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Selective Androgen Receptor Modulator RAD140 Is
Neuroprotective in Cultured Neurons and Kainate-
Lesioned Male Rats
Anusha Jayaraman,* Amy Christensen,* V. Alexandra Moser, Rebekah S. Vest,
Chris P. Miller, Gary Hattersley, and Christian J. Pike
Davis School of Gerontology (A.J., A.C., R.S.V., C.J.P.) and Neuroscience Graduate Program (V.A.M.,
C.J.P.), University of Southern California, Los Angeles, California 90089; and Radius Health, Inc. (C.P.M.,
G.H.), Cambridge, Massachusetts 02139
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. Mech-
anistically, RAD140 neuroprotection was dependent upon MAPK signaling, as evidenced by ele-
vation 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 ex-
periments with gonadectomized, adult male rats, RAD140 was shown to exhibit peripheral tissue-
specific androgen action that largely spared prostate, neural efficacy as demonstrated by activa-
tion 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 Alzhei-
mer’s disease and related neurodegenerative diseases. (Endocrinology 155: 1398–1406, 2014)
The normal age-related decline in testosterone in men
can increase the risks for dysfunction and disease in
several androgen-responsive tissues throughout the body
(1, 2). In brain, low testosterone is an established factor for
the development of Alzheimer’s disease (AD). Circulating
(3, 4) and brain (5, 6) levels of testosterone are lower in
men with AD, and this androgen depletion occurs prior to
clinical (7) and neuropathological (5, 6) diagnoses of the
disease, suggesting that low testosterone contributes to
AD pathogenesis. In transgenic mouse models of AD, de-
pletion of endogenous androgens by surgical (8) or chem-
ical (9) castration accelerates development of AD-like pa-
thology whereas elevation of endogenous testosterone
above normal levels significantly impedes pathology de-
velopment (10).
Androgens induce numerous beneficial neural effects
relevant to a protective role against AD, including reduc-
tion of the AD-related protein

-amyloid (A

) (8, 11, 12)
and promotion of synapse formation (13, 14), neurogen-
esis (15, 16), and specific aspects of cognition (1, 17). An
androgen action particularly important to neurodegen-
erative diseases is neuroprotection. Testosterone can in-
crease neuron survival in several cell culture and animal
models of injury (18). Although testosterone neuropro-
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received August 1, 2013. Accepted December 4, 2013.
First Published Online January 15, 2014
* A.J. and A.C. contributed equally to the study.
Abbreviations: AAII, apoptosis activator II; A

,

-amyloid; AD, Alzheimer’s disease; AR,
androgen receptor; DHT, dihydrotestosterone; E2, 17

-estradiol; ER, estrogen receptor;
GDX, gonadectomized; SARM, selective AR modulator.
NEUROENDOCRINOLOGY
1398 endo.endojournals.org Endocrinology, April 2014, 155(4):1398–1406 doi: 10.1210/en.2013-1725
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tective actions are largely androgen receptor (AR) depen-
dent, testosterone is metabolized to several steroids that
can act through other mechanisms (18). For example, tes-
tosterone is metabolized to dihydrotestosterone (DHT) by
the enzymatic actions of 5
␣
-reductase (19). Because DHT
is a more potent androgen than testosterone at AR, the
conversion of testosterone to DHT results in more robust
androgen signaling in tissues like prostate (20, 21). DHT
is metabolized to other steroids, including 5
␣
-andro-
stane-3
␣
,17

diol, which can reduce some forms of neural
injury (22). Testosterone is also converted by the enzyme
aromatase to 17

-estradiol (E2), which signals through
estrogen receptors (ER) (23). In some paradigms, testos-
terone neuroprotection is dependent upon conversion to
E2 (24–26). Thus, neural benefits of testosterone can be
mediated largely by AR, ER, or a combination of both. For
example, both AR (12) and ER (27, 28) are implicated in
testosterone reduction of A

