAR121 (Tg) is significantly extended by ASO treatment. p< 0.01. The light blue line depicts overlapping curves for wild type, transgenic mice treated with ASO2 @ 50 mg/kg/week for 8 weeks, and transgenic mice treated with ASO2 @ 25mg/kg/week for 8 weeks. (b) Grip strength at 22 weeks, four weeks after final treatment (n=3–5/group, except saline treated BAC fxAR121, where only one mouse survived at this time point). Data are mean +/− SD. ^**p<0.05 compared to saline treated wt males.

ASO treatment rescued polyQ AR mediated pathology in skeletal muscle of both SBMA mouse models. AR113Q knock-in males showed skeletal muscle atrophy at 26 weeks of age, and both tibialis anterior muscle mass (Figure 6a) and quadriceps muscle fiber size (Figure 6b) were increased by ASO1 treatment starting at eight weeks. This rescue of muscle mass was maintained in mice at 36 weeks of age, 10 weeks after termination of treatment, but was lost at 46 weeks, or 20 weeks off treatment. These findings paralleled changes in polyQ AR expression (Figure 2c, d). ASO1 treatment also diminished the frequency of AR immunoreactive intranuclear inclusions in skeletal muscle nuclei of knock-in males (Figure 6c). These inclusions were not detected in quadriceps muscle of treated males at 26 weeks and were present in only occasional nuclei of the levator ani/bulbocavernosus (LA/BC) muscle. As the LA/BC expresses high levels of AR (Jordan et al., 1997; Monks et al., 2004), intranuclear inclusions were frequent in saline treated AR113Q males, and their persistence in some nuclei after ASO treatment indicated that a subpopulation was long-lived. Similar findings have been observed following ASO treatment of Huntington disease mice (Kordasiewicz et al., 2012). As with the LA/BC muscle, the external urethral sphincter (EUS) muscle expressed high levels of AR (data not shown) and exhibited more severe and early onset atrophy than quadriceps muscle (Figure 6d). In BAC fxAR121 males, ASO2 treatment rescued muscle fiber size in both EUS and quadriceps muscles (Figure 6d). In both BAC fxAR121 and AR113Q males, ASO treatment also diminished the levels of acetylcholine receptor-α subunit and myogenin mRNAs, genes that are induced following denervation and are up-regulated in SBMA mice (Figure 6e, f). In both models, this rescue was partially maintained 6–10 weeks post-treatment.

Figure 6. Peripheral ASO administration rescues muscle atrophy and gene expression changes.

(a) Tibialis anterior (TA) muscle mass at 26 weeks from AR113Q or wild type males treated with ASO1 or saline (treated as described in Figure 1c), or post-treatment at age 36 or 46 weeks. Data are mean +/− SEM. ^*p<0.05; n.s., not significant.

(b) Quadriceps muscle fiber size distribution (left panel) and mean +/− SEM (right panel) from AR113Q or wild type males at 26 weeks, treated as described in Figure 1c. ^***p<0.001

(c) AR immunofluorescence (red) of quadriceps and levator ani/bulbocavernosus (LA/BC) muscles of AR113Q and wild type males at 26 weeks, treated as described in Figure 1c. DAPI stains nuclei. Scale bar, 10 μm

(d) External urethral sphincter (upper panel) and quadriceps muscle (lower panel) fiber size were determined from BAC fxAR121 males (n=4/group) that received subcutaneous ASO2 (50 mg/kg/week, starting at 6 weeks) for four weeks. Muscle was harvested from transgenic (Tg) and wild type males at 10 and 16 weeks. Data are mean +/− SD. ^*p<0.05

(e) BAC fxAR121 males (n=3–4/group) received subcutaneous injections of ASO2 (50 mg/kg/week, starting at 6 weeks) or saline for four weeks. Quadriceps muscle was harvested at 10 and 16 weeks, and AChR-α and myogenin mRNA expression determined. Data are mean +/− SD. ^**p<0.01, ^***p<0.001

(f) AChR-α and myogenin mRNA expression in quadriceps muscle of AR113Q or wild type males at 26 weeks (treated as described in Figure 1c), or following termination of treatment at 36 weeks. Data are mean +/− SEM. ^**p<0.01, ^*p<0.05

