Pre-clinical symptoms of SBMA may not be androgen-dependent: implications from two SBMA mouse models

Abstract

A distinguishing aspect of spinal and bulbar muscular atrophy (SBMA) is its androgen-dependence, possibly explaining why only males are clinically affected. This disease, which impairs neuromuscular function, is linked to a polyglutamine expansion mutation in the androgen receptor (AR). In mouse models of SBMA, motor dysfunction is associated with pronounced defects in neuromuscular transmission, including defects in evoked transmitter release (quantal content, QC) and fiber membrane excitability (based on the resting membrane potential, RMP). However, whether such defects are androgen-dependent is unknown. Thus, we recorded synaptic potentials intracellularly from adult muscle fibers of transgenic (Tg) AR97Q male mice castrated pre-symptomatically. Although castration largely protects both QC and the RMP of fibers, correlating with the protective effect of castration on motor function, significant deficits in QC and RMP remained. Surprisingly, comparable defects in QC and RMP were also observed in pre-symptomatic AR97Q males, indicating that such defects emerge early and are pre-clinical. Exposing asymptomatic Tg females to androgens also induces both motor dysfunction and comparable defects in QC and RMP. Notably, asymptomatic Tg females also showed significant deficits in QC and RMP, albeit less severe, supporting their pre-clinical nature, but also raising questions about the androgen-dependence of pre-clinical symptoms. In summary, current evidence indicates that disease progression depends on androgens, but early pathogenic events may be triggered by the mutant AR allele independent of androgens. Such early, androgen-independent disease mechanisms may also be relevant to females carrying the SBMA allele.

Figure 1.

Castration of pre-symptomatic transgenic AR97Q males largely protects synaptic function. (A) Motor function based on hang times for AR97Q males was fully protected by castration pre-symptomatically, confirming previous reports. (B) The RMP of muscle fibers in symptomatic gonadally intact AR97Q males (Tg + S) is significantly depolarized compared with that of WT muscle fibers (WT + S, WT + C). Castrating pre-symptomatic Tg males largely reverses this effect of disease, maintaining membrane excitability at near normal in age-matched castrated Tg (Tg + C) males. Note however a small but significant residual effect on the RMP of AR97Q fibers, despite normal hang times of Tg + C males (A). This residual deficit may be related to the fact that fibers at the time of castration are already significantly depolarized (Fig. 3). Although neither disease or gonadal androgens affects EPP amplitude (C), the cumulative histograms of mEPP amplitude (D) show that disease shifts mEPPs toward significantly larger values (seen as a rightward shift in the curve), an effect prevented by castration (P values <0.05 on the basis of Kolmogorov–Smirnov test). (E) Likewise, disease impairs evoked transmitter release, significantly decreasing QC by more than half in gonadally intact Tg males, an effect largely but not fully prevented by castration. These data suggest that castration also rescued the size of the RRP of synaptic vesicles and/or probability of release. The residual deficit in QC in castrated Tg males may also be related to an early deficit in QC pre-symptomatically (Fig. 3). (F) Representative EPP (average of 10–20 responses) and mEPP traces (average of 2 min recording), normalized and aligned by their peak, from castrated (black) and sham (gray)-operated Tgs reveal visibly prolonged decay times for junctions from diseased gonadally intact Tg males compared with asymptomatic castrated Tg males. (G and H) Averaged values confirm significant increases in both EPP and mEPP decay times for gonadally intact, motor-impaired Tg males, an effect that is completely prevented by castration of pre-symptomatic Tg males. These data suggest that the pathological expression of the gamma subunit of the AChR is also reversed, as we later confirmed (Fig. 3). (I and J) Castration also improves short-term synaptic facilitation in Tg males, reversing a significant disease-related deficit. Representative traces of EPPs (average of 10–20 individual EPPs evoked by trains of five pulses at 100 Hz) in sham-control (black) and castrated (gray) Tg males show that significant decreases in synaptic facilitation emerge quickly, evident even at the second pulse in diseased Tg males (J). Although castration also seemed to enhance synaptic facilitation of WT junctions, this effect was not significant. Values plotted are group means (N for motor function and n for synaptic function) ± SEM (WT sham: n/N = 30/3; WT castrate: n/N = 30/3; Tg sham: n/N = 42/4; Tg castrate: n/N = 52/5) with n indicating the number of endplates/mice and N indicating the number of mice/experimental group. *P < 0.05 from WT sham or WT castrated EDL; ^#P < 0.05 from Tg sham EDL. ‘S’ denotes sham castration; ‘C’ denotes castration.

