Skip to main content

Umbrella menu

  • SfN.org
  • eNeuro
  • The Journal of Neuroscience
  • Neuronline
  • BrainFacts.org

Main menu

  • HOME
  • CONTENT
    • Early Release
    • Featured
    • Latest Articles
    • Issue Archive
    • Editorials
    • Research Highlights
  • TOPICS
    • Cognition and Behavior
    • Development
    • Disorders of the Nervous System
    • History, Teaching and Public Awareness
    • Integrative Systems
    • Neuronal Excitability
    • Novel Tools and Methods
    • Sensory and Motor Systems
  • ALERTS
  • FOR AUTHORS
  • EDITORIAL BOARD
  • BLOG
  • ABOUT
    • Overview
    • For the Media
    • Privacy Policy
    • Contact Us
    • Feedback
  • SfN.org
  • eNeuro
  • The Journal of Neuroscience
  • Neuronline
  • BrainFacts.org

User menu

  • My alerts

Search

  • Advanced search
eNeuro
  • My alerts

eNeuro

Advanced Search

Submit a Manuscript
  • HOME
  • CONTENT
    • Early Release
    • Featured
    • Latest Articles
    • Issue Archive
    • Editorials
    • Research Highlights
  • TOPICS
    • Cognition and Behavior
    • Development
    • Disorders of the Nervous System
    • History, Teaching and Public Awareness
    • Integrative Systems
    • Neuronal Excitability
    • Novel Tools and Methods
    • Sensory and Motor Systems
  • ALERTS
  • FOR AUTHORS
  • EDITORIAL BOARD
  • BLOG
  • ABOUT
    • Overview
    • For the Media
    • Privacy Policy
    • Contact Us
    • Feedback
PreviousNext
Research ArticleNew Research, Integrative Systems

Presence of Androgen Receptor Variant in Neuronal Lipid Rafts

Jo Garza-Contreras, Phong Duong, Brina D. Snyder, Derek A. Schreihofer and Rebecca L. Cunningham
eNeuro 22 August 2017, 4 (4) ENEURO.0109-17.2017; DOI: https://doi.org/10.1523/ENEURO.0109-17.2017
Jo Garza-Contreras
Center for Alzheimer’s and Neurodegenerative Disease Research and Center for Neuroscience Discovery, Institute for Healthy Aging, University of North Texas Health Science Center, Fort Worth, TX 76107
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Phong Duong
Center for Alzheimer’s and Neurodegenerative Disease Research and Center for Neuroscience Discovery, Institute for Healthy Aging, University of North Texas Health Science Center, Fort Worth, TX 76107
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Brina D. Snyder
Center for Alzheimer’s and Neurodegenerative Disease Research and Center for Neuroscience Discovery, Institute for Healthy Aging, University of North Texas Health Science Center, Fort Worth, TX 76107
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Derek A. Schreihofer
Center for Alzheimer’s and Neurodegenerative Disease Research and Center for Neuroscience Discovery, Institute for Healthy Aging, University of North Texas Health Science Center, Fort Worth, TX 76107
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Rebecca L. Cunningham
Center for Alzheimer’s and Neurodegenerative Disease Research and Center for Neuroscience Discovery, Institute for Healthy Aging, University of North Texas Health Science Center, Fort Worth, TX 76107
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF
Loading

Abstract

Fast, nongenomic androgen actions have been described in various cell types, including neurons. However, the receptor mediating this cell membrane–initiated rapid signaling remains unknown. This study found a putative androgen receptor splice variant in a dopaminergic N27 cell line and in several brain regions (substantia nigra pars compacta, entorhinal cortex, and hippocampus) from gonadally intact and gonadectomized (young and middle-aged) male rats. This putative splice variant protein has a molecular weight of 45 kDa and lacks an N-terminal domain, indicating it is homologous to the human AR45 splice variant. Interestingly, AR45 was highly expressed in all brain regions examined. In dopaminergic neurons, AR45 is localized to plasma membrane lipid rafts, a microdomain involved in cellular signaling. Further, AR45 protein interacts with membrane-associated G proteins Gαq and Gαo. Neither age nor hormone levels altered AR45 expression in dopaminergic neurons. These results provide the first evidence of AR45 protein expression in the brain, specifically plasma membrane lipid rafts. AR45 presence in lipid rafts indicates that it may function as a membrane androgen receptor to mediate fast, nongenomic androgen actions.

  • Androgen receptor
  • caveolin
  • G proteins
  • lipid rafts
  • membrane
  • signaling

Significance Statement

Evidence has been building for the existence of a membrane androgen receptor, but the nature of this receptor remains elusive. We predicted that the membrane androgen receptor is a splice variant that is present in lipid raft microdomains within the neuronal plasma membrane. Indeed, an androgen receptor that is lacking an N-terminal domain and complexed to Gαq and Gαo G proteins was highly expressed in lipid rafts in dopaminergic neurons. This putative splice variant provides a potential target for mediating androgen’s nongenomic actions.

Introduction

In addition to peripheral expression in many tissues, the androgen receptor (AR) is expressed in many areas of the brain, including brain regions not involved in regulating classic endocrine functions (e.g., substantia nigra, hippocampus, entorhinal cortex; McEwen, 1980; Song et al., 1991; Kerr et al., 1995; Kritzer, 1997; Kritzer and Creutz, 2008; Sarkey et al., 2008). The classic intracellular AR is involved in genomic transcription (Ozanne et al., 2000; Edwards and Bartlett, 2005; Smith and Toft, 2008; Green et al., 2012). This AR is composed of eight exons that code for a 110-kDa protein belonging to the steroid receptor superfamily of nuclear transcription factors (Claessens et al., 2008). The classic AR has four distinct regions: a regulatory NH2 terminal domain (NTD; Ferro et al., 2002; Ding et al., 2004, 2005; Callewaert et al., 2006; Claessens et al., 2008), a DNA binding domain (DBD), a hinge domain, and a ligand binding domain (LBD) extending to the C-terminal domain (CTD; Claessens et al., 2008).

Evidence for a nongenomic membrane AR (mAR) has been accumulating since the 1990s. Investigators have found that even cell-impermeable androgens can have fast cellular effects (Steinsapir et al., 1991; Gorczynska and Handelsman, 1995; Benten et al., 1999; Estrada et al., 2003, 2006; Holmes et al., 2013). Rapid membrane effects of androgens, via testosterone conjugated to bovine serum albumin (BSA), have been observed in neuronal cell lines (N27 and SH-SY5Y), resulting in an oscillatory pattern of intracellular calcium release that is unaffected by AR antagonists or knockdown of the classic intracellular AR (Estrada et al., 2006; Holmes et al., 2013). Unlike the classic AR, the mAR has not been cloned or purified. Different theories have proposed that the mAR could be the classic AR anchored to the plasma membrane (Pedram et al., 2007), an unknown AR (Hatzoglou et al., 2005), or even a splice variant of the classic AR (Foradori et al., 2008).

Numerous AR splice variants have been found outside the nervous system. These variants originate from alternative splicing at different promoters and thus increase AR complexity and biological functions (Modrek and Lee, 2002; Roberts and Smith, 2002; Stamm, 2002; Ahrens-Fath et al., 2005; Jagla et al., 2007; Hu et al., 2012). Most of these AR splice variants have a truncated LBD in the CTD, which can result in a constitutively active AR (Sun et al., 2010; Watson et al., 2010; Hu et al., 2012). In contrast to the loss of LBD, some of these AR splice variants exhibit partial to full loss of the NH2 regulatory domain. For example, both AR splice variants (AR8 and AR45) have truncated NTD (Jenster et al., 1991; Ikonen et al., 1998; Ahrens-Fath et al., 2005; Yang et al., 2011).

The AR splice variant, AR45, is the least understood variant and has been characterized only in humans. This splice variant lacks the entire NTD (exon 1). This deletion decreases the protein molecular weight of AR from 110 to 45 kDa (Ahrens-Fath et al., 2005). Functionally, in the periphery, AR45 binds androgens via LBD and translocates to the nucleus. It can homodimerize with other AR45 receptors or heterodimerize with classic AR. AR45 can act as a negative modulator of AR activity via competitive inhibition of AREs by homodimers or by interfering with coactivator recruitment necessary for AR activity (Jenster et al., 1991; Ikonen et al., 1998; Ahrens-Fath et al., 2005). AR45 is expressed in multiple tissues, such as muscle, lung, heart, breast, uterus, and prostate (Ahrens-Fath et al., 2005; Weiss et al., 2007; Wu et al., 2008). Although AR45 protein expression was not observed in total brain homogenate (Ahrens-Fath et al., 2005), a recent study found low mRNA expression of AR45 in human brain tissue that was commercially obtained from an aged population (Hu et al., 2014). Neuronal function of AR45 is unknown.

To determine whether the putative mAR could be AR45 in different brain regions (substantia nigra pars compacta, CA1 region of the hippocampus, and the second layer of the entorhinal cortex), we measured protein expression of AR using antibodies targeting the CTD and NTD of the AR. Because neuronal mAR has been associated with intracellular calcium signaling (Steinsapir et al., 1991; Gorczynska and Handelsman, 1995; Benten et al., 1999; Estrada et al., 2003, 2006; Holmes et al., 2013), we examined if the mAR complexed with G proteins.

Materials and Methods

Reagents

Testosterone (A6950-000) was obtained from Steraloids. Goat anti-rabbit (sc-2004), androgen receptor C-19 (sc-815), androgen receptor N-20 (sc-816), AR441 (sc-7305), AR N20P (sc-515856), AR C19P (sc-515863), Gαq (sc-393), Gαs (sc-823), Gαo (sc-393874), Gαi1 (sc-391), Gαi2 (sc-13534), and Gαi3 (sc-262) antibodies were obtained from Santa Cruz Biotechnologies. Flotillin (3253) and caveolin-1 (3267) antibodies were obtained from Cell Signaling Technology. Alexa Fluor 594 antibody was purchased from Jackson ImmunoResearch Laboratories. GAPDH (GT239) antibody was obtained from GeneTex. Biotinylated anti–rabbit IgG (BA-1000) was purchased from Vector Laboratories. Androgen R/NR3C4 (AB58761) was obtained from R&D Systems. DMSO was purchased from VWR. RPMI 1640, penicillin-streptomycin (PS), and trichloroacetic acid (BDH9310) were purchased from VWR. Fetal bovine serum (FBS) and PBS were obtained from Corning. Charcoal-stripped FBS (CS-FBS) was purchased from Atlanta Biologicals. Mounting medium was obtained from Vector Laboratories (H-1200). SuperSignal West Pico/Femto chemiluminescent substrates, dithiothreitol (DTT), and Clean Blot IP Detection Reagent (PI21230) were obtained from Thermo Fisher Scientific. Deoxycholic acid (D-6750), inactin (T133), horse serum (H1138), and Triton X-100 were purchased from Sigma-Aldrich. Tris, Any KD polyacrylamide gel, Tris-glycine buffer, and PVDF membranes were purchased from Bio-Rad. Total testosterone ELISA (RTC001R) was purchased from BioVendor. Testosterone was made using an ethanol vehicle (final concentration of ethanol <0.001%).