Finally, to further assess the relative contribution of peripheral polyQ AR expression to the disease phenotype, we directly compared the efficacy of peripheral versus intraventricular versus combined administration of ASO in BAC fxAR121 mice. For these experiments, we used a third AR-targeted ASO, ASO3, whose chemistry is well tolerated by mice following intraventricular injection (Supplemental Table 1). We found that a single intraventricular administration of ASO3 (100 μg at 8 weeks) diminished human polyQ AR mRNA levels in lumbar spinal cord by ~60% (Figure 7a). This effect was long-lived and was detected up to eight weeks after intraventricular administration. Notably, intraventricular delivery did not alter polyQ AR mRNA levels in quadriceps muscle, which were only diminished in mice receiving subcutaneously delivered ASO2 (Figure 7a). In order to increase the likelihood of detecting an additive benefit from intraventricular administration, we limited the subcutaneous dose to 12.5 mg/kg/week. At this dose, peripheral ASO2 diminished polyQ AR mRNA expression in muscle by ~60–70% (Figure 7a), and partially rescued grip strength and survival (Figure 7b, c). A single bolus intraventricular administration of ASO3 did not alter grip strength or survival in BAC fxAR121 mice, despite lowering polyQ AR mRNA expression in lumbar spinal cord (Figure 7b, c). Furthermore, combined intraventricular and subcutaneous delivery of ASO was no more effective than subcutaneous delivery alone at rescuing the disease phenotype (Figure 7b, c). Taken together, we conclude that peripheral delivery of targeted ASO suppresses AR expression in skeletal muscle but not spinal cord, and that this treatment rescues deficits in grip strength, body weight, survival and skeletal muscle atrophy in two mouse models of SBMA.

Figure 7. Intraventricular ASO administration does not provide added benefit to BAC fxAR121 mice receiving peripheral ASO.

(a) BAC fxAR121 mice were dosed as indicated with saline (sal), intraventricular (ICV) ASO3 or control (cnl) ASO (100 μg at 8 weeks), and subcutaneous (SQ) ASO2 (12.5 mg/kg/week, starting at 8 weeks). Spinal cord and quadriceps muscle were harvested 4 or 8 weeks following ICV administration and analyzed for human AR mRNA levels. Data (mean +/− SEM) are reported relative to mice receiving ICV and SQ saline (n=4/group). ^***p<0.001.

(b, c) Wild type or BAC fxAR121 males (n=8–10/group) were treated as indicated with saline (sal), intraventricular (ICV) ASO3 or control (cnl) ASO (100 μg at 8 weeks), and subcutaneous (SQ) ASO2 (12.5 mg/kg/week, starting at 8 weeks). Age dependent changes in grip strength (b) and survival (c) are shown. Data are mean +/− SEM.

Discussion

Our data demonstrate that polyQ AR suppression outside the CNS is sufficient to ameliorate the disease phenotype in two independent mouse models of SBMA. This strategy is distinct from prior efforts that have focused on targeting polyQ AR toxicity within motor neurons. We show that subcutaneous delivery of ASO suppresses polyQ AR expression in skeletal muscle but not spinal cord, and that this rescues deficits in muscle weight, fiber size and grip strength, reverses changes in muscle gene expression, and extends lifespan of mutant males. Complementary analysis of BAC fxAR121 mice following genetic deletion of the floxed allele specifically in skeletal muscle corroborates our findings (Cortes et al., 2013) and confirms that skeletal muscle is the critical target tissue for this therapeutic intervention.

Our data demonstrate an unexpectedly important contribution of skeletal muscle to the SBMA phenotype. The occurrence of non-cell autonomous neurotoxicity is an increasingly recognized component of degenerative disorders (Ilieva et al., 2009). For motor neuron diseases, models of familial amyotrophic lateral sclerosis caused by mutant superoxide dismutase 1 have uncovered contributions to pathogenesis from astrocytes, oligodendrocytes and microglia (Boillee et al., 2006; Di Giorgio et al., 2007; Kang et al., 2013; Lee et al., 2012; Yamanaka et al., 2008). Similarly, models of spinal muscular atrophy have demonstrated important contributions from skeletal muscle (Cifuentes-Diaz et al., 2001; Mutsaers et al., 2011), and have shown efficacy of peripheral gene targeting using ASO to regulate SMN2 mRNA splicing (Hua et al., 2011). For SBMA, the findings reported here and by Cortes et al (Cortes et al., 2013) indicate that peripheral expression of the polyQ AR in skeletal muscle underlies degenerative changes in the neuromuscular system. This correlates with much higher expression of polyQ AR protein in skeletal muscle than spinal cord of SBMA patients (Tanaka et al., 1999). While the precise mechanism by which skeletal muscle influences disease remains to be defined, impaired trophic support from SBMA muscle may contribute to pathogenesis. Diminished expression of several trophic factors by skeletal muscle in SBMA mouse models has been demonstrated previously, including neurotrophin-4, glial derived neurotrophic factor and vascular endothelial growth factor (Sopher et al., 2004; Yu et al., 2006a). As these and other muscle-derived factors support innervating neurons, their decreased expression could impact the function and viability of lower motor neurons expressing the polyQ AR as well as their resistance to polyQ toxicity (Jordan and Lieberman, 2008). This model of pathogenesis is consistent with the well established role of skeletal muscle in maintaining motor neurons (Jessell and Sanes, 2000), and suggests a mechanism whereby the neuromuscular system may be especially vulnerable to toxicity from the polyQ AR.

