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. 2014 Apr;155(4):1398-406.
doi: 10.1210/en.2013-1725. Epub 2014 Jan 15.

Selective androgen receptor modulator RAD140 is neuroprotective in cultured neurons and kainate-lesioned male rats

Anusha Jayaraman  1 , Amy Christensen, V Alexandra Moser, Rebekah S Vest, Chris P Miller, Gary Hattersley, Christian J Pike
Affiliations

Selective androgen receptor modulator RAD140 is neuroprotective in cultured neurons and kainate-lesioned male rats

Anusha Jayaraman et al. Endocrinology. 2014 Apr.

Abstract

The decline in testosterone levels in men during normal aging increases risks of dysfunction and disease in androgen-responsive tissues, including brain. The use of testosterone therapy has the potential to increase the risks for developing prostate cancer and or accelerating its progression. To overcome this limitation, novel compounds termed "selective androgen receptor modulators" (SARMs) have been developed that lack significant androgen action in prostate but exert agonist effects in select androgen-responsive tissues. The efficacy of SARMs in brain is largely unknown. In this study, we investigate the SARM RAD140 in cultured rat neurons and male rat brain for its ability to provide neuroprotection, an important neural action of endogenous androgens that is relevant to neural health and resilience to neurodegenerative diseases. In cultured hippocampal neurons, RAD140 was as effective as testosterone in reducing cell death induced by apoptotic insults. Mechanistically, RAD140 neuroprotection was dependent upon MAPK signaling, as evidenced by elevation of ERK phosphorylation and inhibition of protection by the MAPK kinase inhibitor U0126. Importantly, RAD140 was also neuroprotective in vivo using the rat kainate lesion model. In experiments with gonadectomized, adult male rats, RAD140 was shown to exhibit peripheral tissue-specific androgen action that largely spared prostate, neural efficacy as demonstrated by activation of androgenic gene regulation effects, and neuroprotection of hippocampal neurons against cell death caused by systemic administration of the excitotoxin kainate. These novel findings demonstrate initial preclinical efficacy of a SARM in neuroprotective actions relevant to Alzheimer's disease and related neurodegenerative diseases.

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Figures

Figure 1.
Figure 1.
RAD140 increases neuron viability against Aβ in a concentration-dependent manner. Neuron survival was measured in cultures pretreated with 0–100 nM T (A) and DHT (B); 0–300 nM RAD140 (C) and RAD192 (D) for 1 hour, followed by 24-hour exposure to 50 μM Aβ1–42 (solid bars). Cell viability data show the mean (± SEM) cell counts of viable cells expressed as percentage of vehicle-treated control group (C, open bar). *, P < .0001 relative to vehicle + AAII condition (0, solid bar); n ≥ 3.
Figure 2.
Figure 2.
RAD140 is neuroprotective against apoptotic insults. Cultures were treated with 50 μM zVAD-fmk (A), 10 nM T (B), 100 nM RAD140 (C), or 100 nM RAD192 (D) for 1 hour, followed by exposure to 50 μM Aβ1–42, 3 μM AAII, or 25 μM H2O2 for 24 hours, and processed for cell viability. Data show mean (± SEM) cell viability expressed as percentage of vehicle-treated control (Veh, open bar). *, P < .001 relative to the corresponding vehicle-treated condition (gray bars); n ≥ 3.
Figure 3.
Figure 3.
Quantitative analyses of MAPK signaling in SARM neuroprotection. A, Pretreatment of cultures with vehicle (top panel) or 10 μM U0126 (middle panel) for 2 hours was followed by exposure to 10 nM T, 100 nM RAD140, or 100 nM RAD192 for 15 minutes, and then examined by Western blot using phosphorylated (top and middle panel) and total (bottom panel) ERK-1/2 antibodies. B, The graph shows the percent phospho-ERK (pERK) expressed as a ratio between phosphorylated to total ERK-1 (44 kDa), normalized to the vehicle-treated control condition. C, Following a 2-hour pretreatment with vehicle or 10 μM of MEK inhibitor U0126 (U), cultures were exposed to 10 nM T, 100 nM RAD140, or 100 nM RAD192 for 1 hour and then treated with 50 μM Aβ1–42 for 24 hours. Cell viability data show mean (± SEM) counts of viable neurons plotted as percentage vehicle-treated control (C, open bar). *, P < .0001 relative to vehicle control (C, open bar); #, P ≤ .001 between each vehicle and the corresponding U0126 treatments (open bar vs solid bars); Ψ, P < .01 relative to U0126-treated control (C, solid bar); n ≥ 3. Veh, vehicle.
Figure 4.
Figure 4.
RAD140 induces tissue-specific androgenic effects. Data show mean (± SEM) tissue weights of (A) seminal vesicles, (B) prostate, and (C) levator ani muscle from male rats in sham-GDX (open bar), GDX (solid bar), GDX+T (gray bar), and GDX+RAD140 (gray bar) conditions. D, Relative mRNA levels of ERα in the hypothalamus were determined across groups by real-time PCR. Data show expression relative to the Sham condition. *, P < .001 vs sham; Ψ, P < .0001 vs T; n ≥ 7 per condition.
Figure 5.
Figure 5.
RAD140 reduces neuronal cell death cause by kainate. The extent of neuronal cell death in the CA2/3 region of the hippocampus following kainate lesion was determined qualitatively and quantitatively across groups. Images show representative thionin-stained sections of CA3 hippocampus from (A) nonlesioned sham-GDX rats and the following kainate-lesioned groups: B, Sham-GDX; C, GDX; D, GDX+T; E, GDX+RAD140. F, Neuron survival was quantified by counts of NeuN-immunoreactive cells. Data show mean (± SEM) counts expressed as a percentage of values from nonlesioned sham animals. *, P < .05 compared with nonlesioned sham-GDX; n ≥ 7 per condition.
Figure 6.
Figure 6.
Kainate-induced seizures are not affected by androgen status Behavioral features of kainate-induced seizures were monitored and quantified for a 3-hour period following the lesion. Data show mean values (± SEM) of (A) latency to seizure onset and (B) seizure severity across groups (n ≥ 7 per condition).
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