Synergic prodegradative activity of Bicalutamide and trehalose on the mutant androgen receptor responsible for spinal and bulbar muscular atrophy

Abstract

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease due to a CAG triplet-repeat expansion in the androgen receptor (AR) gene, which is translated into an elongated polyglutamine (polyQ) tract in AR protein (ARpolyQ). ARpolyQ toxicity is activated by the AR ligand testosterone (or dihydrotestosterone), and the polyQ triggers ARpolyQ misfolding and aggregation in spinal cord motoneurons and muscle cells. In motoneurons, testosterone triggers nuclear toxicity by inducing AR nuclear translocation. Thus, (i) prevention of ARpolyQ nuclear localization, combined with (ii) an increased ARpolyQ cytoplasmic clearance, should reduce its detrimental activity. Using the antiandrogen Bicalutamide (Casodex®), which slows down AR activation and nuclear translocation, and the disaccharide trehalose, an autophagy activator, we found that, in motoneurons, the two compounds together reduced ARpolyQ insoluble forms with higher efficiency than that obtained with single treatments. The ARpolyQ clearance was mediated by trehalose-induced autophagy combined with the longer cytoplasmic retention of ARpolyQ bound to Bicalutamide. This allows an increased recognition of misfolded species by the autophagic system prior to their migration into the nucleus. Interestingly, the combinatory use of trehalose and Bicalutamide was also efficient in the removal of insoluble species of AR with a very long polyQ (Q112) tract, which typically aggregates into the cell nuclei. Collectively, these data suggest that the combinatory use of Bicalutamide and trehalose is a novel approach to facilitate ARpolyQ clearance that has to be tested in other cell types target of SBMA (i.e. muscle cells) and in vivo in animal models of SBMA.

The Role of the Protein Quality Control System in SBMA

“…However, one of the most relevant defects is associated with the fact that ARpolyQ is able to reduce the long-term proteins turnover and to block the cytoplasmic autophagic flux (Carra, et al, 2012,Cortes, et al, 2014b,Giorgetti, et al, 2014,Rusmini, et al, 2010,Rusmini, et al, 2007. As will be discussed in the next sections, the pharmacological restoration of a normal autophagic flux, for example by treatment with trehalose, a TFEB activator (Dehay, et al, 2010), greatly increases the clearance of the mutant misfolded ARpolyQ (Giorgetti, et al, 2014. Defects in autophagy flux have been clearly identified in SBMA mice (Cortes, et al, 2014b), and the importance of autophagy in SBMA is confirmed by several recent studies (Cortes, et al, 2014b,Settembre andBallabio, 2011).…”

“…Using primary cultures of motor neurons derived from SBMA mice, Montie and Merry have shown that both the autophagy activator mTORdependent phenoxazine (or AKTi, a potent Akt inhibitor or rapamicyn), and the mTOR-independent trehalose, were able to rescue motor neurons from ARpolyQ mediated ligand-dependent death (Montie, et al, 2009,Montie andMerry, 2009). In motor neurons, both AKTi and trehalose (Giorgetti, et al, 2014 induced the classical cytoplasmic punctate distribution of the lipidated form of LC3 (LC3-II) which associated with nascent autophagosomes, a clear index of autophagy activation and this correlated with the full removal of aggregated, insoluble ARpolyQ (Giorgetti, et al, 2014,Montie and Merry, 2009. Other small molecules structurally related to trehalose may have the potential to revert ARpolyQ accumulation and aggregation.…”

“…Indeed, since, testosterone activates ARpolyQ toxicity by inducing its nuclear translocation, the prevention of ARpolyQ localization in the nucleus, combined with an increased cytoplasmic clearance may be protective. Notably, cytoplasmic retention of ARpolyQ obtained using the selective AR modulator (SARM) Casodex, not only prevented ARpolyQ aggregation and toxicity, enhancing motor neuronal survival (Orr, et al, 2010,Rusmini, et al, 2007, but also potentiated the pro-autophagic activity of trehalose (Giorgetti, et al, 2014). This synergistic effect may be due to an increased time window for the recognition of misfolded ARpolyQ species in the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64cytoplasm, soon after AR dissociation from HSPs, and their autophagic engulfment prior to migration into the nucleus.…”

Spinal bulbar muscular atrophy, an inherited neurodegenerative disease: Potential mechanisms and therapeutic targets

“…However, trails using antiandrogen therapy, commonly used in the treatment of advanced prostate cancer, yielded disappointing findings, despite highly promising animal studies. Other studies have shown AR:HSP70 complex and small molecules such as trehalose as a potential therapeutic target for SBMA [25,28]. Based on these studies, a potential therapeutic model can be proposed (Figure 2).…”

“…Some of the therapeutic strategies that have been tested in SBMA include gene silencing, protein quality control and/or increased protein degradation, androgen deprivation, and modulation of AR activity and functions. Evidence from various studies that include both in vitro and in vivo models support a role for testosterone binding and nuclear translocation of the AR as the trigger for SBMA [21][22][23][24][25][26][27]. However, trails using antiandrogen therapy, commonly used in the treatment of advanced prostate cancer, yielded disappointing findings, despite highly promising animal studies.…”