Transcriptional Profile of Muscle following Acute Induction of Symptoms in a Mouse Model of Kennedy's Disease/Spinobulbar Muscular Atrophy

<div><p>Background</p><p>Kennedy’s disease/Spinobulbar muscular atrophy (KD/SBMA) is a degenerative neuromuscular disease affecting males. This disease is caused by polyglutamine expansion mutations of the androgen receptor (<i>AR</i>) gene. Although KD/SBMA has been traditionally considered a motor neuron disease, emerging evidence points to a central etiological role of muscle. We previously reported a microarray study of genes differentially expressed in muscle of three genetically unique mouse models of KD/SBMA but were unable to detect those which are androgen-dependent or are associated with onset of symptoms.</p><p>Methodology/Principal Findings</p><p>In the current study we examined the time course and androgen-dependence of transcriptional changes in the HSA-AR transgenic (Tg) mouse model, in which females have a severe phenotype after acute testosterone treatment. Using microarray analysis we identified differentially expressed genes at the onset and peak of muscle weakness in testosterone-treated Tg females. We found both transient and persistent groups of differentially expressed genes and analysis of gene function indicated functional groups such as mitochondrion, ion and nucleotide binding, muscle development, and sarcomere maintenance.</p><p>Conclusions/Significance</p><p>By comparing the current results with those from the three previously reported models we were able to identify KD/SBMA candidate genes that are androgen dependent, and occur early in the disease process, properties which are promising for targeted therapeutics.</p></div

Venn diagram of microarray results in male HSA-AR Tg (M-141) and female HSA-AR Tg after 3 days (F-T-3days) and 7 days (F-T-7days) testosterone treatment.

<p>Diagram representing the number of genes whose expression differed from controls. Gene numbers were obtained by examining gene lists generated by a 2-fold, p<0.05 criterion.</p

Venn diagram of microarray results in female HSA-AR Tg mice after 3 and 7 days testosterone treatment and WT mice after 7 days testosterone treatment.

<p>Diagram representing the number of genes whose expression differed from controls. Gene numbers were obtained by examining gene lists generated by a 2-fold, p < 0.05 criterion.</p

Hierarchical cluster analysis of gene expression in male HSA-AR Tg (M-141) and female HSA-AR Tg after 3 days (F-T3d) and 7 days (F-T7d) testosterone treatment.

<p>Cluster output represents colorimetrically indicated log2 ratio change. Male Tg mice and female Tg mice after 3 and 7 days testosterone treatment are represented by columns and rows represent a single gene. Clusters of genes differentially expressed in a similar pattern are labeled.</p

The list and fold change of differentially expressed genes found in three KD/SBMA mouse models (as compared to WT controls) and female HSA-AR Tg mice with 7 days testosterone treatment (as compared to WT with 7 days testosterone treatment).

<p>Some of these genes were also differentially expressed in other models like Huntington’s disease (HD; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118120#pone.0118120.ref017" target="_blank">17</a>]), AR knock out (ARKO; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118120#pone.0118120.ref018" target="_blank">18</a>]), and Atrophy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118120#pone.0118120.ref019" target="_blank">19</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118120#pone.0118120.ref020" target="_blank">20</a>].</p><p>The list and fold change of differentially expressed genes found in three KD/SBMA mouse models (as compared to WT controls) and female HSA-AR Tg mice with 7 days testosterone treatment (as compared to WT with 7 days testosterone treatment).</p

Validation of the results of microarray experiments by qRT-PCR analysis.

<p>Overall, we find consistency between qPCR and microarray analysis. Genes of varying fold changes were chosen to demonstrate the validity of microarray data. Bars show Tg females treated with testosterone for 3 days as compared to WT treated for 3 days, and Tg females treated for 7 days as compared to female WT treated for 7 days. Error bars represent standard error of the mean.</p

Hierarchical cluster analysis of gene expression in female HSA-AR Tg and WT mice after 3 and 7 days testosterone treatment.

<p>Cluster output represents colorimetrically indicated log2 ratio change. Female HSA-AR Tg and WT mice after 3 and 7 days testosterone treatment are represented by columns and rows represent a single gene. Clusters of genes differentially expressed in a similar pattern are labeled.</p

Antibiotic-induced socio-sexual behavioral deficits are reversed via cecal microbiota transplantation but not androgen treatment

Recent evidence has demonstrated a sex-specific role of the gut microbiome on social behavior such as anxiety, possibly driven by a reciprocal relationship between the gut microbiome and gonadal hormones. For instance, gonadal hormones drive sex differences in gut microbiota composition, and certain gut bacteria can produce androgens from glucocorticoids. We thus asked whether the gut microbiome can influence androgen-dependent socio-sexual behaviors. We first treated C57BL/6 mice with broad-spectrum antibiotics (ABX) in drinking water to deplete the gut microbiota either transiently during early development (embryonic day 16-postnatal day [PND] 21) or in adulthood (PND 60–85). We hypothesized that if ABX interferes with androgens, then early ABX would interfere with critical periods for sexual differentiation of brain and thus lead to long-term decreases in males' socio-sexual behavior, while adult ABX would interfere with androgens’ activational effects on behavior. We found that in males but not females, early and adult ABX treatment decreased territorial aggression, and adult ABX also decreased sexual odor preference. We then assessed whether testosterone and/or cecal microbiota transplantation (CMT) via oral gavage could prevent ABX-induced socio-sexual behavioral deficits in adult ABX-treated males. Mice were treated with same- or other-sex control cecum contents or with testosterone for two weeks. While testosterone was not effective in rescuing any behavior, we found that male CMT restored both olfactory preference and aggression in adult ABX male mice, while female CMT restored olfactory preference but not aggression. These results suggest sex-specific effects of the gut microbiome on socio-sexual behaviors, independent of androgens

Disease Affects Bdnf Expression in Synaptic and Extrasynaptic Regions of Skeletal Muscle of Three SBMA Mouse Models

Spinal bulbar muscular atrophy (SBMA) is a slowly progressive, androgen-dependent neuromuscular disease in men that is characterized by both muscle and synaptic dysfunction. Because gene expression in muscle is heterogeneous, with synaptic myonuclei expressing genes that regulate synaptic function and extrasynaptic myonuclei expressing genes to regulate contractile function, we used quantitative PCR to compare gene expression in these two domains of muscle from three different mouse models of SBMA: the &ldquo;97Q&rdquo; model that ubiquitously expresses mutant human androgen receptor (AR), the 113Q knock-in (KI) model that expresses humanized mouse AR with an expanded glutamine tract, and the &ldquo;myogenic&rdquo; model that overexpresses wild-type rat AR only in skeletal muscle. We were particularly interested in neurotrophic factors because of their role in maintaining neuromuscular function via effects on both muscle and synaptic function, and their implicated role in SBMA. We confirmed previous reports of the enriched expression of select genes (e.g., the acetylcholine receptor) in the synaptic region of muscle, and are the first to report the synaptic enrichment of others (e.g., glial cell line-derived neurotrophic factor). Interestingly, all three models displayed comparably dysregulated expression of most genes examined in both the synaptic and extrasynaptic domains of muscle, with only modest differences between regions and models. These findings of comprehensive gene dysregulation in muscle support the emerging view that skeletal muscle may be a prime therapeutic target for restoring function of both muscles and motoneurons in SBMA