The therapeutic benefits documented here following peripheral administration of ASO provide a compelling rationale for exploring treatments targeted to skeletal muscle in SBMA patients. However, several important questions remain as we work toward translating these findings to patients. The optimal therapeutic dose for alleviating proteotoxicity while maintaining beneficial anabolic effects of AR on skeletal muscle remains to be defined. Figures 1b and 1c show dose-dependent effects on AR mRNA and protein levels over the therapeutic range of ASO used in these studies, and future work will help optimize this approach to treatment. Importantly, no target-related adverse findings were observed in either wild type or SBMA mice following ASO treatment, demonstrating that this treatment approach is feasible, beneficial and lacking untoward consequences. We recognize that the loss of anabolic effects of AR on skeletal muscle may be more pronounced in SBMA patients than in mice and could necessitate concurrent trophic factor support. Furthermore, we acknowledge that both mouse models exhibit marked skeletal muscle pathology and do not display motor neuron loss, a limitation of the available model systems. Nonetheless, the observations reported here establish a foundation for developing disease-modifying therapies targeted to skeletal muscle, and demonstrate the important contribution of peripherally expressed polyQ AR to SBMA pathogenesis.

Experimental Procedures

Mice

Derivation of AR113Q mice with a targeted Ar allele containing 113 CAG repeats in exon 1 was described previously (Yu et al., 2006b). Briefly, mice were generated by recombining a portion of human exon 1 (amino acids 31–484) with the mouse Ar gene in CJ7 embryonic stem cells. Male chimeras were mated with C57BL/6J females, and females heterozygous for the targeted Ar allele were backcrossed to C57BL/6J ten or more generations. BAC fxAR121 mice were derived as described (Cortes et al., 2013). Male offspring housed in SPF facilities and maintained on a constant 12-hour light-dark cycle were used in this study. Subcutaneous administration of ASO or saline was performed according to the indicated treatment schedules. For intraventricular administration, eight-week-old mice were anaesthetised with 2% isoflurane and held by the head in a stereotaxic instrument. Ten μl of ASO (100 μg) in saline was injected into the right lateral ventricle. The coordinates for injection were: 0.3 mm anterior, 1.0 mm lateral and 3.0 mm ventral from the bregma, as described (Sahashi et al., 2013). Body composition was measured by an Echo MRI system (Echo Medical System, Houston, TX). Procedures involving mice were approved by the University of Michigan Committee on Use and Care of Animals, in accord with the NIH Guidelines for the Care and Use of Experimental Animals, or by the Isis Pharmaceuticals Institutional Animal Care and Use Committee.

Oligonucleotides

A series of uniform chimeric 16-mer phosphorothioate oligonucleotides containing constrained ethyl (cEt) groups at positions 1–3 and 14–16 targeted to mouse or human AR and a control ASO (Supplemental Table 1) were synthesized and purified on an automated DNA synthesizer using phosphoramidite chemistry, as previously described (Koller et al., 2011). All ASOs were dissolved in PBS and filtered before injections were performed.

RNA analysis

Total RNA was isolated from tissues of AR113Q knock-in males with Trizol (Invitrogen) following manufacturer’s instruction. RNA (1 μg) was used to synthesize cDNA with the high capacity cDNA archive kit (Applied Biosystems). Gene specific primers (18S rRNA, 4310893E; Ar, Mm00442688_m1; α-acetylcholine receptor, Mm00431627_m1; myogenin, Mm00446194_m1) were purchased from Applied Biosystems, and analyses were performed in duplicate using 10 ng aliquots of cDNA on an ABI 7500 Real Time PCR system. Relative expression levels were calculated comparing with the expression of 18S rRNA.

BAC fxAR121 tissues were homogenized in a guanidine isothiocyanate solution (Invitrogen) supplemented with 8% 2-mercaptoethanol (Sigma-Aldrich). Total RNA was prepared according to the PureLink Total RNA Purification Kit (Invitrogen). The qRT-PCR analyses were done using a StepOne Real-Time PCR System (Applied Biosystems). The sequences of primers and probe used were as follows: Mouse Androgen Receptor (IDT): forward: 5′-CAGCAGAAACGATTGTACCATTG-3′, reverse: 5′-GCTTACGAGCTCCCAGAGTCA-3′, probe: 5′-Fam- AAAATTGCCCATCTTGTCGTCTCCGG-Tamra-3′; Human Androgen Receptor: (IDT) forward: 5′-GCCCCTGGATGGATAGCTACT-3′, reverse: 5′-CCACAGATCAGGCAGGTCTTC-3′, probe: 5′-Fam-ACTGCCAGGGACCATGTTTTGCCC-Tamra-3′; Mouse Cholinergic Receptor Nicotinic, alpha polypeptide1 (Life Technologies) Cat#4331182 Mm00431627_m1; Mouse Myogenin (Life Technologies) Cat#4331182 Mm00446194_m1. PCR results were normalized to total RNA measure by Quant-iT RiboGreen RNA Reagent (Molecular Probes).

