spinal and bulbar muscular atrophy ( sbma ) is a slowly progressive adult onset neurodegenerative disease that affects men in mid - life .
the disease causes degeneration and atrophy of the muscles of the proximal limb girdle and the bulbar muscles of the jaw ( kennedy et al . , 1968 ) , as well as the accumulation of nuclear aggregates of mutant androgen receptor ( ar ) protein , otherwise known as neuronal intranuclear inclusions ( niis ) ( li et al . , 1998 ) .
sbma results from a cag expansion in the first exon of the ar gene , located on the proximal long arm of the x chromosome ( la spada et al . , 1991 ) .
in the rare instances when female carriers manifest disease , their symptoms are mild ( ferlini et al . , 1995 ) .
initially , it was unclear whether female carriers were protected from disease symptoms owing solely to x - inactivation .
however , the finding of homozygous females with very mild symptoms ( schmidt et al . , 2002 ) and evidence from several cell and animal models of sbma indicate that testosterone plays a crucial and causative role in disease pathogenesis .
cellular models of disease show an increase in nuclear inclusion formation upon hormone treatment ( stenoien et al . , 1999
; walcott and merry , 2002 ) , whereas fly models demonstrate that consumption of testosterone induces disease as well as nuclear inclusion formation ( takeyama et al . , 2002 ) .
transgenic mouse models have indicated that both surgical and chemical castration of sbma males alleviates or prevents disease progression , depending on the stage of disease at the time of intervention ( katsuno et al . , 2002 ;
furthermore , administration of testosterone to transgenic female sbma mice exacerbates disease phenotype ( katsuno et al . ,
2002 ) , as does giving testosterone to male sbma mice treated with leuprorelin , a lutenizing hormone - releasing hormone ( lhrh ) analog ( katsuno et al . , 2003 ) . for years , many patients with sbma received testosterone treatment .
the rationale for this treatment was complex , but a partial loss of ar function coupled with the known anabolic effects on muscle suggested that testosterone might prove beneficial ( sheffield - moore , 2000 ) . despite the evidence indicating that testosterone contributes to the progression of sbma , these patients have not reported any negative effects as a result of androgen administration ( goldenberg and bradley , 1996 ; neuschmid - kaspar et al . , 1996 ) . however , one case study reported on the substantial decline of a patient with sbma , with subsequent recovery upon cessation of testosterone treatment ( kinirons and rouleau , 2008 ) .
however , the fact that this patient was taking paroxetine , a selective serotonin reuptake inhibitor ( ssri ) , might complicate the interpretation of this case .
paroxetine has been shown to enhance the conversion of dihydrotestosterone ( dht ) to androstanediol ( griffin and mellon , 1999 ) , and thus there might have been relatively low relative levels of dht within his spinal motor neurons .
treatment of this patient with testosterone might have restored his dht levels and exacerbated disease .
previously , we created and characterized mice that were transgenic for the full - length expanded human ar gene , containing 112 cag repeats , driven by the prion protein ( prp ) promoter ( ar112q mice ) ( chevalier - larsen et al . , 2004 ) .
the disease in this model progresses slowly , with ar112q males beginning to exhibit mild motor dysfunction at 3 months of age and developing late - stage disease by 10 months of age ( chevalier - larsen et al . , 2004 ) .
, we treated a cohort of these ar112q male mice with testosterone , beginning at 2 months of age , for a period of 6 months .
behavioral and histopathological measures of disease indicate no change in severity or age of onset of disease with testosterone treatment ( t - treatment ) .
these data have important clinical implications for patients and suggest that there is a point at which the role of hormone in the pathogenic mechanism of sbma becomes saturated .
( a ) testosterone treatment resulted in elevated serum testosterone levels in transgenic ( tg ) t - treated ( n=8 ) and non - transgenic ( ntg ) t - treated ( n=10 ) animals when compared with tg sham - treated ( n=6 ) mice ( p<0.05 ) , with the exception of months three and six .
although , testosterone levels dropped at the end of pellet life , testosterone levels were still significantly elevated in ntg t - treated mice in month six and there was a trend towards elevated testosterone in tg t - treated animals .