Animals

Experiments were conducted on young adult (3 mo) or middle-aged retired breeder (9–12 mo) male Sprague-Dawley rats (Charles River). Animals were either gonadally intact or were gonadectomized to remove circulating gonadal hormones. Rats were individually housed in a temperature-controlled environment on a 12:12-h light-dark cycle. All rats had ad libitum access to food and water. Animals were weighed at the time of surgery and at death. All experimental procedures were approved by the University of North Texas Health Science Center IACUC in accordance with the guidelines of the Public Health Service, the American Physiologic Society, and the Society for Neuroscience for animal care and use.

Gonadectomy

Under 2.5% isoflurane, a midline scrotal incision was made to expose the spermatic cord. The spermatic cord was tied off with sterile sutures, and the cord was cut distal to the thread to remove the testes. The incision was closed with sterile absorbable sutures (Cunningham et al., 2011).

Micropunch tissue dissection

One week after surgery, each rat was anesthetized with 2.5% isoflurane and decapitated. The brain was removed from the skull, rinsed in ice-cold PBS, and placed into a brain matrix (Braintree Scientific) on ice. Using razor blades, the brain was cut into 1-mm coronal sections. The razor blades were placed on dry ice to freeze the freshly cut brain sections. Punches were obtained from the SN pars compacta (–5.30 mm from bregma), second layer of the entorhinal cortex (ETC; –5.30 mm from bregma), and the CA1 layer of the dorsal hippocampus (–4.52 mm from bregma) using 1-ml syringes with a 20-gauge blunt needle (Snyder et al., 2017). Samples were placed into microcentrifuge tubes, snap-frozen on dry ice, and stored at –80°C or immediately homogenized into whole-cell lysates.

In vitro cell culture

The immortalized neuronal cell line 1RB3AN27 (N27), derived from fetal rat mesencephalic tissue, is positive for tyrosine hydroxylase expression (TH+; Clarkson et al., 1999; Anantharam et al., 2007; Carvour et al., 2008). N27 cells were cultured and maintained at 37°C in 5% CO2. Medium used was RPMI 1640 supplemented with 10% FBS and 1% PS. N27 cells were used only in passages 13–19. Before hormone treatment for whole-cell lysate experiments, the medium was switched to RPMI 1640 with CS-FBS to avoid confounding from the presence of hormones in the serum. Cells were exposed to testosterone (100 nM) or vehicle control for 18 h and collected for protein. The testosterone concentration used in this study was 100 nM, representing the high end of the normal testosterone range in men (Mooradian et al., 1987; Kelly et al., 1999; Smith et al., 2000; Zitzmann et al., 2002).

Whole cell lysates

For in vitro preparations, N27 cells were plated in 100 × 20-mm plates at a density of 8.0 × 104 cells per plate. After treatments, cells were washed with PBS and lysed using a cocktail of NP40 and phosphatase inhibitors (1:100) on ice. For in vivo preparations, brain region micropunches were incubated with RIPA homogenization buffer with DTT (1 µM), EDTA (1 mM), and protease and phosphatase inhibitors (1:200) for 30 min on ice, sonicated (QSonica) at 20% amplitude, and pulsed 3 times for 3 s. Next, lysates were centrifuged at 4°C for 20 min at 12,000 × g. Protein concentrations were determined using the BCA assay (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Detergent-free cellular fractionation and sucrose density analysis of membrane lipid rafts

N27 cells were plated in 100 × 20-mm plates at a density of 8.0 × 104 cells per plate. After treatments, cells were washed with PBS and lysed with a cocktail of hypotonic homogenization buffer and phosphatase inhibitors (1:100) on ice. Each sample (n) consisted of two 100 × 20-mm plates. For micropunches, a total of 0.025 g of SN tissue was homogenized in hypotonic homogenization using a sonicator to homogenize the tissue. After homogenization, cellular fractionation followed by sucrose density analysis of membrane lipid rafts was performed (Jeske et al., 2003, 2004). Cell lysate from either N27 cells or micropunches was centrifuged at 1000 × g for 5 min at 4°C to separate the nuclei. The supernatant was centrifuged at 16,000 × g for 30 min at 4°C to separate the cytosolic proteins from the mitochondria, Golgi fragments, and the plasma membrane. The pellet was then resuspended in homogenization buffer supplemented with 500 mM Na2CO3 (Song et al., 1996). The resuspended membrane pellet was placed into a sucrose flotation-gradient fraction using 5%/35%/45% discontinuous gradient that was spun at 175,000 × g for 18 h at 4°C in an Optima ultracentrifuge Model LE-80K (Beckman Coulter) using a swing bucket rotor (SW 50.1; Beckman Coulter). After the high-speed centrifugation, equal-volume fractions were taken from the top layer of the gradient, resulting in nine fractions (low-density proteins at the top gradient layers to high-density proteins at the bottom gradient layers). Protein was precipitated using the trichloroacetic acid (TCA) method (Link and LaBaer, 2011). The pellet was incubated in 0.15% deoxycholic acid and then 72% trichloroacetic acid, followed by 16,000 × g centrifugation for 30 min at room temperature. The pellet was resuspended in RIPA lysis buffer, Laemmli loading buffer, and 2 M Tris. The sample was loaded into polyacrylamide gels for electrophoresis and Western blot protein analysis. Lipid raft experiments were replicated three times. Only frozen samples were used to decrease AR fragment protein expression (Fig. 1A).

Figure 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 1.

Androgen receptor protein expression. AR protein expression was quantified in N27 cells and SN, ETC, hippocampus, and testes micropunches from young gonadally intact male rats (n = 3). Antibodies targeting either the NTD (AR N20) or the CTD (AR C19) of the AR were used. Low protein expression of full-length AR (110 kDa) was found in all samples, but high protein expression of AR fragments (70 and 30 kDa) was observed in fresh samples and to a lesser extent in frozen samples. Protein expression at 45 kDa (AR45) was observed in all samples and was unaffected by temperature (A). To determine the role of testosterone and age on AR45 expression, different hormone groups were used: N27 cells treated with vehicle control (n = 8) or 100 nM testosterone (n = 8) and SN from young and middle-aged male rats that were either gonadectomized (n = 3) or gonadally intact (n = 3; B, C). Testosterone did not affect AR45 expression in N27 cells. Neither hormones nor age affected AR45 expression in SN (B, C). C, control; T, testosterone; Ts, testes; YI, young gonadally intact male rats; YG, young GDX male rats; MI, middle-aged gonadally intact male rats; MG, middle-aged GDX male rats; SN, substantia nigra pars compacta; ETC, 2nd layer of the entorhinal cortex; Hipp, CA1 region of the dorsal hippocampus.

Coimmunoprecipitation

Because C19+/N20– AR protein expression at 45 kDa was observed only in the membrane fraction (not the cytosol or nuclear fractions), whole-cell lysates were used. Protein (25 μg) was incubated overnight at 4°C in a cocktail containing RIPA lysis buffer and 1 μg primary antibody (AR C-19 or Gαq). A Sepharose bead slurry was coupled to each sample by incubating at 4°C overnight. Samples were washed, eluted, and resuspended in 4× Laemmli buffer. To avoid IgG band interference, the blot was incubated with Clean Blot IP Detection Reagent HRP, per the manufacturer’s instructions. Coimmunoprecipitation experiments were replicated four times. Only frozen samples were used to decrease AR fragment protein expression (Fig. 1A).

Western blot

Equal amounts (20 μg protein) of denatured whole cell lysates, micropunch tissues, cellular fractions, or lipid raft fractions were loaded into a Bio-Rad Any KD polyacrylamide gel, electrophoresed in Tris-glycine buffer, and transferred onto a PVDF membrane. Membranes were blocked for 30 min with 5% nonfat milk in TBS-Tween at room temperature. After blocking, membranes were incubated with specific primary antibodies (AR C-19 1:1000, AR N-20 1:750, AR441 1:1000, Androgen R/NR3C4 1:1000, GAPDH 1:10,000, Gαq 1:1000, Gαs 1:10,000, Gαi1-3 1:10,000, Gαo 1:1000, flotillin 1:1000, and caveolin-1 1:10,000) in TBS-Tween with 1% nonfat milk for 2 h or overnight at 4°C. Afterward, the membranes were washed every 10 min for 30 min and incubated with secondary antibodies at 1:1000 in TBS-Tween with 1% nonfat milk for 30 min at room temperature. Protein bands on the membrane were visualized using an enhanced chemiluminescence detection assay (Thermo Fisher Scientific). Protein band intensities were imaged using GeneSys software corresponding with the G:Box Chemi XRQ system (Syngene). Protein band densities were quantified by NIH ImageJ densitometer software and normalized to GAPDH for whole-cell lysates, using the equation (mean gray value for protein of interest)/(mean gray value of GAPDH) × 100. All antibodies used in this study are commercially available. The specificity of the antibodies was assayed by using primary antibodies that were preabsorbed with blocking peptides (N20P 1:50, C19P 1:20) at 4°C overnight on representative blots. Previous immunohistochemical studies have shown specificity for AR-C19 and AR N-20 (Kritzer and Creutz, 2008; Holmes et al., 2016).

Immunohistochemistry

One week after gonadectomy or sham surgery, each rat was anesthetized with Inactin (100 mg/kg, i.p.; Sigma), transcardially flushed with 0.1 M PBS (100–200 ml), and then perfused with 4% paraformaldehyde in 0.1 M PBS (300–500 ml; Gottlieb et al., 2006; Ji et al., 2007; Cunningham et al., 2011). The brain was removed from the skull and postfixed with 4% paraformaldehyde in 0.1 M PBS for 24 h. Brains were stored in vials containing 30% sucrose in PBS at 4°C for 3–4 d before sectioning. Brains were cut into 3 separate sets of 40-μm coronal sections using a cryostat (CryoStar NX70, Thermo Fisher Scientific). Coronal sections containing the SN (–4.80 to –6.04 mm from bregma) were blocked with 3% PBS diluent (3% horse serum in PBS with 0.25% Triton X-100) for 2 h at room temperature followed by overnight incubation at 4°C with primary antibody (AR C-19 or AR N-20 at 1:500) in 3% PBS diluent. Afterward, the sections were washed with PBS. Serial sections incubated with AR C-19 or AR N-20 antibodies were then incubated with secondary antibodies (Alexa Fluor donkey anti-goat 1:1000) at room temperature for 5 h (Nedungadi et al., 2012). Afterward, sections were washed with PBS. Sections were mounted on slides and sealed with mounting medium. After sealing, the slides were stored at 4°C. Images were captured from each section using an epifluorescent inverted microscope (VWR) equipped with a digital camera (Photometrics Cool Snap Myo; Nikon) and imaging software (NIS Elements, Br 4.50.00; Nikon).

RT-PCR

RNA was extracted from tissue punches of substantia nigra, entorhinal cortex, hippocampus, and confluent N27 cells using a Qiagen RNeasy kit and quantified by spectrophotometry. One microgram of each sample was reverse transcribed using a High Capacity cDNA Reverse Transcription kit from Applied Biosystems. AR intron spanning primers (Table 1) were used for PCR in 100-ng equivalents from each RT reaction over 35 cycles with an annealing temperature of 56°C using 300 nM of each primer and GoTaq polymerase (Promega). Negative controls consisted of RT reactions without template. Samples were separated on 1.2% agarose gels and visualized with ethidium bromide using a Syngene GBox.