levels, whereas AR, but not
ER, mediates testosterone increases in spine density. These
and other experimental data (29) predict that androgen-
based hormone therapy may be an effective approach for
the prevention of AD and related neurodegenerative dis-
orders in aging men.
One significant limitation of androgen therapy is the
potential for increased risk of developing prostate cancer
and or accelerated growth of existing prostate tumors. To
overcome this problem, new classes of synthetic testoster-
one-like compounds, called “selective androgen receptor
modulators” (SARMs), have been developed (30, 31).
SARMs are ligands for AR that exert limited effects in
prostate and other reproductive tissues but have potent
androgenic actions in muscle and bone (32, 33). Although
SARMs such as 7
␣
-methyl-19-nortestosterone undergo
enzymatic aromatization to yield metabolites that bind to
ER (34), most of the currently available SARMs are poor
substrates for aromatase and interact specifically with AR
(35). The possible utility of SARMs for therapeutic use in
AD and other neural disorders has only recently begun to
be investigated.
Clinical utility of SARMs for neural disorders requires
that they mimic androgen actions in brain. Although ef-
ficacy of SARMs for peripheral tissues such as muscle is
well established, the extent to which SARMs exert pro-
tective androgen effects in brain is unclear. To begin ad-
dressing this issue, we evaluated the neuroprotective effi-
cacy of the SARM RAD140 using in vitro and in vivo
paradigms previously demonstrated to be androgen re-
sponsive. RAD140 is a novel SARM with high affinity and
specificity for AR, is orally available, and exhibits potent
anabolic effects in rodents and nonhuman primates (36).
We determined the effects of RAD140 against toxic insults
in both primary neuron cultures and the rat kainate lesion,
an animal model of hippocampal neuron loss relevant to
neurodegenerative diseases (37), which has previously
been established to respond to androgen neuroprotection
(38). These data represent the first preclinical report in-
vestigating the neuroprotective actions of SARMs.
Materials and Methods
Reagents
Testosterone, DHT (Steraloids), and RAD140 and RAD192
(Radius Health Inc) were solubilized in 100% ethanol and then
diluted into culture medium (cell culture experiments; final eth-
anol concentration ⬍0.001%) or in 0.5% methyl cellulose at 1
mg/mL (in vivo experiments). U0126 (EMD Millipore Chemi-
cals) and zVAD-fmk (Sigma-Aldrich) were dissolved in dimeth-
ylsulfoxide and 100% ethanol, respectively, and then diluted
into culture medium. Additional reagents include A

peptide
A

1– 42 (Tocris Bioscience), apoptosis activator II (AAII) (EMD
Millipore Chemicals), and hydrogen peroxide (H
2
O
2
)
(Sigma-Aldrich).
Primary neuron culture
Timed-pregnant female Sprague Dawley rats (Harlan Labo-
ratories, Inc.) were euthanized via CO
2
inhalation, and embry-
onic day 17–18 pups were collected for preparation of neuronal
cultures. All animal procedures were conducted under a protocol
that was approved by the University of Southern California In-
stitutional Animal Care and Use Committee and in accordance
with National Institute of Health standards. Primary rat hip-
pocampal cultures (⬃95% neuronal as determined by immuno-
reactivity with the neuron-specific antibody NeuN) were pre-
pared with some modifications of a previously described
protocol (39). Dissociated hippocampal neurons (n ⱖ6 pups per
preparation) were plated onto poly-L-lysine-coated multiwell
plates at a final density of 2.5 ⫻10
4
cells/cm
2
for cell-viability
assays. Each culture preparation used 12–14 pups to ensure a
mix of male and female pups to control for sex differences. Cul-
tures were maintained at 37°C in a humidified incubator sup-
plemented with 5% CO
2
. All experiments were started after 1–2
days in vitro. All in vitro experiments were repeated in at least 3
independent culture preparations.
Cell-viability assay
Cell viability was determined at the end of the treatment pe-
riod by counts of the number of viable neurons as determined by
staining with the vital dye calcein acetoxymethyl ester (Invitro-
gen), as previously described (40). In brief, cultures were incu-
bated for 5 minutes with 2
M calcein acetoxymethyl ester and
then examined using an inverted fluorescent IX70 Olympus mi-
croscope. The number of healthy, positively stained cells was
counted in 4 separate fields (in a predetermined, regular pattern)
per well, 3 wells per condition in each experiment (n ⱖ3 inde-
pendent culture preparations). Counts of viable neurons in ve-
hicle-treated controls ranged from 250–300 per well.
Western blots
Lysates were collected from treated cultures using a reducing
sample buffer (62.5 mM Tris-HCl, 1% sodium dodecyl sulfate,
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2.5% glycerol, 0.5% 2-

