17-AAG increases autophagic removal of mutant androgen receptor in spinal and bulbar muscular atrophy.

Several types of motorneuron diseases are linked to neurotoxic mutant proteins. These acquire aberrant conformations (misfolding) that trigger deleterious downstream events responsible for neuronal dysfunction and degeneration. The pharmacological removal of misfolded proteins might thus be useful in these diseases. We utilized a peculiar motorneuronal disease model, spinobulbar muscular atrophy (SBMA), in which the neurotoxicity of the protein involved, the mutant androgen receptor (ARpolyQ), can be modulated by its ligand testosterone (T). 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) has already been proven to exert beneficial action in SBMA. Here we demonstrated that 17-AAG exerts its pro-degradative activity on mutant ARpolyQ without impacting on proteasome functions. 17-AAG removes ARpolyQ misfolded species and aggregates by activating the autophagic system. We next analyzed the 17-AAG effects on two proteins (SOD1 and TDP-43) involved in related motorneuronal diseases, such as amyotrophic lateral sclerosis (ALS). In these models 17-AAG was unable to counteract protein aggregation.

Androgens inhibit androgen receptor promoter activation in motor neurons.

The androgen receptor (AR), a ligand-activated transcription factor, has been found mutated in several human diseases. While some mutations reduce, others potentiate AR functions generating different endocrine dysfunctions. A peculiar AR mutation, the CAG-repeat expansion encoding the AR-polyglutamine (polyQ) tract, generates a neurotoxic gain-of-function(s) in this mutant AR (ARpolyQ). This leads to the motor neuronal disease Spinal and Bulbar Muscular Atrophy (SBMA), in which the transcriptional AR down-regulation might have beneficial impacts. We thus analysed the AR-promoter/5'-UTR activation and androgenic regulation, demonstrating that its constitutive activity is considerably high in motor neurons (NSC34). Testosterone, dihydrotestosterone (DHT), but not estradiol, inhibited AR promoter activation. Thus AR establishes a negative control on its own functions, in opposition to that described on classical androgen-responsive elements (ARE) of the AR gene. The AR/DNA interaction is required for this action, since DHT does not inhibit AR expression in presence of an AR (AR_DeltaPhe581) lacking DNA binding activity. The minimal inhibitory region spans from -740/+570 bp, where "in silico" analysis showed a putative AR binding site; deletion studies excluded that this ARE may be involved in this inhibition. A similar effect of DHT has also been observed in AR negative prostate cancer DU145 cell line transfected with the AR. Moreover, androgens down-regulate the expression of the endogenous AR gene in an AR positive prostate cancer LNCaP cell line. Interestingly, in immortalized motor neurons, ARpolyQ was much less effective than wtAR on the positive androgenic control on classical AREs, while ARpolyQ and wtAR had similar inhibitory properties on the AR promoter/5'-UTR activation. This strongly suggests that, in motor neurons, the two types of AR gene androgenic regulation involve different mechanisms. Thus, by acting on the AR promoter it would be possible to reduce AR levels in motor neurons, providing novel approaches to treat SBMA.

The role of the polyglutamine tract in androgen receptor.

The androgen receptor (AR) is a ligand-activated transcription factor which is responsible for the androgen responsiveness of target cells. Several types of mutations have been found in the AR and linked to endocrine dysfunctions. Surprisingly, the polymorphism involving the CAG triplet repeat expansion of the AR gene, coding for a polyglutamine (PolyGln) tract in the N-terminal transactivation domain of the AR protein, has been involved either in endocrine or neurological disorders. For example, among endocrine-related-diseases, the PolyGln size has been proposed to be associated to prostate cancer susceptibility, hirsutism, male infertility, cryptorchidism (in conjunction with polyglycine stretches polymorphism), etc.; the molecular mechanisms of these alterations are thought to involve a modulation of AR transcriptional competence, which inversely correlates with the PolyGln length. Among neurological alterations, a decreased AR function seems to be also involved in depression. Moreover, when the polymorphic PolyGln becomes longer than 35-40 contiguous glutamines (ARPolyGln), the ARPolyGln acquires neurotoxicity, because of an unknown gain-of-function. This mutation has been linked to a rare inherited X-linked motor neuronal disorder, the Spinal and Bulbar Muscular Atrophy, or Kennedy's disease. The disorder is characterized by death of motor neurons expressing high levels of AR. The degenerating motor neurons are mainly located in the anterior horns of the spinal cord and in the bulbar region; some neurons of the dorsal root ganglia may also be involved. Interestingly, the same type of PolyGln elongation has been found in other totally unrelated proteins responsible for different neurodegenerative diseases. A common feature of all these disorders is the formation of intracellular aggregates containing the mutated proteins; at present, but their role in the disease is largely debated. This review will discuss how the PolyGln neurotoxicity of SBMA AR may be either mediated or decreased by aggregates, and will present data on the dual role played by testosterone on motor neuronal functions and dysfunctions.

Aggregation and proteasome: the case of elongated polyglutamine aggregation in spinal and bulbar muscular atrophy.

Aggregates, a hallmark of most neurodegenerative diseases, may have different properties, and possibly different roles in neurodegeneration. We analysed ubiquitin-proteasome pathway functions during cytoplasmic aggregation in polyglutamine (polyQ) diseases, using a unique model of motor neuron disease, the SpinoBulbar Muscular Atrophy. The disease, which is linked to a polyQ tract elongation in the androgen receptor (ARpolyQ), has the interesting feature that ARpolyQ aggregation is triggered by the AR ligand, testosterone. Using immortalized motor neurons expressing ARpolyQ, we found that a proteasome reporter, YFPu, accumulated in absence of aggregates; testosterone treatment, which induced ARpolyQ aggregation, allowed the normal clearance of YFPu, suggesting that aggregation contributed to proteasome de-saturation, an effect not related to AR nuclear translocation. Using AR antagonists to modulate the kinetic of ARpolyQ aggregation, we demonstrated that aggregation, by removing the neurotoxic protein from the soluble compartment, protected the proteasome from an excess of misfolded protein to be processed.