Immunoprecipitation and western blot

Muscle and spinal cord were homogenized in RIPA buffer containing complete protease inhibitor cocktail (Roche) using a motor homogenizer (TH115, OMNI). Lysates were incubated on a rotator at 4°C for 1 hour and then pre-cleared by centrifugation at 13,000 g for 10 min at 4°C. Protein concentration was determined by BCA protein assay (Pierce). Protein lysates (500 μg) were incubated with AR antibody (Millipore, PG-21) or rabbit IgG (Santa Cruz) overnight at 4°C, then with protein A beads (Santa Cruz) for 1 hour at 4°C. Beads were washed and the eluate was resolved by 7.5% SDS-PAGE and then transferred to nitrocellulose membranes (Bio-Rad). Blots were probed with AR antibody (Santa Cruz) and proteins were visualized by chemiluminescence (Thermo Scientific).

Muscle histology and immunofluorescence staining

AR113Q skeletal muscle was frozen in isopentane pre-chilled by liquid nitrogen, sectioned at 5 μm with a cryostat and stained with H&E. For immunofluorescence, frozen sections were stained with an AR antibody (Santa Cruz) and a secondary antibody conjugated to Alexa Fluor 594 (Invitrogen). Confocal images were captured with a Zeiss LSM 510 microscope and a water immersion lens (×63).

BAC fxAR121 muscle was fixed with 10% neutral buffered formalin, embedded in paraffin and sectioned at 4 μm. Antigen retrieval was performed by boiling in Thermo Citrate buffer, pH 6.0 (Thermo Scientific, IL) for 20 min. Slides were blocked by donkey serum (Jackson immunoresearch lab, PA) for 30 min. The primary antibody (AR: sc-816-Santa Cruz, TX; Laminin: ab11575-Abcam, MA) was applied and incubated at RT for one hour. After three washed in TBS, slides were incubated with donkey anti-rabbit HRP (Jackson immunoresearch Lab, PA) at 1:200 for 30 min, then developed with DAB and counterstained with hematoxylin (Surgipath, IL), dehydrated and mounted.

Muscle fiber size quantification

The cross-sectional diameter of BAC fxAR121 muscle fibers was measured by image analysis after staining with laminin to highlight the muscle membrane. The minimal diameter of each muscle fiber was quantified by the muscle fiber algorithm (Indica Lab, NM). Frozen sections of AR113Q muscle (5 μm) were stained by H&E and digital images were captured using a Zeiss Axioplan 2 imaging system. The area of each muscle fiber was defined using Adobe Photoshop CS4, and the pixel number was converted to μm² according to scale, as described (Yu et al., 2011). 100 adjacent fibers from each section were measured.

Grip strength

Forelimb strength of BAC fxAR121 mice was measured with a digital grip strength meter which records the maxiaml strength an animal exerts while trying to resist an opposing pulling force. In brief, each mouse was allowed to grasp the metal rail with it fore limbs and gradually pulled backward in the horizontal plane. The highest reading from 5–10 consecutive trials was recorded by a four channel transducer (TBM4M, WPI, MA) using labScribe2 software (Transonic System, NY).

Testosterone levels

Serum was collected by cardiac puncture. Testosterone levels were determined by radioimmunoassay by the Ligand Assay and Analysis Core Facility at the University of Virginia Center for Research in Reproduction.

Statistics

Statistical significance was assessed by unpaired Student’s t-test (for pair-wise comparisons) or by one-way ANOVA with Newman-Keuls multiple comparison test or Tukey’s post-hoc test (for multiple comparisons). The distribution of muscle fiber size was analyzed by Mann-Whitney test. Effects on survival were assessed by log-rank analysis. All statistics was performed using Prism 5 or 5.04 (GraphPad Software, San Diego, CA). P values less than 0.05 were considered significant.

Highlights.

• We developed antisense oligonucleotides to suppress AR gene expression in mice

• Subcutaneous delivery suppressed AR gene expression in the periphery but not CNS

• Subcutaneous administration rescued disease in two mouse models of SBMA

• Peripherally expressed polyQ AR contributes to disease and is a therapeutic target