-mercaptoethanol), boiled for 5 minutes,
and centrifuged at 13 000 ⫻gfor 10 minutes. The supernatants
were analyzed by immunoblotting using a standard protocol pre-
viously described (39) with 1
g/mL anti-phosphoERK1/2 (EMD
Millipore Chemicals) primary antibody and corresponding horse-
radish peroxidase-conjugated secondary antibody (1:5000) and de-
tected using enhanced luminescence (Amersham). Band densities
were measured using Image J software (version 1.45s) and the rel-
ative percent intensity of the phospho-ERK bands were plotted on
a graph after normalizing to total ERK levels.
In vivo hormone treatments and kainate lesions
Male Sprague Dawley rats (n ⫽8 per group) were purchased
gonadectomized (GDX) and sham-GDX at 3 months of age
(Harlan Laboratories, Inc.). All animals were housed individu-
ally with ad libitum access to food and water under a 12 hour
light, 12 hour-dark cycle. Animals underwent GDX 14 days
prior to the start of treatment, allowing for the depletion of
endogenous hormones. For testosterone (T) treatment, GDX
male rats were implanted with a 30-mm length SILASTIC cap-
sule (1.47 mm inner diameter ⫻1.96 mm outer diameter; Dow
Corning) packed with dry T to a length of 20 mm and capped on
both ends with 5 mm of silicone glue. Vehicle-treated animals
were implanted with an empty capsule with the same dimen-
sions. For SARM treatment, GDX rats were administered 1
mg/kg RAD140 suspended in 0.5% methyl cellulose (1 mg/mL)
by daily oral gavage for 2 weeks. This dose was chosen based on
previous reports for RAD140 efficacy (36). Vehicle-treated an-
imals were gavaged with a similar weight/volume of 0.5%
methyl cellulose. On day 13 of the 2-week hormone treatment
period, kainate (10 mg/kg; Enzo Life Sciences) or sterile water
control was injected ip. Kainate was dissolved immediately prior
to use in sterile water and lightly heated to fully solubilize. On
day 14, SARM-treated rats were administered 1 mg/mL of
RAD140 suspended in safflower oil by sc injection because oral
gavage is difficult following kainate lesion.
At the end of the treatment period, animals were euthanized
by CO
2
inhalation. Brains were removed and hemisected: one
half was immersion fixed for 48 hours in cold 4% paraformal-
dehyde/Sorenson’s phosphate buffer, and the other half was snap
frozen on dry ice and then stored at ⫺80°C for use in RT-PCR
analyses. The prostate, seminal vesicles, and levator ani were
dissected according to standard procedures (41) and weighed.
Seizure assessment
Kainate induces seizures that are characterized in part by ste-
reotypic behaviors (42). To assess seizures, animals were con-
tinuously monitored for 3 hours following kainate injection for
both seizure latency and severity. Latency is defined as the period
from the kainate injection to the appearance of the first “wet dog
shake,” a stereotypic seizure-related behavior. Seizure severity
was behaviorally assessed according to the Racine scale: 0 ⫽no
seizure activity; 1 ⫽occasional wet dog shake; 2 ⫽head nodding
and facial clonus; 3 ⫽unilateral forelimb clonus; 4 ⫽bilateral
clonus with rearing, but without falling; 5 ⫽seizures accompa-
nied by rearing and falling (43). One sham-GDX animal that
reached stage 5 was immediately euthanized and excluded from
analysis.
RT-PCR
For RNA extractions, rat hypothalami were homogenized
using TRIzol reagent (Invitrogen Corp.) and processed for total
RNA extraction as per manufacturer’s protocol. Purified RNA
(1–2
g) was used for reverse transcription using the iScript
cDNA synthesis system (Bio-Rad Laboratories), and the result-
ing cDNA was used for real-time quantitative PCR. Quantitative
PCR was carried out using Bio-Rad CFX Connect (Bio-Rad).
The amplification efficiency was estimated from the standard
curve for each gene. Relative quantification of mRNA levels
from various treated samples was determined by the ⌬⌬Ct
method (44) with

-actin as the normalizing control. The PCR
products were also analyzed qualitatively on a 1% agarose gel.
The following primer pair was used: ER
␣
, forward: 5⬘-CATC-
GATAAGAACCGGAG-3⬘, reverse: 5⬘-AAGGTTGGCAGCT-
CTCAT-3⬘;

