FUS is not dysregulated by the spinal bulbar muscular atrophy androgen receptor polyglutamine repeat expansion

Spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis are two distinct forms of motor neuron disease with different genetic causes, pathology, and clinical course. However, both disorders are characterized by the progressive loss of lower motor neurons and by a similar protective response to growth factors in animal models, therefore raising the possibility of an overlap in the final pathogenic cascade. Mutations in the FUS gene and fused in sarcoma (FUS) protein pathology have now been identified in some amyotrophic lateral sclerosis cases, while a CAG expansion in the androgen receptor gene is known to cause SBMA. Recently, multiple lines of evidence have identified FUS as a major target of the androgen receptor, suggesting that FUS could be dysregulated in SBMA motor neurons. We have investigated this possibility by using a well-established mouse model of SBMA and our analysis of primary motor neuron cultures, spinal cords, and microdissected motor neurons show no evidence for FUS dysregulation.


a b s t r a c t
Spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis are two distinct forms of motor neuron disease with different genetic causes, pathology, and clinical course. However, both disorders are characterized by the progressive loss of lower motor neurons and by a similar protective response to growth factors in animal models, therefore raising the possibility of an overlap in the final pathogenic cascade. Mutations in the FUS gene and fused in sarcoma (FUS) protein pathology have now been identified in some amyotrophic lateral sclerosis cases, while a CAG expansion in the androgen receptor gene is known to cause SBMA. Recently, multiple lines of evidence have identified FUS as a major target of the androgen receptor, suggesting that FUS could be dysregulated in SBMA motor neurons. We have investigated this possibility by using a well-established mouse model of SBMA and our analysis of primary motor neuron cultures, spinal cords, and microdissected motor neurons show no evidence for FUS dysregulation.

Introduction
Mutations in Fused in Sarcoma (FUS) have been found to cause up to 4% of familial amyotrophic lateral sclerosis (Ferraiuolo et al., 2011). Spinal bulbar muscular atrophy (SBMA) is a slowly progressive adult-onset motor neuron (MN) disease characterized by the loss of lower MNs (LMNs) in the spinal cord and brain stem. It is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene which results in an extended polyglutamine tract (AR SBMA poly-Q) (Rhodes et al., 2009). The AR is a ligand-dependant transcription factor and the AR SBMA poly-Q expansion has been shown to cause disease through a variety of mechanisms, including an amplification of native AR interactions and function (Nedelsky et al., 2010).
Amyotrophic lateral sclerosis and SBMA fall under the general class of MN diseases, but have different genetic causes, pathology, and clinical course. Nonetheless, both diseases are associated by the progressive adult onset loss of LMNs, similarities in specific pathology findings, and a positive response of animal models to general protective treatments (Forsberg et al., 2012;Papanikolaou and Ellerby, 2009;Sopher et al., 2004). These findings suggest the possibility that the diseases share features in the pathogenetic cascade that leads to neuronal death (Papanikolaou and Ellerby, 2009).
Two recent independent studies, focusing on prostate cancer cells, have shown that fused in sarcoma (FUS) levels are regulated by the AR in an androgen-dependent fashion (Brooke et al., 2011;Haile et al., 2011). These results raise the possibility that FUS might be misregulated in SBMA, and this might contribute to the MN degeneration in the disease, offering an intriguing link between these 2 distinct forms of MN degeneration. Here we investigate this possible link by determining if FUS expression is dysregulated in a well characterized SBMA mouse model (AR100) carrying a 100 poly-Q expanded mutation in the AR (Sopher et al., 2004).

Methods
Details of mice, embryonic MN culture, laser capture microdissection, real-time quantitative polymerase chain reaction, Western blot, and immunohistochemistry methods are given in the Supplementary data.