Figure 2.

Pre-pubertal castration largely prevents pathological change in mRNA expression in AR97Q adult muscle. (A) The disease-related deficit in adult isoforms, AChR-ε subunit and sodium channel Nav1.4, are completely rescued by castration. Castration also largely reversed the disease-related increase in expression of mRNA for neonatal isoforms AChR-γ and Nav1.5, which underlie the slowed kinetics of EPPs and mEPPs and resistance to µ-CTX in disease, respectively. However, note small but significant increases in the expression of both AChR-γ and Nav1.5, apparently emerging sometime after castration (Fig. 3) independent of circulating gonadal androgens. Evidently the system can tolerate some level of pathological expression of the immature isoforms without affecting mEPP and EPP kinetics, likely reflecting which subunit (adult versus immature) predominates at any given time. (B) Similarly, mRNA expression for the Clcn1, and transcripts for the inward rectifying potassium channel (Kir2.1 and 2.2), and sodium/potassium pump (NKA a1 and a2) were all restored to normal by pre-pubertal castration of Tg males, consistent with the significant rescue of the RMP (Fig. 1). These data show that disease-related changes in the expression of muscle genes for ion channels and ion transporters underlying muscle and synaptic function are largely androgen-dependent, correlating well with the androgen-dependent loss of motor function in this SBMA model. Nonetheless, it also appears that mutant AR has the capacity to trigger subclinical symptoms of disease. Values are mean fold changes ± SEMs (standard errors of the mean) relative to WT + S values based on N/group (WT + S, WT + C and Tg + S: N = 7; Tg + C: N = 8). *P < 0.05 from WT sham or WT castrated; ^#P < 0.05 from Tg sham. ‘S’ denotes sham castration; ‘C’ denotes castration.

Figure 3.

Neuromuscular synapses are defective in pre-symptomatic AR97Q males. (A and B) Body weight and motor function based on hang time is normal in 33- to 40-day-old AR97Q males. (C) However, the RMP of muscle fibers of pre-symptomatic Tg males is already significantly depolarized compared with age-matched WT males, comparable with that of late-stage diseased Tg males (Fig. 1). We also find that quantal size (mEPP amplitude) is significantly larger than normal (D) but EPP amplitude is not similarly affected (E), causing a significant deficit in QC (F) in pre-symptomatic males, also comparable with late-stage, motor impaired AR97Q males (Fig. 1). (G) Spontaneous release of transmitter (mEPP frequency) is also significantly decreased in pre-symptomatic AR97Q mice compared with WT males. (H and I) Unlike diseased adults, the decay time of mEPPs and EPPs is not altered in pre-symptomatic AR97Q NMJs. (J and K) Quantitative (q) PCR revealed that the expression of several critical genes is perturbed in muscle of pre-symptomatic AR97Q males, despite the lack of detectable motor dysfunction. Expression of the embryonic AChR γ-subunit is normal in Tg mice, as expected given the normal decay time of synaptic potentials. However, mRNA for the adult ε-subunit is significantly decreased, indicating an effect of disease already on this measure (J). Likewise, expression of the adult isoform of the sodium channel (Nav1.4) is significantly reduced, as in late-stage muscle, but without the increase in mRNA for the neonatal isoform (Nav1.5) that eventually renders diseased fiber in AR97Q adults appreciably less sensitive to µ-CTX. Expression of the adult isoform of the chloride channel is significantly decreased in pre-symptomatic AR97Q males (K), comparable with late-stage muscle. As expected based on the ∼20 mV depolarizing shift in the RMP, we find defects in the expression of both the inward rectifier potassium channel (Kir2) and the sodium/potassium pump (NKA), two critical determinants of the RMP of skeletal muscle fibers. Both Kir2.1 and NKA α2 mRNAs are significantly downregulated in muscle of pre-symptomatic AR97Q males, whereas Kir2.2 and NKA a1 mRNA levels are unaltered. These data identify several putative pre-clinical markers of disease that represent novel therapeutic targets for halting disease before irreparable damage is done. Findings also begin to characterize the sequelae of pathogenic events that underlie the progressive loss of motor ability. For example, loss of adult isoforms evidently precede, and possibly trigger, the upregulation of neonatal isoforms. A–I: Values plotted are group means (N for motor function and n for synaptic function) ± SEM (WT: n/N = 42/4; pre-symptomatic AR97Q: n/N = 42/4) normalized to WT values with n indicating the number of endplates/mice and N the number of mice per experimental group from the fast twitch EDL muscle. J and K: Values are mean fold changes ± SEMs based on N/group (AR97Q: N = 6; WT: N = 8) from the fast twitch anterior tibialis muscle. *P < 0.05 from WT.