( b ) ability to remain on a rotarod was impaired in ar112q t - treated ( n=10 ) and ar112q sham - treated ( n=12 ) males when compared with t - treated non - transgenics ( n=14 ) ( p<0.05 ) , but rotarod performance was similar between t - treated ar112q males and sham - treated ar112q males ( p>0.05 ) .
( c  e ) grip strength [ using forepaws ( c ) or all paws ( d ) ] and vertical activity ( e ) of all ar112q males was reduced in comparison to t - treated non - transgenic males ( p<0.05 ) , but no significant decrease in any of these measures was observed upon t - treatment of ar112q males ( p>0.05 ) .
( f ) clasping behavior increased with age in all ar112q males and was not affected by t - treatment ; non - transgenic males rarely showed clasping behavior .
transgenic ar112q and non - transgenic mice were implanted with timed - release pellets designed to deliver 46 ng / ml of testosterone for 90 days . over the course of the experiment , implantation of testosterone pellets
increased circulating testosterone an average of threefold , from 0.400.23 ng / ml ( ar112q sham - treated ; n=10 ) to 1.260.48 ng / ml [ non - transgenic ( ntg ) t - treated ; n=8 ] and 1.170.35 ng / ml [ transgenic ( tg ) t - treated ; n=6 ] ; this is a significant elevation of testosterone levels in treated animals ( ntg t - treated and tg t - treated ) over those implanted with placebo pellets ( tg sham - treated ) ( p<0.001 ) . although 90-day testosterone pellets were used , we observed a decline in potency of the pellets by the end of the 90-day period ( months three and six ) ( fig .
implantation of new pellets at the end of 90 days restored circulating testosterone levels ( fig .
although circulating testosterone was elevated in treated mice , motor function assays did not reveal any effect of testosterone treatment on phenotype .
motor function assays were performed monthly for 6 months ; results were consistent for all 6 months of treatment .
beginning at 3 months of age ( 1 month following treatment ) , ar112q males showed decreased rotarod performance , regardless of t - treatment , when compared with non - transgenic t - treated males ( fig .
1b ) . decreased grip strength was apparent in all ar112q animals , regardless of t - treatment , when animals gripped with either forepaws only ( fig .
2004 ) , weakness was more pronounced when assessing all paws , implying greater weakness in the hindlimbs .
vertical activity , or rearing behavior , was also reduced in ar112q males ( fig .
1e ) , although no deficits were seen in t - treated ar112q males when compared with sham - treated ar112q males .
clasping behavior was greater in both t - treated and sham - treated ar112q males when compared with t - treated non - transgenic mice and increased steadily with time ( fig .
t - treatment had no effect on weight or survival and no gross alterations in muscle fiber morphology were observed ( data not shown ) .
cellular markers of disease were also examined in spinal cord and brain tissue to determine whether t - treatment expedited cellular dysfunction that might not be detected by behavioral changes . as with the behavioral analyses
, no changes were detected between the t - treated and sham - treated ar112q males .
niis were present in both t - treated and sham - treated males ; nii size and frequency were similar between these two groups ( fig .
a total of 49.66.5% of motor neurons from lumbar spinal cord contained niis in tg sham - treated animals , whereas niis were seen in 48.510.5% of motor neurons in tg t - treated mice ( fig .
similarly , the level of unphosphorylated neurofilament heavy chain ( nf - h ) was reduced in the neuronal soma of all ar112q animals when compared with non - transgenic mice ( fig .