-actin, forward: 5⬘-AGCCATGTACGTAGC-
CATCC-3⬘, reverse: 5⬘-CTCTCAGCTGTGGTGGTGAA-3⬘.
Immunohistochemistry
Fixed brains were sectioned exhaustively in the horizontal
plane at 40
m using a vibratome (Leica Microsystems) and
stored at 4°C in PBS with 0.03% sodium azide until use. Every
eighth section was immunostained with NeuN antibody (1:250;
Millipore) using standard avidin-biotinylated enzyme complex
immunohistochemistry with Vectastain Elite ABC kit (Vector
Laboratories), as previously described (38). Stained sections
were mounted on slides, allowed to dry overnight, and then cov-
erslipped with Krystalon (EMD Chemicals) without further de-
hydration. A second set of tissue was mounted and dried and
underwent a routine thionin stain for comparison and imaging.
The number of NeuN immunoreactive cells in the CA2/3 re-
gion of the hippocampus was estimated by 2-dimensional cell
counts using random sampling based on the optical dissector
technique, which has been previously used to estimate the num-
ber of total cells in the hippocampus (38, 45). Briefly, an Olym-
pus BX50 microscope equipped with a motorized stage and com-
puter guided CASTGrid software (Olympus) was used for
unbiased sampling. In every eighth section of hippocampus
(8 –12 sections per brain), the CA2/CA3 region of the hippocam-
pus was outlined, and a randomly oriented counting frame (476
m
2
) with an X-Y step of 150
m⫻150
m was used for cell
counts. Only cells with positively stained nuclei were counted,
not dead or dying cells or cells that were on the upper or lower
edge of the section the cytoplasm of which was stained but ap-
peared without a nucleus. To control for variability in the num-
ber of sections analyzed, the total number of NeuN-immunore-
active nuclei in each animal was divided by the number of
sections assessed and then expressed as a percentage of neurons
counted in the sham-GDX, nonlesioned group. Of the 24 rats
that received kainate and survived the kainate-induced seizures,
2 were excluded from analyses because they showed extensive
loss of CA1 neurons indicating hypoxic injury, which is known
to occur in a subset of lesioned animals (46).
Statistical analyses
Raw data were statistically assessed using ANOVA followed
by between-group comparisons using Fisher’s least significant
different test. Significance was indicated by Pⱕ.05.
1400 Jayaraman et al Neuroprotectiveness of SARM RAD140 Endocrinology, April 2014, 155(4):1398 –1406
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Results
RAD140 protects cultured neurons against A

in a
dose-dependent manner
As an initial step to evaluate the neuroprotective po-
tential of RAD140, we compared RAD140 with the en-
dogenous androgens T and DHT for their relative abilities
to reduce neuron death induced by
aggregated A

1–42, the peptide im-
plicated in AD neurodegeneration.
We found that 24 hours’ exposure to
A

decreased the number of viable
neurons by approximately 50%, as
compared to vehicle treatment. Con-
sistent with previous observations
(47), treatment with T and DHT be-
ginning 1 hour prior to A

signifi-
cantly reduced cell death (Figure 1, A
and B). In comparison, treatment of
cultures with increasing doses of
RAD140 (Figure 1C) or the related
SARM RAD192 (Figure 1D) pro-
vided similar levels of neuroprotec-
tion. The minimum effective concen-
tration of both SARMs was 30 nM
whereas T and DHT yielded signifi-
cant protection at 10 nM.
RAD140 protects cultured
neurons against apoptotic insults
We previously showed that androgen neuroprotection
is limited to insults that involve apoptosis (48). To inves-
tigate whether the SARMs mimic this established andro-
gen-protective pathway, we assessed their abilities to re-
duce cell death induced by 3 insults: A

, apoptosis
activator II (AAII), and hydrogen peroxide (H
2
O
2
). To
confirm our prior observations that
A

and AAII, but not H
2
O
2
, induce
cell death by caspase-dependent ap-
optosis in our culture system, we
evaluated the ability of the caspase
inhibitor zVAD to attenuate cell
death. Exposure of cultures to 50
M zVAD-fmk for 2 hours prior to
insult exposure significantly attenu-
ated cell death due to 50
MA

and
3
M AAII but did not significantly
affect cell loss caused by 25
M
H
2
O
2
(Figure 2A). Next, we com-
pared the pattern of protection
against the 3 insults by T and the 2
SARMs. Cultures were pretreated
for 1 hour with 10 nM testosterone,
100 nM RAD140, or 100 nM
RAD192, and then exposed for 24
hours to A

