Androgen receptor antagonism accelerates disease onset in the SOD1^G93A mouse model of amyotrophic lateral sclerosis

Abstract

Background and Purpose Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease typically more common in males, implicating androgens in progression of both patients and mouse models. Androgen effects are mediated by androgen receptor which is highly expressed in spinal motor neurons and skeletal muscles. To clarify the role of androgen receptors in ALS, we therefore examined the effect of androgen receptor antagonism in the SOD1G93A mouse model. Experimental Approach The androgen receptor antagonist, flutamide, was administered to presymptomatic SOD1G93A mice as a slow‐release subcutaneous implant (5 mg·day−1). Testosterone, flutamide, and metabolite levels were measured in blood and spinal cord tissue by LC–MS–MS. Effects on disease onset and progression were assessed using motor function tests, survival, muscle, and neuropathological analyses. Key Results Flutamide was metabolised to 2‐hydroxyflutamide achieving steady‐state plasma levels across the study duration and reached the spinal cord at pharmacologically active concentrations. Flutamide treatment accelerated disease onset and locomotor dysfunction in male SOD1G93A mice, but not female mice, without affecting survival. Analysis of hindlimb muscles revealed exacerbation of myofibre atrophy in male SOD1G93A mice treated with flutamide, although motor neuron pathology was not affected. Conclusion and Implications The androgen receptor antagonist accelerated disease onset in male SOD1G93A mice, leading to exacerbated muscle pathology, consistent with a role of androgens in modulating disease severity, sexual dimorphism, and peripheral pathology in ALS. These results also demonstrate a key contribution of skeletal muscle pathology to disease onset, but not outcome, in this mouse model of ALS.

Dissociation of disease onset, progression and sex differences from androgen receptor levels in a mouse model of amyotrophic lateral sclerosis

“…Male vs. female WT levels were not significantly different using two-way ANOVA analysis due to the dominating effect of the AR Q24 levels. When analysed separately, female WT mice had 40% less AR in lumbar spinal cord compared to males and 54% less AR in gastrocnemius, consistent with our previous study 20 . AR protein overexpression was reflected in ventral horn spinal cord immunohistochemistry (Fig.…”

“…Alternatively, the adrenal glands provide another source of androgens 19 . Blockade of AR by the anti-androgen, flutamide, exacerbated muscle atrophy and induced an earlier disease onset in male SOD1 G93A mice 20 . Administration of the androgen, dihydrotestosterone (DHT), was neuroprotective in SOD1 G93A male mice 21 , while the anabolic steroid nandrolone was detrimental 22 .…”

Exploring germline recombination in Nestin‐Cre transgenic mice using floxed androgen receptor

“…Primer sequences were: Ar ex1 forward 5 0 -AAG CAG GTA GCT CTG GGA CA-3 0 , Ar ex1 reverse 5 0 -GAG CCA GCG GAA AGT TGT AG-3 0 ; and internal control DAG1 forward 5 0 -CCA AGG AGC AGA TCA TAG GGC-3 0 , DAG1 reverse 5 0 -AGA GCA TTG GAG AAG GCA GG-3 0 . RT-qPCR was performed as previously described (McLeod et al, 2019). Relative expression of the Ar gene was deter-…”

“…Data were analyzed by Student's t test comparing genotype using GraphPad Prism 8.0 software (San Diego, CA) with p < .05 taken as statistical significance. Data are presented as mean ± SEM.1.4 | ImmunoblottingWhole brain, testis and ovary lysates were prepared using previously described methods(McLeod et al, 2019). Protein lysates from brain (50 μg), testis (20 μg) and ovary (20 μg) were electrophoresed throughT A B L E 1 Breeding capacity of AR flox ::NesCre Parent Mice Free™ FastCast™ Acrylamide Kit handcast gels (Bio-Rad Laboratories, NSW, Australia) and transferred onto PVDF membrane at 20 V overnight at 4 C. Total protein transferred onto the membrane was imaged on a ChemiDoc™ MP Imaging System (Bio-Rad Laboratories) before 1 hr blocking in 5% low-fat milk powder in TBST.…”

Dihydrotestosterone in Amyotrophic lateral sclerosis—The missing link?

“…However, studies using ultra‐sensitive assay methods have shown that circulating gonadotrophin concentrations and diurnal Rhythms of Luteinizing Hormone, Follicle‐Stimulating Hormone, Testosterone, and Estradiol Secretion exist and are well established in prepubertal children as young as 4.6 years One is justified in making an assumption based on these findings that probably biorhythms of these hormones exist in younger children too. Studies on animal models of ALS (McLeod et al., 2019; Yoo & Ko, 2012) have also shown androgens deficiency/receptor antagonism may contribute to ALS.…”