2a ) , and the extent to which unphosphorylated nf - h immunoreactivity was reduced was similar between all ar112q animals , regardless of t - treatment ( fig . 2c ) . in summary ,
signs of cellular pathogenesis were consistent with those observed during characterization of this disease model and were unchanged by long - term treatment with testosterone .
although it is clear that testosterone plays a key role in sbma pathogenesis , the mechanism leading to neurodegeneration remains unknown , although an increased understanding of the specific aspects of ar metabolism that contribute to its misfolding , aggregation and toxicity have come to light in recent years ( montie et al .
the data from this study suggest that , whatever the mechanism that leads to neuronal dysfunction , there is a threshold at which the effect of testosterone on this pathway plateaus .
it is therefore likely that testosterone would have a greater capacity to exert its pathogenic effects in ar112q mice than in sbma patients expressing normal levels of ar . despite their increased capability to bind ligand , testosterone in excess of endogenous levels
niis were detected with ar - h280 antibody in spinal cord of all ar112q males ( a , top ) ; no differences in nii frequency were detected upon t - treatment ( b ; n=2 ) .
nuclei of non - transgenic ( ntg ) t - treated males show diffuse nuclear ar and no niis ( a ) .
unphosphorylated nf - h , as detected by smi32 immunostaining ( a , bottom ) , was abundant in neuronal soma of the non - transgenic spinal cord but was reduced in ar112q tissue .
( c ) quantification of smi32 intensity did not reveal any differences between transgenic ( tg ) t - treated and tg sham - treated males ( n=2 ) .
panels showing niis ( a , top ) and smi32 ( a , bottom ) are not from the same fields .
insets in a represent the location within the anterior horn from which higher - magnification images were taken ( yellow box ) .
the red oval outlines the central canal , with the dorsal aspect to the left and the ventral aspect to the right .
several previous studies have demonstrated that hormone withdrawal can ameliorate disease onset and/or progression ( katsuno et al . , 2002 ; katsuno et al . , 2003 ; chevalier - larsen et al .
it is possible that the brief decline in elevated testosterone levels seen at the end of month three of these studies might alleviate any adverse effects of hormone treatment .
this seems unlikely , however , because behavioral improvement due to androgen ablation was not observed in mice of a comparable age until 3 months following castration ( chevalier - larsen et al . , 2004 ) and
a caveat to the studies reported here is that our transgenic mice express the mutant ar primarily in the nervous system , through the use of the prp promoter , and thus the evaluation of the mutant protein ( and added testosterone ) in muscle , which plays an important role in disease pathogenesis ( yu et al . ,
expression of the prp - promoter - driven transgene in muscle was observed , albeit at levels lower than those in neural tissue ( chevalier - larsen et al . , 2004 ) , and nuclear inclusions in muscle were also noted ( data not shown ) . whether testosterone treatment would exacerbate disease through effects on muscle is unknown ,
although we find the possibility of different pathogenic processes in motor neurons and muscle unlikely .
mutant ar protein might exert its toxic effects on neurons through multiple pathways , including disruption of protein folding homeostasis , interference with protein degradation , transcriptional dysregulation and/or by inhibition of axonal transport .
it is possible that a threshold level of mutant ar present in the nucleus would lead to maximal disruption of neuronal function . in motor neurons , testosterone and dht
; thus , an excess of these ligands might actually moderate the prevalence of the mutant ar .
it is likely that dht , not testosterone , is the ligand responsible for neuronal pathology in sbma , given the high motor neuron levels of the testosterone - to - dht converting enzyme 5-reductase ( pozzi et al . , 2003 ) .
in this case , the conversion of testosterone to dht via 5-reductase could represent a point of saturation in the pathological pathway .
this hypothesis is particularly attractive because it would suggest a mechanism for cell - type specificity in sbma .
however , manipulation of 5-reductase activity with the inhibitor dutasteride failed to modulate disease in sbma patients ( fernandez - rhodes et al . , 2011 ) or in a transgenic mouse model of sbma
regardless of the molecular mechanism by which this saturation occurs , our data are encouraging in that sbma patients who had previously taken supplemental testosterone are unlikely to have exacerbated their disease .
genotyping was performed as described previously ( chevalier - larsen et al . , 2004 ) .
