Disruption of nongenomic testosterone signaling in a model of spinal and bulbar muscular atrophy

As one of the nine hereditary neurodegenerative polyQ disorders, spinal and bulbar muscular atrophy (SBMA) results from a polyQ tract expansion in androgen receptor (AR). Although protein aggregates are the pathological hallmark of many neurodegenerative diseases, their direct role in the neurodegeneration is more and more questioned. To determine the early molecular mechanisms causing motor neuron degeneration in SBMA, we established an in vitro system based on the tetracycline-inducible expression of normal (AR20Q), the mutated, 51 glutamine-extended (AR51Q), or polyQ-deleted (AR0Q) AR in NSC34, a motor neuron-like cell line lacking endogenous AR. Although no intracellular aggregates were formed, the expression of the AR51Q leads to a loss of function characterized by reduced neurite outgrowth and to a toxic gain of function resulting in decreased cell viability. In this study, we show that both AR20Q and AR51Q are recruited to lipid rafts in response to testosterone stimulation. However, whereas testosterone induces the activation of the c-jun N-terminal kinase/c-jun pathway via membrane-associated AR20Q, it does not so in NSC34 expressing AR51Q. Phosphorylation of c-jun N-terminal kinase plays a crucial role in AR20Q-dependent survival and differentiation of NSC34. Moreover, c-jun protein levels decrease more slowly in AR20Q- than in AR51Q-expressing NSC34 cells. This is due to a rapid and transient inhibition of glycogen synthase kinase 3α occurring in a phosphatidylinositol 3-kinase-independent manner. Our results demonstrate that the deregulation of nongenomic AR signaling may be involved in SBMA establishment, opening new therapeutic perspectives.     

Leuprorelin rescues polyglutamine-dependent phenotypes in a transgenic mouse model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an adult-onset motor neuron disease that affects males. It is caused by the expansion of a polyglutamine (polyQ) tract in androgen receptors. Female carriers are usually asymptomatic. No specific treatment has been established. Our transgenic mouse model carrying a full-length human androgen receptor with expanded polyQ has considerable gender-related motor impairment. This phenotype was abrogated by castration, which prevented nuclear translocation of mutant androgen receptors. We examined the effect of androgen-blockade drugs on our mouse model. Leuprorelin, a lutenizing hormone-releasing hormone (LHRH) agonist that reduces testosterone release from the testis, rescued motor dysfunction and nuclear accumulation of mutant androgen receptors in male transgenic mice. Moreover, leuprorelin treatment reversed the behavioral and histopathological phenotypes that were once caused by transient increases in serum testosterone. Flutamide, an androgen antagonist promoting nuclear translocation of androgen receptors, yielded no therapeutic effect. Leuprorelin thus seems to be a promising candidate for the treatment of SBMA.     

Testosterone reduction prevents phenotypic expression in a transgenic mouse model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is a polyglutamine disease caused by the expansion of a CAG repeat in the androgen receptor (AR) gene. We generated a transgenic mouse model carrying a full-length AR containing 97 CAGs. Three of the five lines showed progressive muscular atrophy and weakness as well as diffuse nuclear staining and nuclear inclusions consisting of the mutant AR. These phenotypes were markedly pronounced in male transgenic mice, and dramatically rescued by castration. Female transgenic mice showed only a few manifestations that markedly deteriorated with testosterone administration. Nuclear translocation of the mutant AR by testosterone contributed to the phenotypic difference with gender and the effects of hormonal interventions. These results suggest the therapeutic potential of hormonal intervention for SBMA.     

Androgen receptor and Kennedy disease/spinal bulbar muscular atrophy

Kennedy Disease/Spinal Bulbar Muscular Atrophy (KD/SBMA) is a progressive neurodegenerative disease caused by genetic polyglutamine expansion of the androgen receptor. We have recently found that overexpression of wildtype androgen receptor in skeletal muscle of transgenic mice results in a KD/SBMA phenotype. This surprising result challenges the orthodox view that KD/SBMA requires expression of polyglutamine expanded androgen receptor within motoneurons. Theories relating to the etiology of this disease drawn from studies of human patients, cellular and mouse models are considered with a special emphasis on potential myogenic contributions to as well as the molecular etiology of KD/SBMA.     