View this table:
  • View inline
  • View popup
Table 1.

Primer sequences

Bioassays

During the first 2 h of the circadian light phase, each rat was anesthetized with 2.5% isoflurane and decapitated. Prostate and seminal vesicle wet tissue weights were measured. Trunk blood (5–7 ml) was obtained in EDTA-coated tubes (13 × 100 mm, Covidien) on ice. The blood was centrifuged (Allegra X-30R, Beckman Coulter) at 2000 × g for 10 min at 4°C, then separated plasma was placed in microcentrifuge tubes and stored at –80°C until ELISA analysis for total testosterone. Plasma testosterone levels were assayed according to manufacturer’s instructions. The intra-assay coefficient of variation was 8.54%, and the interassay coefficient of variation was 9.97%. The sensitivity of this assay was 0.066 ng/ml testosterone. The specificity of this assay is 100% testosterone, 69.6% dihydrotestosterone (DHT), 7.4% dihydroxyandrosterone, and <0.1% for androstenedione, androsterone, epiandrosterone, dihydroandrosterone, estrone, estradiol, estriol, cortisol, 11-deoxycortisol, progesterone, and 17OH-progesterone.

Statistical analysis

Analysis was performed using IBM SPSS Statistics, version 21. Data were expressed as mean Embedded Image SEM. Significance (p Embedded Image 0.05) was determined by ANOVA.

Results

Bioassays

Body weights, prostate weights, seminal vesicle weights, and total testosterone levels were quantified (Table 2). Hormone treatment did not have a significant impact on body weight, regardless of age. However, middle-aged rats (9–12 mo) were significantly heavier than young rats (3 mo; F1,45 = 233.284, p < 0.05). One week after gonadectomy (GDX), plasma testosterone levels were significantly decreased compared with gonadally intact rats, regardless of age (F1,45 = 54.221, p < 0.05). No differences in testosterone levels between young and middle-aged gonadally intact rats were observed. Consistent with a decline in testosterone, GDX rats exhibited a significant decrease in weights for androgen-sensitive accessory organs: prostate (F1,45 = 74.117, p < 0.05) and seminal vesicles (F1,45 = 33.088, p < 0.05). Middle-aged retired breeders, regardless of hormone condition, had significantly heavier prostate (F1,45 = 23.577, p < 0.05) and seminal vesicle (F1,45 = 42.929, p < 0.05) weights than young sexually naive rats. These results are consistent with prior studies showing that sexual experience increases testosterone and androgen-sensitive accessory organ weights (Drori and Folman, 1964; Thomas and Neiman, 1968; Aumüller et al., 1985; Cummings et al., 2013).

View this table:
  • View inline
  • View popup
Table 2.

Bioassays

Androgen receptor expression

Androgen receptor expression was quantified in a dopaminergic N27 cell line, along with brain tissue from the substantia nigra pars compacta (SN), second layer of the entorhinal cortex (ETC), and CA1 layer of the dorsal hippocampus from young and middle-aged male rats that were either gonadally intact or GDX (Fig. 1). Frozen and fresh tissue were used, since prior reports found that low temperatures can increase full-length AR fragments (Kemppainen et al., 1992; Gregory et al., 2001). Antibodies targeting either the NTD or CTD of the AR were used to examine AR protein expression. Protein from testes was used as a positive control for AR expression. Low protein expression of full-length AR (110 kDa) was found in all samples using both NTD (AR N20) and CTD (AR C-19) AR antibodies. High protein expression of AR fragments (70 and 30 kDa) were observed in fresh samples and to a lesser extent in frozen samples (Fig. 1A). These likely represent calpain-dependent proteolysis, as described by Pelley et al. (2006), in human prostate cancer cells. Protein expression at 45 kDa was observed in all samples and was unaffected by temperature. Further, neither hormone nor age altered AR45 protein expression in N27 cells, SN tissue, and testes (Fig. 1B, C). Interestingly, this 45-kDa band was evident only when using a CTD-targeted antibody for the AR and not an NTD antibody, consistent with the AR splice variant AR45 that lacks an NTD. Similar results were observed using different antibodies (AR441 and R/NR3C4) targeting the NTD and CTD of the AR (data not shown).

Widespread CTD AR immunoreactivity (and not NTD AR–positive cells) was observed throughout the SN in young and middle-aged rats, respective of hormone and age status (Fig. 2A, B). AR immunoreactivity included extranuclear staining, suggesting membrane or cytosolic localization. In contrast, the hippocampus and the ETC express both CTD- and NTD-positive AR within the nucleus in young intact males (Fig. 2C), consistent with prior immunohistochemical studies using AR antibodies (Sar et al., 1990; Xiao and Jordan, 2002; DonCarlos et al., 2003; Kritzer, 2004).

Figure 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 2.

Widespread AR45 distribution in the substantia nigra. AR immunoreactivity using a CTD targeted antibody (AR C19) was observed throughout the SN in all age and hormone groups (A). No AR immunoreactivity was observed using a NTD targeted antibody in the SN (B). Both the hippocampus and the ETC express CTD and NTD AR immunoreactivity that is present within the nucleus, as evidenced by DAPI colocalization (C). SN, substantia nigra pars compacta; ETC, 2nd layer of the entorhinal cortex; Hipp, CA1 region of the dorsal hippocampus. Scale bar = 200 μm.

Expression profile of androgen receptors

N27 cells and SN brain tissue were split into membrane, cytosol, and nuclear fractions. The membrane portion of both N27 cells and SN brain tissue was further separated into nine fractions using a sucrose gradient (Figs. 3–5). Full-length AR at 110 kDa was not observed in any membrane fraction. Although full-length AR was not expressed, a 45-kDa protein corresponding to AR45 was observed in all samples. In N27 cells, regardless of testosterone exposure, AR45 expression was evident in fractions 3–6, which are lipid rafts as shown by caveolin-1 and flotillin immunoreactivity (Fig. 3A, B). Similarly, AR45 immunoreactivity was present in caveolin- and flotillin-enriched membrane lipid rafts in both young and middle-aged rats in all hormone groups (Figs. 4–5). AR45 immunoreactivity was not found in any non–lipid raft portion of the membrane, nor was it observed in cytosolic or nuclear fractions.

Figure 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 3.

N27 cells express AR45 protein in membrane lipid rafts. N27 cells were treated with either vehicle control (A) or 100 nM testosterone for 24 h (B). The membrane portion of the cells were further separated into nine fractions using a sucrose gradient and ultracentrifugation to examine lipid rafts. Primary antibodies targeting AR45 (AR-C19 antibody), G protein Gαq, and lipid raft markers (caveolin-1 and flotillin) were used. AR45 and Gαq expression were observed only in lipid raft fractions, as evidenced by caveolin-1 and flotillin expression. Hormone treatment did not alter AR45 and Gαq expression in lipid rafts. Full-length (110-kDa) AR, Gαo, Gαi1-3, and Gαs G proteins were not observed in the membranes of N27 cells (data not shown). n = 3 per treatment group.

Figure 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 4.

Young male rats express AR45 protein in membrane lipid rafts. Young rats (3 mo old) were either gonadectomized (A) or gonadally intact (B). Micropunches of substantia nigra tissue were collected, and the membrane was isolated and then separated into nine fractions using a sucrose gradient to examine lipid rafts. Primary antibodies targeting AR45 (AR-C19 antibody), G proteins Gαq and Gαo, and lipid raft markers (caveolin-1 and flotillin) were used. AR45 expression was observed only in lipid raft fractions, as evidenced by caveolin-1 and flotillin expression. Hormone status did not alter AR45 expression in lipid rafts. Gαq and Gαo was observed throughout the membrane, regardless of hormone status. No protein expression of Gαi1-3 and Gαs G proteins or full-length (100-kDa) AR was observed in the substantia nigral membranes of young male rats (data not shown). GDX, gonadectomy; intact, gonadally intact. n = 3 per treatment group.

Figure 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 5.

Aged male rats express AR45 protein in membrane lipid rafts. Middle-aged rats (9- to 12-mo old retired breeders) were either gonadectomized (A) or gonadally intact (B). Membrane portion of the substantia nigra micropunch tissue was isolated, and then separated into nine fractions using a sucrose gradient. AR45 (AR-C19 antibody), Gαq and Gαo, and lipid raft markers (caveolin-1 and flotillin) were examined. AR45 expression was observed only in lipid raft fractions, regardless of hormone status. Gαq and Gαo was observed throughout the membrane in both gonadectomized and gonadally intact males. Protein expression of Gαi1-3, Gαs, or full-length (110-kDa) AR were not observed in the substantia nigral membranes of aged male rats (data not shown). GDX, gonadectomy; intact, gonadally intact. n = 3 per treatment group.

Androgen receptor mRNA expression

RT-PCR for AR was performed to determine whether full-length transcripts were present in SN and N27 cells (Fig. 6). Several intron-spanning primer sets were used to amplify equivalent amounts of cDNA under identical conditions. In the brain, we detected positive signals from all regions examined (SN, ETC, Hipp), confirming the presence of full-length AR mRNA (Fig. 6A, B). However, in N27 cells, we detected no product from an exon 1 to exon 3 primer pair. Exon 2–7 amplicons were detected in N27 cells (Fig. 6B), although the signal was less distinct than in brain samples. These data suggest that full-length AR mRNA is not present in N27 cells, or is present only at very low levels, below detectability under our conditions (35 cycles, EtBr detection).

Figure 6.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 6.

Androgen receptor RT-PCR. Intron-spanning primer sets for rat AR were used to amplify cDNA from young intact male rat substantia nigra pars compacta (SN), 2nd layer of the entorhinal cortex (ETC), CA1 region of the hippocampus (Hip), and N27 cells (A and B). Note the lack of amplification product for the 3′ region of AR in N27 cells (A). C, Targets of the antibodies used to detect AR and their epitopes in parentheses aligned with the AR domain structure (top), and the relative amplification product locations in the AR mRNA (bottom). Exons 2 and 3 code for the DBD. M denotes a 100-bp DNA ladder, and negative controls are marked with –.

Expression profile of GPCRs in membrane lipid rafts

Because neuronal mAR has been associated with intracellular calcium signaling (Steinsapir et al., 1991; Gorczynska and Handelsman, 1995; Benten et al., 1999; Estrada et al., 2003, 2006; Holmes et al., 2013), we examined whether G proteins were present in lipid rafts. In both N27 cells and SN tissue, the G protein Gαq was expressed in the membrane fraction and in lipid rafts, but Gαi1-3 and Gαs were not expressed in any of the membrane fractions. Interestingly, the G protein Gαo was expressed only in SN tissue and not in the N27 cell line (Figs. 3–5).