, AAII, or H
2
O
2
. T (Fig-
ure 2B), RAD140 (Figure 2C), and
RAD192 (Figure 2D) shared similar
protective profiles of significantly
Figure 1.RAD140 increases neuron viability against A

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
MA

1–42
(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 ⱖ3.
Figure 2.RAD140 is neuroprotective against apoptotic insults. Cultures were treated with 50
M zVAD-fmk (A), 10 nM T (B), 100 nM RAD140 (C), or 100 nM RAD192 (D) for 1 hour,
followed by exposure to 50
MA

1–42, 3
M AAII, or 25
MH
2
O
2
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 ⱖ3.
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protecting against neuronal death induced by A

and
AAII, but not H
2
O
2
.
MAPK signaling is involved in SARM-mediated
neuroprotection
Androgen-mediated neuroprotection against apoptosis
is dependent upon activation of a MAPK/ERK signaling
pathway (40). To evaluate the role of MAPK/ERK signal-
ing in mediating the observed neuroprotective effects of
SARMs, we first determined whether RAD140 and
RAD192 activate MAPK/ERK signaling. Neuronal cul-
tures were exposed for 15 minutes to 10 nM T, 100 nM
RAD140, or 100 nM RAD192 in the presence or absence
of pretreatment with 10
M U0126, a MEK inhibitor that
blocks MAPK/ERK signaling (49). Western blots with
phospho-specific and pan-ERK antibodies show that T,
RAD140, and RAD192 induce a significant increase in
levels of phosphorylated but not total ERK (Figure 3, A
and B). Both the basal levels of ERK phosphorylation and
the androgen-mediated increases were significantly atten-
uated by U0126 (Figure 3, A and B).
We then examined the effect of U0126 inhibition of
MAPK/ERK signaling on the extent of neuroprotection by
SARMs against A

-induced cell death. Pretreatment with
10
M U0126 completely blocked androgen protection
by T, RAD140, and RAD192 (Figure 3C). U0126 alone
had no effect on either basal cell viability or the magnitude
of A

toxicity (Figure 3C).
RAD140 has tissue-specific effects in vivo
Based on the cell culture observations, we investigated the
neuroprotective potential of RAD140 in vivo. Young adult
male rats were randomly assigned to sham-GDX, GDX,
GDX⫹T, and GDX⫹RAD140 groups that were exposed to
the neurotoxin kainate 2 weeks after the initiation of andro-
gen treatments. Following the lesion, seminal vesicles, pros-
tate, and levator ani were removed and weighed to confirm
the reported tissue-selective androgen effects of RAD140
(36). GDX resulted in significantly reduced weight of all 3
androgen-responsive tissues, but tissue weights were re-
stored to sham-GDX levels in the GDX⫹T group (Figure 4,
A–C). RAD140 treatment resulted in a nonsignificant in-
crease in the weights of seminal vesicles and prostate, which
were still significantly lower than weights observed in the sham-
GDX and GDX⫹T groups. On the other hand, RAD140 sig-
nificantly increased the weight of the levator ani, an androgen-
responsive muscle, to levels similar to those observed in the
sham-GDX and GDX⫹T groups (Figure 4, A–C).
We next investigated whether RAD140 exerts androgenic
effects in brain. To accomplish this, we examined androgen
regulation of ER
␣
mRNA expression in the hypothalamus,
a brain region largely unaffected by kainate. Consistent with
prior observations (50), we observed that GDX resulted in
increased ER
␣
mRNA expression in comparison to sham-
GDX animals. Both GDX⫹T and GDX⫹RAD140 groups
exhibited significantly decreased ER
␣
mRNA expression rel-
ative to the GDX animals (Figure 4D).
RAD140 is neuroprotective against kainate-
induced hippocampal neuron loss
To investigate the neuroprotective effects of RAD140
in vivo, the extent of kainate-induced neuron death was
assessed by counts of surviving cells immunostained with
the neuron-specific antibody NeuN in the CA2/3 region of
hippocampus. Relative to vehicle treatment, kainate in-
duced approximately 20% cell loss in the sham-GDX an-
Figure 3.Quantitative analyses of MAPK signaling in SARM
neuroprotection. A, Pretreatment of cultures with vehicle (top panel)
or 10
M 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
M 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
MA