Transgenic mouse models of spinal and bulbar muscular atrophy (SBMA)

Spinal and bulbar muscular atrophy (SBMA) is a late-onset motor neuron disease characterized by proximal muscle atrophy, weakness, contraction fasciculations, and bulbar involvement. Only males develop symptoms, while female carriers usually are asymptomatic. A specific treatment for SBMA has not been established. The molecular basis of SBMA is the expansion of a trinucleotide CAG repeat, which encodes the polyglutamine (polyQ) tract, in the first exon of the androgen receptor (AR) gene. The pathologic hallmark is nuclear inclusions (NIs) containing the mutant and truncated AR with expanded polyQ in the residual motor neurons in the brainstem and spinal cord as well as in some other visceral organs. Several transgenic (Tg) mouse models have been created for studying the pathogenesis of SBMA. The Tg mouse model carrying pure 239 CAGs under human AR promoter and another model carrying truncated AR with expanded CAGs show motor impairment and nuclear NIs in spinal motor neurons. Interestingly, Tg mice carrying full-length human AR with expanded polyQ demonstrate progressive motor impairment and neurogenic pathology as well as sexual difference of phenotypes. These models recapitulate the phenotypic expression observed in SBMA. The ligand-dependent nuclear localization of the mutant AR is found to be involved in the disease mechanism, and hormonal therapy is suggested to be a therapeutic approach applicable to SBMA.     

Preimplantation genetic diagnosis for spinal and bulbar muscular atrophy (SBMA)

X-linked spinal and bulbar muscular atrophy is characterized by adult onset motor neuron disease and results from a defect in the androgen receptor. The disease is caused by a dynamic mutation in the first exon of the androgen receptor gene, involving a CAG trinucleotide repeat. We have developed a single-cell polymerase chain reaction assay for the androgen receptor gene and describe the application of this assay for preimlantation genetic diagnosis (PGD) in a couple at risk, where the female partner is a carrier of 47 repeats. Diagnosis was based on the detection of both normal and expanded alleles. Allele dropout of the expanded allele was observed in only 1 of 25 lymphoblasts of the carrier and of a non-expanded allele in 1 of 20 research blastomeres tested before the actual PGD. One contraction of four repeats was also found in the carrier's lymphoblasts. Neither expansions nor contractions were observed in the blastomeres biopsied from 11 embryos. Two embryos were unaffected, eight were female carriers and one was an affected male embryo.     

Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

Establishing human cell models of spinal muscular atrophy (SMA) to mimic motor neuron-specific phenotypes holds the key to understanding the pathogenesis of this devastating disease. Here, we developed a closely representative cell model of SMA by knocking down the disease-determining gene, survival motor neuron (SMN), in human embryonic stem cells (hESCs). Our study with this cell model demonstrated that knocking down of SMN does not interfere with neural induction or the initial specification of spinal motor neurons. Notably, the axonal outgrowth of spinal motor neurons was significantly impaired and these disease-mimicking neurons subsequently degenerated. Furthermore, these disease phenotypes were caused by SMN-full length (SMN-FL) but not SMN-Δ7 (lacking exon 7) knockdown, and were specific to spinal motor neurons. Restoring the expression of SMN-FL completely ameliorated all of the disease phenotypes, including specific axonal defects and motor neuron loss. Finally, knockdown of SMN-FL led to excessive mitochondrial oxidative stress in human motor neuron progenitors. The involvement of oxidative stress in the degeneration of spinal motor neurons in the SMA cell model was further confirmed by the administration of N-acetylcysteine, a potent antioxidant, which prevented disease-related apoptosis and subsequent motor neuron death. Thus, we report here the successful establishment of an hESC-based SMA model, which exhibits disease gene isoform specificity, cell type specificity, and phenotype reversibility. Our model provides a unique paradigm for studying how motor neurons specifically degenerate and highlights the potential importance of antioxidants for the treatment of SMA.     