Androgen receptor variant association with GPCR subunits

To determine whether AR45 interacts with G proteins, coimmunoprecipitation was performed. Antibodies targeting the CTD of the AR were used to pull down proteins. In a reciprocal fashion to ensure specificity, Gαq-containing proteins were immunoprecipitated and then immunoblotted for AR45 with an AR C19 antibody. After electrophoresis, membranes were probed for AR45 (C-19 antibody) or G protein immunoreactivity. In N27 cells, AR45 interacted with Gαq, regardless of testosterone exposure (Fig. 7A). Furthermore, in SN tissue, AR45 was associated with Gαq and Gαo in all hormone states and ages (Fig. 7B).

Figure 7.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 7.

Coimmunoprecipitation (IP) of the AR45-G protein complex. Whole-cell lysates of N27 cells treated with either vehicle or 100 nM testosterone (A) and substantia nigral tissue from young and middle-aged male rats that were either gonadectomized or gonadally intact (B) were used. AR CTD-containing AR45 proteins were immunoprecipitated and then immunoblotted (WB) for Gαq and Gαo. In a reciprocal fashion, Gαq-containing proteins were immunoprecipitated and then immunoblotted for AR45. In N27 cells, bands corresponding to AR45 (AR-C19) and Gαq were detectable. In substantia nigral tissue, bands corresponding to AR45 (AR-C19), Gαq, and Gαo were observed. C, control; T, testosterone; YI, young gonadally intact male rats; YG, young GDX male rats; MI, middle-aged gonadally intact male rats; MG, middle-aged GDX male rats. n = 4 per treatment group.

Discussion

This study found AR protein expression at 45-kDa molecular weight in all samples examined: N27 cells, SNpc, hippocampus, ETC, and testes. Furthermore, this AR variant is present in plasma membrane lipid rafts from N27 cells and SN brain tissue from young and middle-aged rats with and without sex hormones. The 45-kDa membrane protein was evident only using a CTD-targeted antibody for AR, consistent with the AR45 splice variant. Interestingly, protein expression of AR45 was not altered by steroid hormones, age, or temperature. Although full-length AR mRNA was found in all brain samples examined, little to no AR immunoreactivity using the NTD-targeted AR antibody was observed in SNpc tissue, unlike the ETC and hippocampus. Expression profiles for AR immunoreactivity for these brain regions are different, indicating different mechanisms of androgen action in SN versus hippocampus and ETC. This result, coupled with a lack of a detectable exon 1–containing transcript in N27 cells, suggests that the immunoreactive AR45 may represent a bona fide splice variant rather than posttranslational processing of the full-length AR.

Prior studies in young adult and adolescent male Sprague-Dawley rats described AR immunoreactivity in the SNpc using AR antibodies targeting the NTD of the receptor (Kritzer, 1997; Kritzer and Creutz, 2008; Purves-Tyson et al., 2012). Our results are consistent with the Kritzer laboratory’s findings of low NTD-AR immunoreactivity within the SNpc (only the dorsomedial region) but no AR expression in the rest of the SNpc obtained from young adult male rats. The highest AR immunoreactivity was observed in SN pars lateralis, wherein >50% of the neurons are AR+ (Kritzer, 1997; Kritzer and Creutz, 2008). These findings indicate that full-length AR is not highly expressed in the SNpc of young adult male rats. In contrast, adolescent male rats have high NTD-AR immunoreactivity in the SNpc, as evidenced by >65% of the TH+ neurons immunoreactive for AR (Purves-Tyson et al., 2012). This difference in NTD-AR immunoreactivity between young adult and adolescent male rats could be due to adolescent pruning of TH+ neurons in the SNpc (Kuhn et al., 2010). To the best of the authors’ knowledge, there are no prior immunohistochemical studies using CTD-AR antibodies in the SN.

Because classic full-length AR protein was not highly present in SNpc and N27 cells, it is possible that the AR45 splice variant is mediating androgen’s actions in this brain region by acting as an mAR. Prior studies, using nonneuronal cells, have linked AR to membrane lipid rafts (Lu et al., 2001; Cinar et al., 2007; Pedram et al., 2007). Lipid rafts are low-density microdomains, enriched with cholesterol and lipids, and insoluble in non-ionic detergents (Brown and Rose, 1992; Brown and London, 2000). The proteins flotillin and caveolin are integral components of lipid rafts. Neuronal lipid rafts generally are planar and composed of flotillin, unlike nonneuronal cells that contain rafts with invaginations composed of caveolin (Murata et al., 1995; Bickel et al., 1997; Lang et al., 1998). However, studies have shown that caveolin-1 can be present in neurons under certain conditions, such as oxidative stress and aging (Volonté et al., 2001; Kang et al., 2006; Marquet-de Rougé et al., 2013). One of the brain regions with the highest expression of caveolin-1 is the SN (Galbiati et al., 1998), which is composed mainly of dopaminergic TH+ neurons that have increased oxidative stress from dopamine metabolism (Lotharius et al., 2002; Hastings, 2009).

Our data showed that AR45 is present in membrane fractions from N27 cells and SN tissue. Specifically, AR45 was present only in caveolin- and flotillin-enriched lipid rafts, indicating localization to lipid rafts that contain invaginations. Although AR45 was expressed only in caveolin+ lipid rafts, differences were observed in which membrane fractions these proteins localized to. Caveolin was more widespread in the membrane fractions from the N27 cell line, wherein caveolin were observed in lower-density protein fractions. Notably, testosterone appeared to shift caveolin expression to higher-density protein fractions in both N27 cells and SN tissue. It is possible that caveolin is undergoing posttranslational modification, resulting in higher-density proteins. Posttranslational modifications (e.g., palmitoylation and phosphorylation) commonly occur in caveolin proteins and steroid receptors (Parat and Fox, 2001; Fukata and Fukata, 2010; Kim et al., 2011). A common initiator of posttranslational modifications in caveolin includes oxidative stress (Kim et al., 2000; Volonté et al., 2001; Wehinger et al., 2015), and testosterone can act as an oxidative stressor (Holmes et al., 2013, 2016). Therefore, it is possible that testosterone, via oxidative stress, is increasing posttranslational modifications in caveolin protein, resulting in the caveolin/AR45 complex shifting to higher-density protein fractions.

Numerous signaling proteins, such as receptor tyrosine kinases, GPCRs, and G proteins, reside in lipid rafts and play a pivotal role in signal transduction (Mineo et al., 1999; Toki et al., 1999; Brown and London, 2000; Lasley et al., 2000; Rybin et al., 2000; Rebois and Hébert, 2003; Pucadyil and Chattopadhyay, 2004; Allen et al., 2005; Xu et al., 2006). Prior studies have shown that Gαq mainly localizes in caveolae lipid rafts, unlike Gαs and Gαi proteins (Murthy and Makhlouf, 2000; Oh and Schnitzer, 2001; Sengupta et al., 2008). Indeed, the results from this study show that Gαq and Gαo proteins are present in lipid rafts, along with AR45. Furthermore, AR45 coimmunoprecipitates with Gαq and Gαo proteins, indicating that AR45 interacts with G proteins in lipid rafts in dopaminergic cells.

Gαq has been well established as an activator of intracellular calcium release from the endoplasmic reticulum (Rhee, 2001; Shi and Kehrl, 2001), which can affect dopaminergic neuronal function in the SNpc (Surmeier et al., 2012). Much less is known about the function of Gαo proteins that are highly present in frontal cortex, cerebellum, hypothalamus, hippocampus, and SN (Worley et al., 1986). Gαo can couple to receptors that decrease intracellular calcium release (Lewis et al., 1986; Hescheler et al., 1987), and in the brain Gαo is predominantly coupled to inhibitory D2 dopamine receptors (Jiang et al., 2001). Interestingly, this association with D2 receptors may explain the lack of Gαo expression in the N27 cells, as D2 receptors are not expressed in this cell line (Urban and Mailman, 2005). Further supporting the role of Gαo in SN dopaminergic neuronal involvement, Gαo knockout mice exhibit poor motor coordination (Jiang et al., 1998). Although data about Gαo is sparse, Gαo is linked with motor function and calcium signaling.

Because AR45 appears to be the predominant AR in the SNpc, it may have clinical relevance in the progressive motor disorder Parkinson’s disease (PD), resulting from the loss of TH+ neurons in the SNpc (Pike et al., 2006, 2009; Cunningham et al., 2009, 2011; Rosario et al., 2011; Holmes et al., 2013, 2016; Verdile et al., 2014). Men have a twofold increased risk for PD than women (Baldereschi et al., 2000). Similarly, postmenopausal women have a greater risk for PD than age-matched premenopausal women (Currie et al., 2004; Ragonese et al., 2006a, 2006b), which may be due to the higher circulating androgen-to-estrogen state during menopause (Vermeulen, 1976; Laughlin et al., 2000; Liu et al., 2001; Handelsman et al., 2016; Labandeira-Garcia et al., 2016). It is unknown what mechanisms underlie this PD sex difference, but it is possible that AR45 located in dopaminergic membrane caveolin lipid rafts may be involved, especially as upregulated caveolin expression and lipid rafts have been linked with PD (Hashimoto et al., 2003; Trushina et al., 2006; Schengrund, 2010; Sonnino et al., 2014; Cha et al., 2015). Furthermore, recent in vitro studies support the involvement of an mAR in mediating cell viability, oxidative stress generation, and calcium signaling in dopaminergic cells (Estrada et al., 2006; Holmes et al., 2013, 2016), which are key characteristics and processes observed in PD pathology (Jenner et al., 1992; Sherer et al., 2002; Dickson, 2007). Interestingly, both of the in vitro cell lines (N27 and SH-SY5Y cells) are female-derived cell lines, indicating that these fast, nongenomic androgen actions in neurons are (1) applicable to both males and females and (2) dependent on the hormonal and AR expression milieu.

This is the first study to show the presence of a putative AR splice variant protein in the SNpc, hippocampus, and ETC brain regions. Specifically, our results show that AR45 localizes in the membrane lipid rafts from N27 cells and SN dopaminergic neurons. Furthermore, AR45 interacts with Gαq and Gαo G proteins, which can impact intracellular calcium signaling (Fig. 8). More research needs to be conducted to further determine the function and role of this AR splice variant in dopaminergic neuronal function and dysfunction.

Figure 8.
  • Download figure
  • Open in new tab
  • Download powerpoint
Figure 8.

Model. Caveolae lipid rafts are flask-shaped invaginations made up of caveolin (purple) and flotillin (blue) proteins that function by organizing signaling complexes in the plasma membrane. Proteins present in caveolae include AR45. Furthermore, AR45 proteins interact with Gαq and Gαo proteins, which are involved in modulating intracellular calcium levels. Gαq can increase intracellular calcium release via the PLC/IP3 pathway, whereas Gαo can inhibit intracellular calcium. PLC, phospholipase C, IP3: inositol trisphosphate.

Acknowledgments

Acknowledgments: We thank Drs. J. Thomas Cunningham, Robert Barber, and Robert Luedtke for their technical assistance.

Footnotes

  • Authors report no conflicts of interest.