1–42 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); #, Pⱕ.001 between each vehicle and the
corresponding U0126 treatments (open bar vs solid bars); ⌿,P⬍.01
relative to U0126-treated control (C, solid bar); n ⱖ3. Veh, vehicle.
1402 Jayaraman et al Neuroprotectiveness of SARM RAD140 Endocrinology, April 2014, 155(4):1398 –1406
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imals (Figure 5, A and B). Neuron survival was significantly
reduced in the GDX group, an effect that was prevented by
T treatment (Figure 5, C and D; F
(3, 29)
⫽6.93, P⬍.001).
Notably, RAD140 was as effective as T in protecting GDX
rats from kainate-induced neuron loss (Figure 5, E and F).
Because kainate induces seizures,
we also assessed the effects of the
hormonal manipulations on both the
latency to seizure onset and maxi-
mum seizure severity. The intensity
of seizure behavior is known to affect
the degree of hippocampal neuron
loss. All kainate-treated animals
achieved at least level 1 seizure
within the 3-hour observation pe-
riod but none of the vehicle-injected
animals showed any seizure-related
behaviors. Among the lesioned ani-
mals, there was no significant differ-
ence across groups in seizure latency
(Figure 6A; F
(3, 24)
⫽0.75 P⫽.53).
Similarly, seizure severity did not sig-
nificantly vary by treatment group (F
(3, 26)
⫽1.36 P⫽.28), although there
was a trend toward reduced severity
in the GDX⫹T and GDX⫹RAD140
groups (Figure 6B).
Discussion
In this study, we report the first findings of neuroprotec-
tive actions by SARMs in both cell culture and in vivo. Our
results show in primary neuron cul-
tures that the SARM RAD140 in-
creases cell viability against A

tox-
icity in a concentration-dependent
manner. The neuroprotective effects
of RAD140 are specific to apoptotic
insults and dependent upon a
MAPK-signaling pathway. In GDX
rats treated with RAD140, RAD140
induces androgenic responses in
muscle and brain, but not in repro-
ductive tissues. Moreover, RAD140
treatment significantly protects hip-
pocampal neurons from kainate
lesion.
SARMs are steroidal and non-
steroidal ligands for AR capable of
activating androgen signaling in a
tissue-specific manner (35, 51).
Many factors contribute to the tissue
specificity of SARMs. For example,
unlike testosterone, nonsteroidal
SARMs are not substrates for aro-
matase and 5
␣
-reductase and thus
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 ER
␣
in the hypothalamus were determined across groups by real-time PCR. Data show
expression relative to the Sham condition. *, P⬍.001 vs sham; ⌿,P⬍.0001 vs T; n ⱖ7 per condition.
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 ⱖ7 per condition.
doi: 10.1210/en.2013-1725 endo.endojournals.org 1403
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do not yield potent estrogen and androgen metabolites
(35, 52). Tissue specificity of SARMs can also be related
to tissue-specific expression of AR coregulators and pro-
tein-protein interactions associated with SARM binding
that can differ across tissues (51, 53). The promise of se-
lective androgen treatment has been realized in animal
models, with SARMs shown to promote muscle and bone
health in the absence of prostate growth (33, 52, 54, 55).
These advances have encouraged evaluation of SARMs in
clinical trials for disorders including cachexia (clinical
trial NCT00467844) (55) and osteoporosis (51). The data
presented here are among the first highlighting the poten-
tial efficacy of SARMs for neural endpoints. Our finding
that RAD140 can exert androgenic actions in brain at a
dose that retains peripheral tissue selectivity is consistent
with prior observations in rodents that the SARM ACP-
105 can ameliorate cognitive deficits associated with apo-
lipoprotein E (56) and irradiation (57).
Androgens exert numerous beneficial actions in brain
by several distinct mechanisms (18). Many, but not all,
neural androgen actions involve AR activation, which
triggers a wide range of rapid cell-signaling pathways as
well as classic genomic regulation (58). Because SARMs
can interact with AR differently than endogenous andro-
gens, the efficacy of specific SARMs in activating defined
androgenic pathways is a key consideration in pursuing
translational goals. In terms of androgen neuroprotection,
our prior work has defined a mechanism that is both de-
pendent upon MAPK/ERK signaling pathway (40) and lim-
ited in protective efficacy to apoptosis (48). Our findings
with RAD140 and the related compound RAD192 demon-
strate that both SARMs mimic this established mechanism of
neuroprotection in cultured neurons: they activate MAPK/
ERK signaling as evidenced by ERK
phosphorylation, their neuroprotec-
tion is blocked by pharmacologic in-
hibition of MAPK signaling, and they
protect against 2 apoptotic insults but
not a nonapoptotic insult. MAPK/
ERK signaling is also known to con-
tribute to androgen protection in non-
neural cells (59, 60). In adult male rats
depleted of endogenous androgens by
GDX, RAD140 matched the neuro-
protection observed with T against
kainate, a neurotoxin known to kill
hippocampal neurons via apoptosis
(61). Because neither T nor RAD140
significantly affected kainate-induced
seizure behavior, a variable that can
alter lesion severity (38), these obser-
vations suggest a direct mechanism of
androgen neuroprotection consistent with our prior obser-
vations (39).
Still unclear is the extent to which RAD140 mimics
activation of other beneficial androgen pathways in brain.
MAPK/ERK signaling is involved in several important
functions in the brain including neurogenesis, differenti-
ation, synaptic plasticity, memory formation, and cell sur-
vival (62–65). However, additional signaling pathways
likely contribute to androgen actions in brain. in addition
to MAPK/ERK, androgens also rapidly activate cAMP re-
sponse element binding protein signaling in neurons by a
protein kinase C-dependent mechanism (66). In addition
to rapid cell-signaling pathways, many neural androgen
effects involve classic genomic responses. For example,
androgen regulation of gene expression is implicated in
protecting against AD pathology by reducing A