Figure 4.

Reduced probability of transmitter release underlies enhanced short-term synaptic facilitation in pre-symptomatic AR97Q NMJs. (A) Synapses in pre-symptomatic AR97Q males exhibit a significant increase in facilitation (based on average EPP responses to trains of five pulses at 100 Hz) compared with junctions from WT males of the same age, suggesting a reduced probability of transmitter release. (B) Representative traces of evoked EPP (average of 10–20 individual EPPs) from WT and pre-symptomatic AR97Q NMJs showing the enhanced size of EPPs with repeated stimulation for Tg junctions. (C and D) The size of the RRP for pre-symptomatic AR97Q males is unaffected (C). The same x-intercept extrapolated from the initial phase of rapid decline in QC for Tg and WT synapses indicates that the maximum number of immediately releasable quanta (RRP size) without replenishment is comparable, with mean (± SEM) values in (D). (E) Pr, on the other hand, is significantly decreased in motor nerve terminals of pre-symptomatic AR97Q males, indicating that early deficits in QC reflect deficits in Pr with the size of the RRP eventually also reduced as disease progresses. Pr was calculated as initial QC/RRP. Values plotted are group means ± SEM (WT: n/N = 42/4 and pre-symptomatic AR97Q: n/N = 42/4). n is the number of endplates per experimental group and N is the number of mice per experimental group. *P < 0.05 from WT.

Figure 5.

Acute androgen treatment induces comparable synaptic and motor dysfunction in two female models of SBMA, the myogenic (Myo) and AR97Q models. (A) Only 5 days of testosterone treatment drives rapid loss of motor function in myogenic females (expressing WT androgen receptor only in muscle fibers), whereas androgen-dependent demise of motor function in AR97Q females takes a full 2 weeks. (B) The RMP of EDL muscle fibers is significantly depolarized in testosterone (T)-treated females of both models, comparable with that of diseased males. Note that the RMP in asymptomatic blank (B)-treated Tg females in both models is also significantly depolarized compared with WT controls, akin to that of pre-symptomatic AR97Q males (Fig. 3). These data indicate that muscle fibers in SBMA models are diseased prior to overt motor dysfunction and independent of male levels of androgens. T has no such effect on the RMP of WT fibers. (C) Only junctions in diseased AR97Q females show significant increases in mEPP amplitude triggered by T, comparable with that of symptomatic AR97Q males (Fig. 3). (D) On the other hand, EPP amplitude is significantly reduced by T treatment in myogenic females. (E) The net effect of T on the size of EPPs and mEPPs in the two models is a significant reduction in QC for symptomatic Tg females of both models. Also note that QC is significantly reduced in asymptomatic AR97Q females, again mirroring defects observed in pre-symptomatic AR97Q males. These data also underscore the possibility that some effects of mutant AR may be independent of androgens. Moreover, synaptic weakening is likely a core trait of SBMA that begins early and precedes overt motor dysfunction. (F) Androgen treatment also reduced mEPP frequency, although this effect does not depend on the disease-causing transgene, because T also has comparable effects in WTs, suggesting that this effect is not a product of disease. (G–I) T prolongs decay time of EPPs (G and H) in both Tg models, correlating with motor dysfunction, and comparable with chronically diseased males in both models. This defect may mark the progression of disease from asymptomatic to symptomatic, because it is not evident in asymptomatic Tg females nor in pre-symptomatic AR97Q males (Fig. 3). Representative average EPP traces from both T-treated (gray) and B-treated (black) Tg females which have been normalized and aligned by their peak to compare decay rates (H). Note that although mEPP decay time (I) is prolonged only in T-treated AR97Q females, T prolongs EPP decay time (G) for diseased junctions of both myogenic and AR97Q females. We also find that T treatment induced a slight, but significant prolongation of EPP decay in WT control females after two weeks of treatment but not after five days (WTs for AR97Q model versus WTs for myogenics), indicating that T can also affect EPP kinetics in WT muscle. These data show that although considerable pathology in synaptic and muscle functions is driven by the combination of androgens and a disease allele, some aspects of SBMA appear to be driven by a toxic AR independent of androgens whereas others by native androgen-dependent AR function. Values plotted are group means (N for motor function and n = synaptic function) ± SEM. Myogenic group: WT + B: n/N = 41/4 and WT + T: n/N = 29/3; Tg + B: n/N = 30/3 and Tg + T: n/N = 40/4. AR97Q group: WT + B: n/N = 29/3 and WT + T: n/N = 30/3; Tg + B: n/N = 41/4 and Tg + T: n/N = 40/4. n is the number of endplates per experimental group and N is the number of mice per experimental group. *P < 0.05 from WT +B or WT + T; ^#P < 0.05 from Tg + B. ‘B’ denotes Blank -treated; ‘T’ Testosterone-treated.