ASC-J9 ameliorates spinal and bulbar muscular atrophy phenotype via degradation of androgen receptor

Motor neuron degeneration resulting from the aggregation of the androgen receptor with an expanded polyglutamine tract (AR-polyQ) has been linked to the development of spinal and bulbar muscular atrophy (SBMA or Kennedy disease). Here we report that adding 5-hydroxy-1,7-bis(3,4-dimethoxyphenyl)-1,4,6-heptatrien-3-one (ASC-J9) disrupts the interaction between AR and its coregulators, and also increases cell survival by decreasing AR-polyQ nuclear aggregation and increasing AR-polyQ degradation in cultured cells. Intraperitoneal injection of ASC-J9 into AR-polyQ transgenic SBMA mice markedly improved disease symptoms, as seen by a reduction in muscular atrophy. Notably, unlike previous approaches in which surgical or chemical castration was used to reduce SBMA symptoms, ASC-J9 treatment ameliorated SBMA symptoms by decreasing AR-97Q aggregation and increasing VEGF164 expression with little change of serum testosterone. Moreover, mice treated with ASC-J9 retained normal sexual function and fertility. Collectively, our results point to a better therapeutic and preventative approach to treating SBMA, by disrupting the interaction between AR and AR coregulators.     

Androgen induced cell death in SHSY5Y neuroblastoma cells expressing wild-type and spinal bulbar muscular atrophy mutant androgen receptors

Spinal bulbar muscular atrophy (SBMA) is one of a family of inherited neurodegenerative diseases caused by expansion of CAG encoding polyglutamine repeats; in SBMA the affected gene is the androgen receptor. To understand further the mechanisms that lead to neuronal cell death in SBMA, we generated SHSY5Y neuroblastoma cell lines that stably express identical levels of wild-type (19 polyglutamine repeat) or SBMA (52 polyglutamine repeat) androgen receptor. Parental SHSY5Y cells do not express detectable levels of the androgen receptor. In the absence of androgen, the transfected cell lines have similar phenotypes and growth characteristics to parental SHSY5Y cells. However, upon treatment with androgen, both cell lines undergo a marked dose-dependent loss of viability; this loss was significantly greater in cells expressing the SBMA receptor. Morphological analyses of the androgen treated cells revealed that cell death bore hallmarks of apoptosis involving altered nuclear morphology and cleavage of poly(ADP-ribose) polymerase and of caspase 3 in both wild-type and SBMA cell lines. The caspase inhibitor VAD-fmk was able to decrease loss of viability of both cell lines on exposure to androgen.     

Inhibition of apoptosis blocks human motor neuron cell death in a stem cell model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a genetic disorder caused by a deletion of the survival motor neuron 1 gene leading to motor neuron loss, muscle atrophy, paralysis, and death. We show here that induced pluripotent stem cell (iPSC) lines generated from two Type I SMA subjects-one produced with lentiviral constructs and the second using a virus-free plasmid-based approach-recapitulate the disease phenotype and generate significantly fewer motor neurons at later developmental time periods in culture compared to two separate control subject iPSC lines. During motor neuron development, both SMA lines showed an increase in Fas ligand-mediated apoptosis and increased caspase-8 and-3 activation. Importantly, this could be mitigated by addition of either a Fas blocking antibody or a caspase-3 inhibitor. Together, these data further validate this human stem cell model of SMA, suggesting that specific inhibitors of apoptotic pathways may be beneficial for patients.     

Synaptic defects in the spinal and neuromuscular circuitry in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a major genetic cause of death in childhood characterized by marked muscle weakness. To investigate mechanisms underlying motor impairment in SMA, we examined the spinal and neuromuscular circuitry governing hindlimb ambulatory behavior in SMA model mice (SMNΔ7). In the neuromuscular circuitry, we found that nearly all neuromuscular junctions (NMJs) in hindlimb muscles of SMNΔ7 mice remained fully innervated at the disease end stage and were capable of eliciting muscle contraction, despite a modest reduction in quantal content. In the spinal circuitry, we observed a ～28% loss of synapses onto spinal motoneurons in the lateral column of lumbar segments 3-5, and a significant reduction in proprioceptive sensory neurons, which may contribute to the 50% reduction in vesicular glutamate transporter 1(VGLUT1)-positive synapses onto SMNΔ7 motoneurons. In addition, there was an increase in the association of activated microglia with SMNΔ7 motoneurons. Together, our results present a novel concept that synaptic defects occur at multiple levels of the spinal and neuromuscular circuitry in SMNΔ7 mice, and that proprioceptive spinal synapses could be a potential target for SMA therapy.     