  • This study was funded by the National Institute of Neurological Disorders and Stroke (R01 NS088514) to RLC.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

References

  1. ↵
    Ahrens-Fath I, Politz O, Geserick C, Haendler B (2005) Androgen receptor function is modulated by the tissue-specific AR45 variant. FEBS J 272:74–84. doi:10.1111/j.1742-4658.2004.04395.x pmid:15634333
    OpenUrlCrossRefPubMed
  2. ↵
    Allen JA, Yu JZ, Donati RJ, Rasenick MM (2005) Beta-adrenergic receptor stimulation promotes G alpha s internalization through lipid rafts: a study in living cells. Mol Pharmacol 67:1493–1504. doi:10.1124/mol.104.008342
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Anantharam V, Lehrmann E, Kanthasamy A, Yang Y, Banerjee P, Becker KG, Freed WJ, Kanthasamy AG (2007) Microarray analysis of oxidative stress regulated genes in mesencephalic dopaminergic neuronal cells: relevance to oxidative damage in Parkinson’s disease. Neurochem Int 50:834–847. doi:10.1016/j.neuint.2007.02.003 pmid:17397968
    OpenUrlCrossRefPubMed
  4. ↵
    Aumüller G, Braun BE, Seitz J, Müller T, Heyns W, Krieg M (1985) Effects of sexual rest or sexual activity on the structure and function of the ventral prostate of the rat. Anat Rec 212:345–352. doi:10.1002/ar.1092120404 pmid:4073550
    OpenUrlCrossRefPubMed
  5. ↵
    Baldereschi M, Di Carlo A, Rocca WA, Vanni P, Maggi S, Perissinotto E, Grigoletto F, Amaducci L, Inzitari D (2000) Parkinson’s disease and parkinsonism in a longitudinal study: two-fold higher incidence in men. ILSA Working Group. Italian Longitudinal Study on Aging. Neurology 55:1358–1363. doi:10.1212/WNL.55.9.1358
    OpenUrlCrossRef
  6. ↵
    Benten WP, Lieberherr M, Stamm O, Wrehlke C, Guo Z, Wunderlich F (1999) Testosterone signaling through internalizable surface receptors in androgen receptor-free macrophages. Mol Biol Cell 10:3113–3123. pmid:10512854
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Bickel PE, Scherer PE, Schnitzer JE, Oh P, Lisanti MP, Lodish HF (1997) Flotillin and epidermal surface antigen define a new family of caveolae-associated integral membrane proteins. J Biol Chem 272:13793–13802. pmid:9153235
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Brown DA, Rose JK (1992) Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68:533–544. pmid:1531449
    OpenUrlCrossRefPubMed
  9. ↵
    Brown DA, London E (2000) Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem 275:17221–17224. doi:10.1074/jbc.R000005200 pmid:10770957
    OpenUrlFREE Full Text
  10. ↵
    Callewaert L, Van Tilborgh N, Claessens F (2006) Interplay between two hormone-independent activation domains in the androgen receptor. Cancer Res 66:543–553. doi:10.1158/0008-5472.CAN-05-2389 pmid:16397271
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Carvour M, Song C, Kaul S, Anantharam V, Kanthasamy A (2008) Chronic low-dose oxidative stress induces caspase-3-dependent PKCdelta proteolytic activation and apoptosis in a cell culture model of dopaminergic neurodegeneration. Ann N Y Acad Sci 1139:197–205. doi:10.1196/annals.1432.020
    OpenUrlCrossRefPubMed
  12. ↵
    Cha SH, Choi YR, Heo CH, Kang SJ, Joe EH, Jou I, Kim HM, Park SM (2015) Loss of parkin promotes lipid rafts-dependent endocytosis through accumulating caveolin-1: implications for Parkinson’s disease. Mol Neurodegener 10:63. doi:10.1186/s13024-015-0060-5 pmid:26627850
    OpenUrlCrossRefPubMed
  13. ↵
    Cinar B, Mukhopadhyay NK, Meng G, Freeman MR (2007) Phosphoinositide 3-kinase-independent non-genomic signals transit from the androgen receptor to Akt1 in membrane raft microdomains. J Biol Chem 282:29584–29593. doi:10.1074/jbc.M703310200 pmid:17635910
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Claessens F, Denayer S, Van Tilborgh N, Kerkhofs S, Helsen C, Haelens A (2008) Diverse roles of androgen receptor (AR) domains in AR-mediated signaling. Nucl Recept Signal 6:e008. doi:10.1621/nrs.06008 pmid:18612376
    OpenUrlCrossRefPubMed
  15. ↵
    Clarkson ED, Edwards-Prasad J, Freed CR, Prasad KN (1999) Immortalized dopamine neurons: a model to study neurotoxicity and neuroprotection. Proc Soc Exp Biol Med 222:157–163. pmid:10564540
    OpenUrlCrossRefPubMed
  16. ↵
    Cummings JA, Clinton SM, Perry AN, Akil H, Becker JB (2013) Male rats that differ in novelty exploration demonstrate distinct patterns of sexual behavior. Behav Neurosci 127:47–58. doi:10.1037/a0031528 pmid:23398441
    OpenUrlCrossRefPubMed
  17. ↵
    Cunningham RL, Giuffrida A, Roberts JL (2009) Androgens induce dopaminergic neurotoxicity via caspase-3-dependent activation of protein kinase Cdelta. Endocrinology 150:5539–5548. doi:10.1210/en.2009-0640 pmid:19837873
    OpenUrlCrossRefPubMed
  18. ↵
    Cunningham RL, Macheda T, Watts LT, Poteet E, Singh M, Roberts JL, Giuffrida A (2011) Androgens exacerbate motor asymmetry in male rats with unilateral 6-hydroxydopamine lesion. Horm Behav 60:617–624. doi:10.1016/j.yhbeh.2011.08.012 pmid:21907204
    OpenUrlCrossRefPubMed
  19. ↵
    Currie LJ, Harrison MB, Trugman JM, Bennett JP, Wooten GF (2004) Postmenopausal estrogen use affects risk for Parkinson disease. Arch Neurol 61:886–888. doi:10.1001/archneur.61.6.886 pmid:15210525
    OpenUrlCrossRefPubMed
  20. ↵
    Dickson DW (2007) Linking selective vulnerability to cell death mechanisms in Parkinson’s disease. Am J Pathol 170:16–19. doi:10.2353/ajpath.2007.061011 pmid:17200178
    OpenUrlCrossRefPubMed
  21. ↵
    Ding D, Xu L, Menon M, Reddy GP, Barrack ER (2004) Effect of a short CAG (glutamine) repeat on human androgen receptor function. Prostate 58:23–32. doi:10.1002/pros.10316
    OpenUrlCrossRefPubMed
  22. ↵
    Ding D, Xu L, Menon M, Reddy GP, Barrack ER (2005) Effect of GGC (glycine) repeat length polymorphism in the human androgen receptor on androgen action. Prostate 62:133–139. doi:10.1002/pros.20128 pmid:15389799
    OpenUrlCrossRefPubMed
  23. ↵
    DonCarlos LL, Garcia-Ovejero D, Sarkey S, Garcia-Segura LM, Azcoitia I (2003) Androgen receptor immunoreactivity in forebrain axons and dendrites in the rat. Endocrinology 144:3632–3638. doi:10.1210/en.2002-0105 pmid:12865346
    OpenUrlCrossRefPubMed
  24. ↵
    Drori D, Folman Y (1964) Effects of cohabitation on the reproductive system, kidneys and body composition of male rats. J Reprod Fertil 8:351–359. pmid:14248595
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Edwards J, Bartlett JM (2005) The androgen receptor and signal-transduction pathways in hormone-refractory prostate cancer. Part 1: Modifications to the androgen receptor. BJU Int 95:1320–1326. doi:10.1111/j.1464-410X.2005.05526.x pmid:15892825
    OpenUrlCrossRefPubMed
  26. ↵
    Estrada M, Uhlen P, Ehrlich BE (2006) Ca2+ oscillations induced by testosterone enhance neurite outgrowth. J Cell Sci 119:733–743. doi:10.1242/jcs.02775 pmid:16449326
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Estrada M, Espinosa A, Müller M, Jaimovich E (2003) Testosterone stimulates intracellular calcium release and mitogen-activated protein kinases via a G protein-coupled receptor in skeletal muscle cells. Endocrinology 144:3586–3597. doi:10.1210/en.2002-0164
    OpenUrlCrossRefPubMed
  28. ↵
    Ferro P, Catalano MG, Dell’Eva R, Fortunati N, Pfeffer U (2002) The androgen receptor CAG repeat: a modifier of carcinogenesis? Mol Cell Endocrinol 193:109–120. pmid:12161010
    OpenUrlCrossRefPubMed
  29. ↵
    Foradori CD, Weiser MJ, Handa RJ (2008) Non-genomic actions of androgens. Front Neuroendocrinol 29:169–181. doi:10.1016/j.yfrne.2007.10.005 pmid:18093638
    OpenUrlCrossRefPubMed
  30. ↵
    Fukata Y, Fukata M (2010) Protein palmitoylation in neuronal development and synaptic plasticity. Nat Rev Neurosci 11:161–175. doi:10.1038/nrn2788 pmid:20168314
    OpenUrlCrossRefPubMed
  31. ↵
    Galbiati F, Volonté D, Gil O, Zanazzi G, Salzer JL, Sargiacomo M, Scherer PE, Engelman JA, Schlegel A, Parenti M, Okamoto T, Lisanti MP (1998) Expression of caveolin-1 and -2 in differentiating PC12 cells and dorsal root ganglion neurons: caveolin-2 is up-regulated in response to cell injury. Proc Natl Acad Sci U S A 95:10257–10262. pmid:9707634
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Gorczynska E, Handelsman DJ (1995) Androgens rapidly increase the cytosolic calcium concentration in Sertoli cells. Endocrinology 136:2052–2059. doi:10.1210/endo.136.5.7720654 pmid:7720654
    OpenUrlCrossRefPubMed
  33. ↵
    Gottlieb HB, Ji LL, Jones H, Penny ML, Fleming T, Cunningham JT (2006) Differential effects of water and saline intake on water deprivation-induced c-Fos staining in the rat. Am J Physiol Regul Integr Comp Physiol 290:R1251–R1261.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Green SM, Mostaghel EA, Nelson PS (2012) Androgen action and metabolism in prostate cancer. Mol Cell Endocrinol 360:3–13. doi:10.1016/j.mce.2011.09.046 pmid:22453214
    OpenUrlCrossRefPubMed
  35. ↵
    Gregory CW, He B, Wilson EM (2001) The putative androgen receptor-A form results from in vitro proteolysis. J Mol Endocrinol 27:309–319. pmid:11719283
    OpenUrlAbstract
  36. ↵
    Handelsman DJ, Sikaris K, Ly LP (2016) Estimating age-specific trends in circulating testosterone and sex hormone-binding globulin in males and females across the lifespan. Ann Clin Biochem 53:377–384. doi:10.1177/0004563215610589 pmid:26438522
    OpenUrlCrossRefPubMed
  37. ↵
    Hashimoto M, Takenouchi T, Rockenstein E, Masliah E (2003) Alpha-synuclein up-regulates expression of caveolin-1 and down-regulates extracellular signal-regulated kinase activity in B103 neuroblastoma cells: role in the pathogenesis of Parkinson’s disease. J Neurochem 85:1468–1479. pmid:12787066
    OpenUrlCrossRefPubMed
  38. ↵
    Hastings TG (2009) The role of dopamine oxidation in mitochondrial dysfunction: implications for Parkinson’s disease. J Bioenerg Biomembr 41:469–472. doi:10.1007/s10863-009-9257-z pmid:19967436
    OpenUrlCrossRefPubMed
  39. ↵
    Hatzoglou A, Kampa M, Kogia C, Charalampopoulos I, Theodoropoulos PA, Anezinis P, Dambaki C, Papakonstanti EA, Stathopoulos EN, Stournaras C, Gravanis A, Castanas E (2005) Membrane androgen receptor activation induces apoptotic regression of human prostate cancer cells in vitro and in vivo. J Clin Endocrinol Metab 90:893–903. doi:10.1210/jc.2004-0801 pmid:15585562
    OpenUrlCrossRefPubMed
  40. ↵
    Hescheler J, Rosenthal W, Trautwein W, Schultz G (1987) The GTP-binding protein, Go, regulates neuronal calcium channels. Nature 325:445–447. doi:10.1038/325445a0 pmid:2433590
    OpenUrlCrossRefPubMed
  41. ↵
    Holmes S, Singh M, Su C, Cunningham RL (2016) Effects of oxidative stress and testosterone on pro-inflammatory signaling in a female rat dopaminergic neuronal cell line. Endocrinology 157:2824–2835. doi:10.1210/en.2015-1738
    OpenUrlCrossRef
  42. ↵
    Holmes S, Abbassi B, Su C, Singh M, Cunningham RL (2013) Oxidative stress defines the neuroprotective or neurotoxic properties of androgens in immortalized female rat dopaminergic neuronal cells. Endocrinology 154:4281–4292. doi:10.1210/en.2013-1242 pmid:23959938
    OpenUrlCrossRefPubMed
  43. ↵