Results
To assess the effect of the AR SBMA poly-Q repeat expansion and dihydrotestosterone (DHT) on FUS regulation in MNs, we initially determined FUS messenger RNA (mRNA) levels in purified embryonic MN (eMN) cultures obtained from AR100 and wild type (WT) littermate embryos. Taqman analysis showed that DHT treatment increases FUS levels [F(1,8) ¼ 10.108; p ¼ 0.013], but there was no significant difference between AR100 and WT eMNs in both DHT-treated and untreated conditions. (Fig. 1A and B).
To assess if the presence of the AR SBMA poly-Q repeat expansion caused a change in FUS levels in the context of the mature adult central nervous system, we tested FUS protein levels in spinal cord lysates of presymptomatic 3-month-old mice. The results showed no significant difference in FUS protein levels between AR100 mice and littermate WT controls (Fig. 1C).
FUS protein levels in the spinal cord come from an extremely diverse population of cells and this might mask possible differences present in specific cell types. We therefore analyzed FUS mRNA (C) Protein levels in 3-month-old AR100 (n ¼ 6) and littermate control (WT) (n ¼ 5) spinal cords normalized to b-actin (ns, by 2-tailed unpaired t test; p ¼ 0.62). (D) Protein levels in 12-month-old AR100 (n ¼ 3) and WT (n ¼ 3) spinal cords normalized to b-actin (ns, by 2-tailed unpaired t test; p ¼ 0.49). FUS mRNA expression in laser capture microdissection motor neurons (MNs) of AR100 and WT mice, normalized to Actb (E) and Gapdh (F) (ns, by 2-tailed unpaired t test; p ¼ 0.30, p ¼ 0.63, respectively). Fused in sarcoma protein (Fus) localization is normal in 18-month-old AR100 mice MNs. Confocal images from lumbar spinal cord sections of WT (GeJ) and AR100 (KeN) mice. Choline acetyltransferase (ChAT) (green), Fus (red), DAPI (blue). Fus appears to localize mainly in the nucleus of MNs and other cell types in both genotypes. levels specifically in LMNs isolated by laser capture microdissection. However, no significant changes were observed in AR100 LMNs ( Fig. 1E and F).
Finally, to test if FUS dysregulation plays a role during the active disease process, we analyzed FUS protein levels in spinal cords of aged AR100 and control mice and found no significant differences (Fig. 1D). We also investigated FUS localization in lumbar spinal cord sections from end-stage AR100 and control mice. The staining revealed that FUS was localized in the nucleus throughout cells in the spinal cord and also specifically in MNs, in both genotypes (Fig. 1GeN).

Discussion
FUS, which when mutated can be causative of amyotrophic lateral sclerosis and is found to mislocalize in patients' spinal cords (Ferraiuolo et al., 2011), is known to interact with nuclear hormone receptors and the AR (Haile et al., 2011;Powers et al., 1998). FUS is regulated at both the protein and mRNA levels through the AR in response to androgen stimuli (Brooke et al., 2011;Haile et al., 2011). These findings, along with the recent evidence that the mutant expanded poly-Q AR exerts part of its pathogenicity through misappropriation of native function (Nedelsky et al., 2010), have raised the question as to whether FUS could be misregulated in SBMA.
Our results show that FUS mRNA levels are increased in response to DHT in both AR100 and control purified eMNs, but no difference between AR100 and controls was found. We then analyzed if any pathologic changes occurred in vivo and, to study the direct effect of the poly-Q expanded AR, and not secondary disease changes, we chose to conduct our initial analysis at the presymptomatic age of 3 months. Both FUS protein levels in spinal cord lysates and FUS mRNA levels in microdissected MNs showed no changes between AR100 and controls. Finally, we investigated the involvement of FUS in the active phases of disease by testing mice at advanced and endstage time points. However, even at these symptomatic stages of disease, protein levels were unchanged and FUS localization was also normal.
In conclusion, our results show that FUS expression is not altered in the spinal cord or in MNs in AR100 mice and is therefore not influenced by the presence of the poly-Q expanded AR or by secondary changes occurring in active SBMA.

Disclosure statement
The authors declare no actual of potential conflicts of interest. Mice were housed in controlled conditions in accordance with guidance issued by the Medical Research Council in Responsibility in the Use of Animals for Medical Research (1993) and all experi-ments were carried out under License from the UK Home Office and with Local Ethical Review panel approval.