the area between the shoulder blades was shaved and the surgical area sterilized with alcohol . a small ( 12 cm ) dorsal midline incision was made .
a pellet containing a 90-day - release 12.5 mg testosterone pellet or a placebo pellet ( both from innovative research of america ) was placed into the pocket and the incision was closed with a wound clip .
mice were re - implanted with hormone or sham pellet once during the course of this study .
all animal surgeries were performed according to the guidelines of the office of laboratory animal welfare ( olaw ) and the institutional animal care and use committee ( iacuc ) at thomas jefferson university .
sera were assessed for circulating testosterone levels using a colorimetric eia kit ( caymen chemical ) according to the manufacturer s instructions .
testing was carried out as previously described ( chevalier - larsen et al . , 2004 ) .
immunofluorescence of brain and spinal cord tissue was carried out as previously described ( chevalier - larsen et al . , 2004 ) .
antibodies used include ar h280 ( 1:100 ; santa cruz ) and smi32 ( 1:1000 ; sternberger monoclonal ) . motor neurons of the lumbar spinal cord were scored as inclusion - positive whether they had several punctate or single , large inclusions .
unphosphorylated neurofilament immunoreactivity was quantified using imagej software ; integrated density values were acquired for regions of interest delineated around motor neuron soma of the lumbar spinal cord .
spinal and bulbar muscular atrophy ( sbma ) is a neurodegenerative disorder that is caused by a polyglutamine expansion in the androgen receptor ( ar ) and affects approximately 1 in every 40,000 men .
affected men typically develop muscle weakness of the proximal limbs and bulbar muscle , tremors and fasciculations in mid - life .
signs of androgen insensitivity , such as gynecomastia and reduced fertility , often accompany muscle deficits .
female carriers of the disease are protected both by x - inactivation of the gene encoding the mutant ar and by lower levels of circulating ar ligands ( testosterone and dihydrotestosterone ) .
prior to the discovery that disease pathogenesis is androgen - dependent , many individuals with sbma received androgens therapeutically , based on the assumption at the time that a loss of ar function contributed to disease symptoms .
this study examines the impact of exogenous testosterone on disease progression in a mouse model of sbma to determine whether testosterone treatment of the patient population might in fact have exacerbated the disease .
the data indicate that the addition of exogenous testosterone does not exacerbate disease progression in intact male mice that are transgenic for a mutant ar . behavioral measures of disease in testosterone - treated transgenic males are indistinguishable from transgenic males that are sham treated . additionally , cellular markers of disease in testosterone - treated transgenic males are comparable to those in sham - treated transgenic males . implications and future directions these data suggest that it is unlikely that androgen therapy has hastened the progress of disease in sbma patients .
they also suggest that there is a threshold at which the contribution of testosterone to disease pathogenesis becomes saturated .
 summaryevidence from multiple animal models demonstrates that testosterone plays a crucial role in the progression of symptoms in spinal and bulbar muscular atrophy ( sbma ) , a condition that results in neurodegeneration and muscle atrophy in affected men . 
 mice bearing a transgene encoding a human androgen receptor ( ar ) that contains a stretch of 112 glutamines ( expanded polyglutamine tract ; ar112q mice ) reproduce several aspects of the human disease . 
 we treated transgenic male ar112q mice with testosterone for 6 months . 
 surprisingly , testosterone treatment of ar112q males did not exacerbate the disease . 
 although transgenic ar112q males exhibited functional deficits when compared with non - transgenics , long - term testosterone treatment had no effect on motor function . 
 testosterone treatment also failed to affect cellular markers of disease , including inclusion formation ( the accumulation of large nuclear aggregates of mutant ar protein ) and levels of unphosphorylated neurofilament heavy chain . 
 these data suggest that the mechanism of disease in sbma saturates at close to endogenous hormone levels and that individuals with sbma who take , or have taken , testosterone for its putative therapeutic properties are unlikely to suffer adverse effects . 
INTRODUCTION
RESULTS
DISCUSSION
METHODS