    Hu DG, Hickey TE, Irvine C, Wijayakumara DD, Lu L, Tilley WD, Selth LA, Mackenzie PI (2014) Identification of androgen receptor splice variant transcripts in breast cancer cell lines and human tissues. Horm Cancer 5:61–71. doi:10.1007/s12672-014-0171-4 pmid:24570075
    OpenUrlCrossRefPubMed
  44. ↵
    Hu R, Lu C, Mostaghel EA, Yegnasubramanian S, Gurel M, Tannahill C, Edwards J, Isaacs WB, Nelson PS, Bluemn E, Plymate SR, Luo J (2012) Distinct transcriptional programs mediated by the ligand-dependent full-length androgen receptor and its splice variants in castration-resistant prostate cancer. Cancer Res 72:3457–3462. doi:10.1158/0008-5472.CAN-11-3892 pmid:22710436
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Ikonen T, Palvimo JJ, Jänne OA (1998) Heterodimerization is mainly responsible for the dominant negative activity of amino-terminally truncated rat androgen receptor forms. FEBS Lett 430:393–396. pmid:9688578
    OpenUrlCrossRefPubMed
  46. ↵
    Jagla M, Fève M, Kessler P, Lapouge G, Erdmann E, Serra S, Bergerat JP, Céraline J (2007) A splicing variant of the androgen receptor detected in a metastatic prostate cancer exhibits exclusively cytoplasmic actions. Endocrinology 148:4334–4343. doi:10.1210/en.2007-0446
    OpenUrlCrossRefPubMed
  47. ↵
    Jenner P, Dexter DT, Sian J, Schapira AH, Marsden CD (1992) Oxidative stress as a cause of nigral cell death in Parkinson’s disease and incidental Lewy body disease. The Royal Kings and Queens Parkinson’s Disease Research Group. Ann Neurol 32(Suppl):S82–S87. pmid:1510385
    OpenUrlCrossRefPubMed
  48. ↵
    Jenster G, van der Korput HA, van Vroonhoven C, van der Kwast TH, Trapman J, Brinkmann AO (1991) Domains of the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization. Mol Endocrinol 5:1396–1404. doi:10.1210/mend-5-10-1396 pmid:1775129
    OpenUrlCrossRefPubMed
  49. ↵
    Jeske NA, Glucksman MJ, Roberts JL (2003) EP24.15 is associated with lipid rafts. J Neurosci Res 74:468–473. doi:10.1002/jnr.10778 pmid:14598323
    OpenUrlCrossRefPubMed
  50. ↵
    Jeske NA, Glucksman MJ, Roberts JL (2004) Metalloendopeptidase EC3.4.24.15 is constitutively released from the exofacial leaflet of lipid rafts in GT1-7 cells. J Neurochem 90:819–828. doi:10.1111/j.1471-4159.2004.02557.x
    OpenUrlCrossRefPubMed
  51. ↵
    Ji LL, Gottlieb HB, Penny ML, Fleming T, Toney GM, Cunningham JT (2007) Differential effects of water deprivation and rehydration on Fos and FosB/DeltaFosB staining in the rat brainstem. Exp Neurol 203:445–456. doi:10.1016/j.expneurol.2006.08.020 pmid:17027755
    OpenUrlCrossRefPubMed
  52. ↵
    Jiang M, Spicher K, Boulay G, Wang Y, Birnbaumer L (2001) Most central nervous system D2 dopamine receptors are coupled to their effectors by Go. Proc Natl Acad Sci U S A 98:3577–3582. doi:10.1073/pnas.051632598 pmid:11248120
    OpenUrlAbstract/FREE Full Text
  53. ↵
    Jiang M, Gold MS, Boulay G, Spicher K, Peyton M, Brabet P, Srinivasan Y, Rudolph U, Ellison G, Birnbaumer L (1998) Multiple neurological abnormalities in mice deficient in the G protein Go. Proc Natl Acad Sci U S A 95:3269–3274. doi:10.1073/pnas.95.6.3269
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Kang MJ, Chung YH, Hwang CI, Murata M, Fujimoto T, Mook-Jung IH, Cha CI, Park WY (2006) Caveolin-1 upregulation in senescent neurons alters amyloid precursor protein processing. Exp Mol Med 38:126–133. doi:10.1038/emm.2006.16 pmid:16672766
    OpenUrlCrossRefPubMed
  55. ↵
    Kelly SJ, Ostrowski NL, Wilson MA (1999) Gender differences in brain and behavior: hormonal and neural bases. Pharmacol Biochem Behav 64:655–664. pmid:10593187
    OpenUrlCrossRefPubMed
  56. ↵
    Kemppainen JA, Lane MV, Sar M, Wilson EM (1992) Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. J Biol Chem 267:968–974. pmid:1730684
    OpenUrlAbstract/FREE Full Text
  57. ↵
    Kerr JE, Allore RJ, Beck SG, Handa RJ (1995) Distribution and hormonal regulation of androgen receptor (AR) and AR messenger ribonucleic acid in the rat hippocampus. Endocrinology 136:3213–3221. doi:10.1210/endo.136.8.7628354 pmid:7628354
    OpenUrlCrossRefPubMed
  58. ↵
    Kim W, Bennett EJ, Huttlin EL, Guo A, Li J, Possemato A, Sowa ME, Rad R, Rush J, Comb MJ, Harper JW, Gygi SP (2011) Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell 44:325–340. doi:10.1016/j.molcel.2011.08.025 pmid:21906983
    OpenUrlCrossRefPubMed
  59. ↵
    Kim YN, Wiepz GJ, Guadarrama AG, Bertics PJ (2000) Epidermal growth factor-stimulated tyrosine phosphorylation of caveolin-1. Enhanced caveolin-1 tyrosine phosphorylation following aberrant epidermal growth factor receptor status. J Biol Chem 275:7481–7491. pmid:10713051
    OpenUrlAbstract/FREE Full Text
  60. ↵
    Kritzer M (2004) The distribution of immunoreactivity for intracellular androgen receptors in the cerebral cortex of hormonally intact adult male and female rats: localization in pyramidal neurons making corticocortical connections. Cereb Cortex 14:268–280. pmid:14754867
    OpenUrlCrossRefPubMed
  61. ↵
    Kritzer MF (1997) Selective colocalization of immunoreactivity for intracellular gonadal hormone receptors and tyrosine hydroxylase in the ventral tegmental area, substantia nigra, and retrorubral fields in the rat. J Comp Neur 379:247–260. doi:10.1002/(SICI)1096-9861(19970310)379:2<247::AID-CNE6>3.0.CO;2-3
    OpenUrlCrossRefPubMed
  62. ↵
    Kritzer MF, Creutz LM (2008) Region and sex differences in constituent dopamine neurons and immunoreactivity for intracellular estrogen and androgen receptors in mesocortical projections in rats. J Neurosci 28:9525–9535. doi:10.1523/JNEUROSCI.2637-08.2008 pmid:18799684
    OpenUrlAbstract/FREE Full Text
  63. ↵
    Kuhn C, Johnson M, Thomae A, Luo B, Simon SA, Zhou G, Walker QD (2010) The emergence of gonadal hormone influences on dopaminergic function during puberty. Horm Behav 58:122–137. doi:10.1016/j.yhbeh.2009.10.015 pmid:19900453
    OpenUrlCrossRefPubMed
  64. ↵
    Labandeira-Garcia JL, Rodriguez-Perez AI, Valenzuela R, Costa-Besada MA, Guerra MJ (2016) Menopause and Parkinson’s disease. Interaction between estrogens and brain renin-angiotensin system in dopaminergic degeneration. Front Neuroendocrinol 43:44–59. doi:10.1016/j.yfrne.2016.09.003 pmid:27693730
    OpenUrlCrossRefPubMed
  65. ↵
    Lang DM, Lommel S, Jung M, Ankerhold R, Petrausch B, Laessing U, Wiechers MF, Plattner H, Stuermer CA (1998) Identification of reggie-1 and reggie-2 as plasmamembrane-associated proteins which cocluster with activated GPI-anchored cell adhesion molecules in non-caveolar micropatches in neurons. J Neurobiol 37:502–523. pmid:9858255
    OpenUrlCrossRefPubMed
  66. ↵
    Lasley RD, Narayan P, Uittenbogaard A, Smart EJ (2000) Activated cardiac adenosine A(1) receptors translocate out of caveolae. J Biol Chem 275:4417–4421. pmid:10660613
    OpenUrlAbstract/FREE Full Text
  67. ↵
    Laughlin GA, Barrett-Connor E, Kritz-Silverstein D, von Muhlen D (2000) Hysterectomy, oophorectomy, and endogenous sex hormone levels in older women: the Rancho Bernardo Study. J Clin Endocrinol Metab 85:645–651. doi:10.1210/jcem.85.2.6405
    OpenUrlCrossRefPubMed
  68. ↵
    Lewis DL, Weight FF, Luini A (1986) A guanine nucleotide-binding protein mediates the inhibition of voltage-dependent calcium current by somatostatin in a pituitary cell line. Proc Natl Acad Sci U S A 83:9035–9039. doi:10.1073/pnas.83.23.9035
    OpenUrlAbstract/FREE Full Text
  69. ↵
    Link AJ, LaBaer J (2011) Trichloroacetic acid (TCA) precipitation of proteins. Cold Spring Harb Protoc 2011:993–994. doi:10.1101/pdb.prot5651 pmid:21807853
    OpenUrlCrossRefPubMed
  70. ↵
    Liu Y, Ding J, Bush TL, Longenecker JC, Nieto FJ, Golden SH, Szklo M (2001) Relative androgen excess and increased cardiovascular risk after menopause: a hypothesized relation. Am J Epidemiol 154:489–494. pmid:11549553
    OpenUrlCrossRefPubMed
  71. ↵
    Lotharius J, Barg S, Wiekop P, Lundberg C, Raymon HK, Brundin P (2002) Effect of mutant alpha-synuclein on dopamine homeostasis in a new human mesencephalic cell line. J Biol Chem 277:38884–38894. doi:10.1074/jbc.M205518200
    OpenUrlAbstract/FREE Full Text
  72. ↵
    Lu ML, Schneider MC, Zheng Y, Zhang X, Richie JP (2001) Caveolin-1 interacts with androgen receptor. A positive modulator of androgen receptor mediated transactivation. J Biol Chem 276:13442–13451. doi:10.1074/jbc.M006598200 pmid:11278309
    OpenUrlAbstract/FREE Full Text
  73. ↵
    Marquet-de Rougé P, Clamagirand C, Facchinetti P, Rose C, Sargueil F, Guihenneuc-Jouyaux C, Cynober L, Moinard C, Allinquant B (2013) Citrulline diet supplementation improves specific age-related raft changes in wild-type rodent hippocampus. Age (Dordr) 35:1589–1606. doi:10.1007/s11357-012-9462-2 pmid:22918749
    OpenUrlCrossRefPubMed
  74. ↵
    McEwen BS (1980) Binding and metabolism of sex steroids by the hypothalamic-pituitary unit: Physiological implications. Ann Rev Physiol 42:97–110. doi:10.1146/annurev.ph.42.030180.000525 pmid:6996603
    OpenUrlCrossRefPubMed
  75. ↵
    Mineo C, Gill GN, Anderson RG (1999) Regulated migration of epidermal growth factor receptor from caveolae. J Biol Chem 274:30636–30643. pmid:10521449
    OpenUrlAbstract/FREE Full Text
  76. ↵
    Modrek B, Lee C (2002) A genomic view of alternative splicing. Nat Genet 30:13–19. doi:10.1038/ng0102-13 pmid:11753382
    OpenUrlCrossRefPubMed
  77. ↵
    Mooradian AD, Morley JE, Korenman SG (1987) Biological actions of androgens. Endocr Rev 8:1–28. doi:10.1210/edrv-8-1-1 pmid:3549275
    OpenUrlCrossRefPubMed
  78. ↵
    Murata M, Peränen J, Schreiner R, Wieland F, Kurzchalia TV, Simons K (1995) VIP21/caveolin is a cholesterol-binding protein. Proc Natl Acad Sci U S A 92:10339–10343. pmid:7479780
    OpenUrlAbstract/FREE Full Text
  79. ↵
    Murthy KS, Makhlouf GM (2000) Heterologous desensitization mediated by G protein-specific binding to caveolin. J Biol Chem 275:30211–30219. doi:10.1074/jbc.M002194200
    OpenUrlAbstract/FREE Full Text
  80. ↵
    Nedungadi TP, Dutta M, Bathina CS, Caterina MJ, Cunningham JT (2012) Expression and distribution of TRPV2 in rat brain. Exp Neurol 237:223–237. doi:10.1016/j.expneurol.2012.06.017 pmid:22750329
    OpenUrlCrossRefPubMed
  81. ↵
    Oh P, Schnitzer JE (2001) Segregation of heterotrimeric G proteins in cell surface microdomains. G(q) binds caveolin to concentrate in caveolae, whereas G(i) and G(s) target lipid rafts by default. Mol Biol Cell 12:685–698. doi:10.1091/mbc.12.3.685
    OpenUrlAbstract/FREE Full Text
  82. ↵
    Ozanne DM, Brady ME, Cook S, Gaughan L, Neal DE, Robson CN (2000) Androgen receptor nuclear translocation is facilitated by the f-actin cross-linking protein filamin. Mol Endocrinol 14:1618–1626. doi:10.1210/mend.14.10.0541 pmid:11043577
    OpenUrlCrossRefPubMed
  83. ↵
    Parat MO, Fox PL (2001) Palmitoylation of caveolin-1 in endothelial cells is post-translational but irreversible. J Biol Chem 276:15776–15782. doi:10.1074/jbc.M006722200 pmid:11278313
    OpenUrlAbstract/FREE Full Text
  84. ↵
    Pedram A, Razandi M, Sainson RC, Kim JK, Hughes CC, Levin ER (2007) A conserved mechanism for steroid receptor translocation to the plasma membrane. J Biol Chem 282:22278–22288. doi:10.1074/jbc.M611877200 pmid:17535799
    OpenUrlAbstract/FREE Full Text
  85. ↵
