Supplementary Material for: Prenatal Flutamide Enhances Survival in a Myogenic Mouse Model of Spinal Bulbar Muscular Atrophy

Background: Spinal bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion mutation in the androgen receptor (AR) gene, and mutant AR is presumed to act in motoneurons to cause SBMA. However, we found that mice overexpressing wild-type (wt) AR solely in skeletal muscle fibers display the same androgen-dependent disease phenotype as when mutant AR is broadly expressed, challenging the assumptions that only an expanded AR can induce disease and that SBMA is strictly neurogenic. We have previously reported that AR toxicity was ligand dependent in our model, and that very few transgenic (tg) males survived beyond birth. Methods: We tested whether the AR antagonist flutamide could block perinatal toxicity. tg males were treated prenatally with flutamide and assessed for survival and motor behavior in adulthood. Results: Prenatal treatment with flutamide rescued tg male pups from perinatal death, and, as adults, such perinatally rescued tg males showed an SBMA phenotype that was comparable to that of previously described untreated tg males. Moreover, tg males carrying a mutant endogenous allele for AR – the testicular feminization mutation (tfm) – and thus having functional AR only in muscle fibers nevertheless displayed the same androgen-dependent disease phenotype as adults. Conclusions: These mice represent an excellent model to study the myogenic contribution to SBMA as they display many of the core features of disease as other mouse models. These data demonstrate that AR acting exclusively in muscle fibers is sufficient to induce SBMA symptoms and that flutamide is protective perinatally.

背景：脊髓延髓肌萎缩症（Spinal bulbar muscular atrophy，SBMA）是由雄激素受体（androgen receptor，AR）基因的CAG重复扩增突变所引发的疾病，此前学界普遍推测突变型雄激素受体（mutant AR）通过作用于运动神经元导致SBMA。然而本研究发现，仅在骨骼肌纤维中过表达野生型（wild-type，wt）AR的小鼠，会表现出与广泛表达突变型AR时相同的雄激素依赖性疾病表型，这对“仅扩增型AR才可致病”以及“SBMA严格属于神经源性疾病”的两项假设均提出了挑战。本团队此前已报道，在该模型中AR毒性具有配体依赖性，且仅有极少数转基因（transgenic，tg）雄性小鼠可存活至出生后。

方法：本研究测试了AR拮抗剂氟他胺（flutamide）是否能够阻断围产期毒性。对转基因雄性小鼠产前给予氟他胺处理，并评估其成年后的存活率与运动行为表现。

结果：产前氟他胺处理可挽救转基因雄性幼鼠免于围产期死亡；成年后，经围产期挽救的转基因雄性小鼠所表现出的SBMA表型，与此前报道的未处理转基因雄性小鼠相当。此外，携带突变型内源性AR等位基因——即睾丸女性化突变（testicular feminization mutation，tfm）——因而仅在肌纤维中具有功能性AR的转基因雄性小鼠，成年后仍表现出相同的雄激素依赖性疾病表型。

结论：此类小鼠是研究SBMA肌源性致病贡献的优质模型，它们展现出了其他SBMA小鼠模型所具备的诸多核心疾病特征。本研究数据证实，仅在肌纤维中发挥作用的AR足以诱导SBMA症状，且氟他胺在围产期具有保护作用。