accu-
mulation. Specifically, androgens reduce the expression of
the proamyloidogenic enzyme

-secretase (10) and in-
crease expression of the A

degrading enzyme neprilysin
by an AR-dependent genomic mechanism (12). Androgen-
induced neurogenesis is also AR dependent in male rats (67)
and, at least in songbirds, involves genomic mechanisms in-
cluding up-regulation of matrix metalloproteinases (68).
Similarly, androgen-mediated increases in dendritic spines in
mouse hippocampus has been linked to up-regulation of
brain-derived neurotrophic factor and postsynaptic density
protein 95 (69). It is important to note that several neural
actions of testosterone, including its effects on sexual behav-
ior and some aspects of neuroprotection, involve its conver-
sion to E2 (24, 70). Because RAD140 and related SARMs are
not aromatase substrates and are not known to signal
through estrogen pathways, there are limitations in their abil-
ities to fully mimic T actions in brain.
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 ⱖ7 per condition).
1404 Jayaraman et al Neuroprotectiveness of SARM RAD140 Endocrinology, April 2014, 155(4):1398 –1406
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 September 2014. at 21:59 For personal use only. No other uses without permission. . All rights reserved.
The loss of androgens with normal aging can negatively
impact androgen-responsive tissues, including the brain,
and has been shown to be a significant risk factor for
development of neurodegenerative disorders including
AD (6, 7). The increased risk for prostate cancer makes T
therapy a risky treatment option (71). Moreover, even in
neurons, high doses of T can be harmful rather than ben-
eficial (72, 73). In this regard, SARMs like RAD140 can be
better alternatives to T because they are only partial ago-
nists or antagonists to androgenic regulation of prostate
while still having androgenic effects on other tissues such
as muscle and brain. In addition, higher doses of SARMs
as compared with T might still promote neuron viability
and not induce apoptosis, making them a suitable thera-
peutic strategy against age-related disorders such as AD.
Acknowledgments
Address all correspondence and requests for reprints to: Christian
J. Pike, University of Southern California, 3715 McClintock Ave-
nue, Los Angeles, CA 90089-0191. E-mail: cjpike@usc.edu.
This study was supported by grants from the Alzheimer’s
Association (IIRG-10–174301) and National Institutes of
Health (P50 AG05142) (to C.J.P.).
Disclosure Summary: A.J., A.C., V.A.M., R.V., and C.J.P.
have nothing to disclose and no conflicts of interest with the
information presented in this manuscript including any personal,
financial, or other conflicts. G.H. is an employee and stockholder
and C.P.M. is a stockholder of Radius Health, Inc.
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