    Pelley RP, Chinnakannu K, Murthy S, Strickland FM, Menon M, Dou QP, Barrack ER, Reddy GP (2006) Calmodulin-androgen receptor (AR) interaction: calcium-dependent, calpain-mediated breakdown of AR in LNCaP prostate cancer cells. Cancer Res 66:11754–11762. doi:10.1158/0008-5472.CAN-06-2918 pmid:17178871
    OpenUrlAbstract/FREE Full Text
  86. ↵
    Pike CJ, Rosario ER, Nguyen TV (2006) Androgens, aging, and Alzheimer’s disease. Endocrine 29:233–241. doi:10.1385/ENDO:29:2:233 pmid:16785599
    OpenUrlCrossRefPubMed
  87. ↵
    Pike CJ, Carroll JC, Rosario ER, Barron AM (2009) Protective actions of sex steroid hormones in Alzheimer’s disease. Front Neuroendocrinol 30:239–258. doi:10.1016/j.yfrne.2009.04.015 pmid:19427328
    OpenUrlCrossRefPubMed
  88. ↵
    Pucadyil TJ, Chattopadhyay A (2004) Cholesterol modulates ligand binding and G-protein coupling to serotonin(1A) receptors from bovine hippocampus. Biochim Biophys Acta 1663:188–200. doi:10.1016/j.bbamem.2004.03.010 pmid:15157621
    OpenUrlCrossRefPubMed
  89. ↵
    Purves-Tyson TD, Handelsman DJ, Double KL, Owens SJ, Bustamante S, Weickert CS (2012) Testosterone regulation of sex steroid-related mRNAs and dopamine-related mRNAs in adolescent male rat substantia nigra. BMC Neurosci 13:95doi:10.1186/1471-2202-13-95 pmid:22867132
    OpenUrlCrossRefPubMed
  90. ↵
    Ragonese P, D’Amelio M, Savettieri G (2006a) Implications for estrogens in Parkinson’s disease: an epidemiological approach. Ann N Y Acad Sci 1089:373–382. doi:10.1196/annals.1386.004 pmid:17261781
    OpenUrlCrossRefPubMed
  91. ↵
    Ragonese P, D’Amelio M, Callari G, Salemi G, Morgante L, Savettieri G (2006b) Age at menopause predicts age at onset of Parkinson’s disease. Mov Disord 21:2211–2214. doi:10.1002/mds.21127 pmid:17029261
    OpenUrlCrossRefPubMed
  92. ↵
    Rebois RV, Hébert TE (2003) Protein complexes involved in heptahelical receptor-mediated signal transduction. Receptors Channels 9:169–194. pmid:12775338
    OpenUrlCrossRefPubMed
  93. ↵
    Rhee SG (2001) Regulation of phosphoinositide-specific phospholipase C. Annu Rev Biochem 70:281–312. doi:10.1146/annurev.biochem.70.1.281 pmid:11395409
    OpenUrlCrossRefPubMed
  94. ↵
    Roberts GC, Smith CW (2002) Alternative splicing: combinatorial output from the genome. Curr Opin Chem Biol 6:375–383. pmid:12023119
    OpenUrlCrossRefPubMed
  95. ↵
    Rosario ER, Chang L, Head EH, Stanczyk FZ, Pike CJ (2011) Brain levels of sex steroid hormones in men and women during normal aging and in Alzheimer’s disease. Neurobiol Aging 32:604–613. doi:10.1016/j.neurobiolaging.2009.04.008 pmid:19428144
    OpenUrlCrossRefPubMed
  96. ↵
    Rybin VO, Xu X, Lisanti MP, Steinberg SF (2000) Differential targeting of beta-adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway. J Biol Chem 275:41447–41457. doi:10.1074/jbc.M006951200 pmid:11006286
    OpenUrlAbstract/FREE Full Text
  97. ↵
    Sar M, Lubahn DB, French FS, Wilson EM (1990) Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 127:3180–3186. doi:10.1210/endo-127-6-3180 pmid:1701137
    OpenUrlCrossRefPubMed
  98. ↵
    Sarkey S, Azcoitia I, Garcia-Segura LM, Garcia-Ovejero D, DonCarlos LL (2008) Classical androgen receptors in non-classical sites in the brain. Horm Behav 53:753–764. doi:10.1016/j.yhbeh.2008.02.015 pmid:18402960
    OpenUrlCrossRefPubMed
  99. ↵
    Schengrund CL (2010) Lipid rafts: keys to neurodegeneration. Brain Res Bull 82:7–17. doi:10.1016/j.brainresbull.2010.02.013 pmid:20206240
    OpenUrlCrossRefPubMed
  100. ↵
    Sengupta P, Philip F, Scarlata S (2008) Caveolin-1 alters Ca(2+) signal duration through specific interaction with the G alpha q family of G proteins. J Cell Sci 121:1363–1372. doi:10.1242/jcs.020081
    OpenUrlAbstract/FREE Full Text
  101. ↵
    Sherer TB, Betarbet R, Stout AK, Lund S, Baptista M, Panov AV, Cookson MR, Greenamyre JT (2002) An in vitro model of Parkinson’s disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage. J Neurosci 22:7006–7015.
    OpenUrlAbstract/FREE Full Text
  102. ↵
    Shi CS, Kehrl JH (2001) PYK2 links G(q)alpha and G(13)alpha signaling to NF-kappa B activation. J Biol Chem 276:31845–31850. doi:10.1074/jbc.M101043200 pmid:11435419
    OpenUrlAbstract/FREE Full Text
  103. ↵
    Smith DF, Toft DO (2008) The intersection of steroid receptors with molecular chaperones: observations and questions. Mol Endocrinol 22:2229–2240. doi:10.1210/me.2008-0089 pmid:18451092
    OpenUrlCrossRefPubMed
  104. ↵
    Smith KW, Feldman HA, McKinlay JB (2000) Construction and field validation of a self-administered screener for testosterone deficiency (hypogonadism) in ageing men. Clin Endocrinol (Oxf) 53:703–711. pmid:11155092
    OpenUrlCrossRefPubMed
  105. ↵
    Snyder B, Shell B, Cunningham JT, Cunningham RL (2017) Chronic intermittent hypoxia induces oxidative stress and inflammation in brain regions associated with early stage neurodegeneration. Physiol Rep 5:e13258.
  106. ↵
    Song CS, Rao TR, Demyan WF, Mancini MA, Chatterjee B, Roy AK (1991) Androgen receptor messenger ribonucleic acid (mRNA) in the rat liver: changes in mRNA levels during maturation, aging, and calorie restriction. Endocrinology 128:349–356. doi:10.1210/endo-128-1-349 pmid:1986927
    OpenUrlCrossRefPubMed
  107. ↵
    Song KS, Li S, Okamoto T, Quilliam LA, Sargiacomo M, Lisanti MP (1996) Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains. J Biol Chem 271:9690–9697. doi:10.1074/jbc.271.16.9690
    OpenUrlAbstract/FREE Full Text
  108. ↵
    Sonnino S, Aureli M, Grassi S, Mauri L, Prioni S, Prinetti A (2014) Lipid rafts in neurodegeneration and neuroprotection. Mol Neurobiol 50:130–148. doi:10.1007/s12035-013-8614-4 pmid:24362851
    OpenUrlCrossRefPubMed
  109. ↵
    Stamm S (2002) Signals and their transduction pathways regulating alternative splicing: a new dimension of the human genome. Hum Mol Genet 11:2409–2416. pmid:12351576
    OpenUrlCrossRefPubMed
  110. ↵
    Steinsapir J, Socci R, Reinach P (1991) Effects of androgen on intracellular calcium of LNCaP cells. Biochem Biophys Res Commun 179:90–96. pmid:1883394
    OpenUrlCrossRefPubMed
  111. ↵
    Sun S, Sprenger CC, Vessella RL, Haugk K, Soriano K, Mostaghel EA, Page ST, Coleman IM, Nguyen HM, Sun H, Nelson PS, Plymate SR (2010) Castration resistance in human prostate cancer is conferred by a frequently occurring androgen receptor splice variant. J Clin Invest 120:2715–2730. doi:10.1172/JCI41824 pmid:20644256
    OpenUrlCrossRefPubMed
  112. ↵
    Surmeier DJ, Guzman JN, Sanchez J, Schumacker PT (2012) Physiological phenotype and vulnerability in Parkinson’s disease. Cold Spring Harb Perspect Med 2:a009290. doi:10.1101/cshperspect.a009290 pmid:22762023
    OpenUrlAbstract/FREE Full Text
  113. ↵
    Thomas TR, Neiman CN (1968) Aspects of copulatory behavior preventing atrophy in male rats’ reproductive system. Endocrinology 83:633–635. doi:10.1210/endo-83-3-633 pmid:5674032
    OpenUrlCrossRefPubMed
  114. ↵
    Toki S, Donati RJ, Rasenick MM (1999) Treatment of C6 glioma cells and rats with antidepressant drugs increases the detergent extraction of G(s alpha) from plasma membrane. J Neurochem 73:1114–1120. doi:10.1046/j.1471-4159.1999.0731114.x
    OpenUrlCrossRefPubMed
  115. ↵
    Trushina E, Du Charme J, Parisi J, McMurray CT (2006) Neurological abnormalities in caveolin-1 knock out mice. Behav Brain Res 172:24–32. doi:10.1016/j.bbr.2006.04.024 pmid:16750274
    OpenUrlCrossRefPubMed
  116. ↵
    Urban JD, Mailman RB (2005) Characterization of the N27 dopaminergic cell line as a potential model for elucidating the mechanisms of action of functionally selective dopamienrgic ligands. In: Society for Neuroscience, Program No. 33.21. Washington DC: Society for Neuroscience.
  117. ↵
    Verdile G, Laws SM, Henley D, Ames D, Bush AI, Ellis KA, Faux NG, Gupta VB, Li QX, Masters CL, Pike KE, Rowe CC, Szoeke C, Taddei K, Villemagne VL, Martins RN (2014) Associations between gonadotropins, testosterone and beta amyloid in men at risk of Alzheimer’s disease. Mol Psychiatry 19:69–75. doi:10.1038/mp.2012.147
    OpenUrlCrossRefPubMed
  118. ↵
    Vermeulen A (1976) The hormonal activity of the postmenopausal ovary. J Clin Endocrinol Metab 42:247–253. doi:10.1210/jcem-42-2-247 pmid:177438
    OpenUrlCrossRefPubMed
  119. ↵
    Volonté D, Galbiati F, Pestell RG, Lisanti MP (2001) Cellular stress induces the tyrosine phosphorylation of caveolin-1 (Tyr(14)) via activation of p38 mitogen-activated protein kinase and c-Src kinase. Evidence for caveolae, the actin cytoskeleton, and focal adhesions as mechanical sensors of osmotic stress. J Biol Chem 276:8094–8103. doi:10.1074/jbc.M009245200 pmid:11094059
    OpenUrlAbstract/FREE Full Text
  120. ↵
    Watson PA, Chen YF, Balbas MD, Wongvipat J, Socci ND, Viale A, Kim K, Sawyers CL (2010) Constitutively active androgen receptor splice variants expressed in castration-resistant prostate cancer require full-length androgen receptor. Proc Natl Acad Sci U S A 107:16759–16765. doi:10.1073/pnas.1012443107 pmid:20823238
    OpenUrlAbstract/FREE Full Text
  121. ↵
    Wehinger S, Ortiz R, Díaz MI, Aguirre A, Valenzuela M, Llanos P, Mc Master C, Leyton L, Quest AF (2015) Phosphorylation of caveolin-1 on tyrosine-14 induced by ROS enhances palmitate-induced death of beta-pancreatic cells. Biochim Biophys Acta 1852:693–708. doi:10.1016/j.bbadis.2014.12.021
    OpenUrlCrossRef
  122. ↵
    Weiss B, Faus H, Haendler B (2007) Phylogenetic conservation of the androgen receptor AR45 variant form in placental mammals. Gene 399:105–111. doi:10.1016/j.gene.2007.04.037 pmid:17574777
    OpenUrlCrossRefPubMed
  123. ↵
    Worley PF, Baraban JM, Van Dop C, Neer EJ, Snyder SH (1986) Go, a guanine nucleotide-binding protein: immunohistochemical localization in rat brain resembles distribution of second messenger systems. Proc Natl Acad Sci U S A 83:4561–4565. pmid:3086888
    OpenUrlAbstract/FREE Full Text
  124. ↵
    Wu ZY, Chen K, Haendler B, McDonald TV, Bian JS (2008) Stimulation of N-terminal truncated isoform of androgen receptor stabilizes human ether-a-go-go-related gene-encoded potassium channel protein via activation of extracellular signal regulated kinase 1/2. Endocrinology 149:5061–5069. doi:10.1210/en.2007-1802 pmid:18599551
    OpenUrlCrossRefPubMed
  125. ↵
    Xiao L, Jordan CL (2002) Sex differences, laterality, and hormonal regulation of androgen receptor immunoreactivity in rat hippocampus. Horm Behav 42:327–336. pmid:12460592
    OpenUrlCrossRefPubMed
  126. ↵
    Xu W, Yoon SI, Huang P, Wang Y, Chen C, Chong PL, Liu-Chen LY (2006) Localization of the kappa opioid receptor in lipid rafts. J Pharmacol Exp Ther 317:1295–1306. doi:10.1124/jpet.105.099507 pmid:16505160
    OpenUrlAbstract/FREE Full Text
  127. ↵
    Yang X, Guo Z, Sun F, Li W, Alfano A, Shimelis H, Chen M, Brodie AM, Chen H, Xiao Z, Veenstra TD, Qiu Y (2011) Novel membrane-associated androgen receptor splice variant potentiates proliferative and survival responses in prostate cancer cells. J Biol Chem 286:36152–36160. doi:10.1074/jbc.M111.265124
    OpenUrlAbstract/FREE Full Text
  128. ↵
    Zitzmann M, Brune M, Nieschlag E (2002) Vascular reactivity in hypogonadal men is reduced by androgen substitution. J Clin Endocrinol Metab 87:5030–5037. doi:10.1210/jc.2002-020504 pmid:12414868
    OpenUrlCrossRefPubMed