Figure 6.

Muscle from acutely diseased females show comparable pathological changes in mRNA expression as chronically diseased males. (A and B) Acute disease in both models is associated with a significant reduction in the amount of mRNA for the adult isoform AChR ε-subunit. A complementary increased expression of the neonatal isoform AChR γ-subunit occurred only in AR97Q females with 2 weeks of treatment, but not in myogenic females, aligning well with prolonged mEPP decay times in diseased AR97Q females but not in diseased myogenic females (Fig. 5I). AChR ε-subunit is also reduced in asymptomatic control females of both models, like for pre-symptomatic AR97Q males (Fig. 3). Because controls females are not exposed to exogenous androgens, the milder but nonetheless significant effects on AChR ε-subunit suggest that the mutant AR may drive pathological expression of some genes in SBMA muscle independent of androgens, with androgens further exacerbating AR’s toxic effects. Only diseased myogenic females show reduced expression of the adult isoform Nav1.4 of the voltage-dependent sodium channel, whereas expression of the immature isoform Nav1.5 is increased significantly by disease in both models. Such changes in mRNA expression for the sodium channel and AChR also occur in chronically diseased males (Fig. 2) (17). These data suggest that the sodium channel and AChR are each early targets of disease. (C and D) As expected, mRNA expression of the inward rectifying potassium channel (Kir), the Cl^– ion channel and the NKA pump were affected in both acute models, comparable with chronically diseased males (Fig. 2), and likely underlying the reduced RMP of muscle fibers in each model. Note that Kir2.1 mRNA is significantly down-regulated in both symptomatic and asymptomatic Tg females of both models, but that the magnitude of the change is smaller in asymptomatic control Tg females. These changes correlate well with the magnitude of change in the RMP for symptomatic and asymptomatic myogenic females (Fig. 5B), arguing that changes in Kir2.1 heavily influence the RMP. Kir2.2 is also significantly reduced in acutely diseased AR97Q females and thus, may also contribute to the lowered RMP in this model. Expression of the neonatal isoform of the sodium/potassium pump NKA α1 is up-regulated by disease in both acute models, whereas the adult NKA α2 isoform is significantly reduced only in acutely diseased AR97Q females. Clcn1 mRNA expression is significantly reduced in both acute models, paralleling the effects of disease on Clcn1 expression in chronically diseased males (Fig. 2). These data provide further evidence that disease affects specific aspects of synaptic and muscle function, and that these disease events occur early but with a distinct time line of progression, with some representing pre-clinical aspects of the disease. Values are mean fold changes ± SEMs normalized to control WTs (WT + B) based on N/group. Myogenic group: WT + B, WT + T, Tg + B and Tg + T: N = 6. AR97Q group: WT + B and WT + T: N = 5; Tg + B and Tg + T: N = 4. *P < 0.05 from WT +B or WT + T; ^#P < 0.05 from Tg + B. ‘B’ denotes Blank -treated; ‘T’ Testosterone-treated.