Modeling spinal muscular atrophy in Drosophila

Spinal Muscular Atrophy (SMA), a recessive hereditary neurodegenerative disease in humans, has been linked to mutations in the survival motor neuron (SMN) gene. SMA patients display early onset lethality coupled with motor neuron loss and skeletal muscle atrophy. We used Drosophila, which encodes a single SMN ortholog, survival motor neuron (Smn), to model SMA, since reduction of Smn function leads to defects that mimic the SMA pathology in humans. Here we show that a normal neuromuscular junction (NMJ) structure depends on SMN expression and that SMN concentrates in the post-synaptic NMJ regions. We conducted a screen for genetic modifiers of an Smn phenotype using the Exelixis collection of transposon-induced mutations, which affects approximately 50% of the Drosophila genome. This screen resulted in the recovery of 27 modifiers, thereby expanding the genetic circuitry of Smn to include several genes not previously known to be associated with this locus. Among the identified modifiers was wishful thinking (wit), a type II BMP receptor, which was shown to alter the Smn NMJ phenotype. Further characterization of two additional members of the BMP signaling pathway, Mothers against dpp (Mad) and Daughters against dpp (Dad), also modify the Smn NMJ phenotype. The NMJ defects caused by loss of Smn function can be ameliorated by increasing BMP signals, suggesting that increased BMP activity in SMA patients may help to alleviate symptoms of the disease. These results confirm that our genetic approach is likely to identify bona fide modulators of SMN activity, especially regarding its role at the neuromuscular junction, and as a consequence, may identify putative SMA therapeutic targets.     

Early functional impairment of sensory-motor connectivity in a mouse model of spinal muscular atrophy

To define alterations of neuronal connectivity that occur during motor neuron degeneration, we characterized the function and structure of spinal circuitry in spinal muscular atrophy (SMA) model mice. SMA motor neurons show reduced proprioceptive reflexes that correlate with decreased number and function of synapses on motor neuron somata and proximal dendrites. These abnormalities occur at an early stage of disease in motor neurons innervating proximal hindlimb muscles and medial motor neurons innervating axial muscles, but only at end-stage disease in motor neurons innervating distal hindlimb muscles. Motor neuron loss follows afferent synapse loss with the same temporal and topographical pattern. Trichostatin A, which improves motor behavior and survival of SMA mice, partially restores spinal reflexes, illustrating the reversibility of these synaptic defects. Deafferentation of motor neurons is an early event in SMA and may be a primary cause of motor dysfunction that is amenable to therapeutic intervention.     

Programmed cell death and the gene behind spinal muscular atrophy.

A gene involved in the development of spinal muscular atrophy (SMA) has been found on human chromosome 5 after a 4-year search. Named the neuronal apoptosis inhibitor protein (NAIP) gene, it is believed to inhibit the normal process of apoptosis--the disintegration of single cells that results from programmed cell death--in motor neurons. The researchers who found the NAIP gene also discovered that healthy people carry one complete copy of the gene along with many other partial copies. Many children with SMA have the partial copies but not the complete gene. This discovery facilitates the accurate genetic diagnosis of SMA. But gene therapy for SMA will not be possible until researchers find a suitable vector to stably introduce activated and intact copies of the gene into the motor neurons of children with SMA in time to stop motor neuron loss.     

A novel primer extension method to detect the number of CAG repeats in the androgen receptor gene in families with X-linked spinal and bulbar muscular atrophy.

X-linked spinal and bulbar muscular atrophy (SBMA), an adult-onset form of motor neuron disease, was recently reported to be caused by amplification of the CAG repeats in the androgen receptor gene. We report here a simple and rapid strategy to detect the precise number of the CAGs. After the DNA fragment containing the CAG repeats is amplified by the polymerase chain reaction, a primer extension is carried out; the extension of the end-labelled reverse primer adjacent to 3' end of CAG repeats stops at the first T after CAG repeats with the incorporation of dideoxy ATP in the reaction mixture. The resultant primer products are analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. This method could be quite useful to detect not only CAG repeats in SBMA but also other polymorphic dinucleotide and trinucleotide repeats.