Synthesis

Reviewing Editor: Jeffrey Blaustein, University of Massachusetts

Decisions are customarily a result of the Reviewing Editor and the peer reviewers coming together and discussing their recommendations until a consensus is reached. When revisions are invited, a fact-based synthesis statement explaining their decision and outlining what is needed to prepare a revision will be listed below. The following reviewer(s) agreed to reveal their identity: Toni Pak

Although I am accepting as is, when you get the proofs, please make the following corrections:

1. line 245, identify syngene Gbox.

2. line 259: estrone misspelled

I think this will be more expedient than sending it back to you now.

Back to top

In this issue

eneuro: 4 (4)
eNeuro
Vol. 4, Issue 4
July/August 2017
  • Table of Contents
  • Index by author
Email

Thank you for sharing this eNeuro article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Presence of Androgen Receptor Variant in Neuronal Lipid Rafts
(Your Name) has forwarded a page to you from eNeuro
(Your Name) thought you would be interested in this article in eNeuro.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Print
View Full Page PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Citation Tools
Presence of Androgen Receptor Variant in Neuronal Lipid Rafts
Jo Garza-Contreras, Phong Duong, Brina D. Snyder, Derek A. Schreihofer, Rebecca L. Cunningham
eNeuro 22 August 2017, 4 (4) ENEURO.0109-17.2017; DOI: 10.1523/ENEURO.0109-17.2017

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Respond to this article
Share
Presence of Androgen Receptor Variant in Neuronal Lipid Rafts
Jo Garza-Contreras, Phong Duong, Brina D. Snyder, Derek A. Schreihofer, Rebecca L. Cunningham
eNeuro 22 August 2017, 4 (4) ENEURO.0109-17.2017; DOI: 10.1523/ENEURO.0109-17.2017
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Significance Statement
    • Introduction
    • Materials and Methods
    • Results
    • Discussion
    • Acknowledgments
    • Footnotes
    • References
    • Synthesis
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF

Keywords

  • Androgen Receptor
  • Caveolin
  • G Proteins
  • Lipid Rafts
  • Membrane
  • signaling

Responses to this article

Respond to this article

Jump to comment:

No eLetters have been published for this article.

Related Articles

Cited By...

More in this TOC Section

New Research

  • Postnatal fluoxetine treatment alters perineuronal net formation and maintenance in the hippocampus
  • The Contribution of Environmental Enrichment to Phenotypic Variation in Mice and Rats
  • Unique effects of social defeat stress in adolescent male mice on the Netrin-1/DCC pathway, prefrontal cortex dopamine and cognition (Social stress in adolescent vs. adult male mice)
Show more New Research

Integrative Systems

  • Postnatal fluoxetine treatment alters perineuronal net formation and maintenance in the hippocampus
  • The Contribution of Environmental Enrichment to Phenotypic Variation in Mice and Rats
  • Unique effects of social defeat stress in adolescent male mice on the Netrin-1/DCC pathway, prefrontal cortex dopamine and cognition (Social stress in adolescent vs. adult male mice)
Show more Integrative Systems

Subjects

  • Integrative Systems
  • Home
  • Alerts
  • Visit Society for Neuroscience on Facebook
  • Follow Society for Neuroscience on Twitter
  • Follow Society for Neuroscience on LinkedIn
  • Visit Society for Neuroscience on Youtube
  • Follow our RSS feeds

Content

  • Early Release
  • Current Issue
  • Latest Articles
  • Issue Archive
  • Blog
  • Browse by Topic

Information

  • For Authors
  • For the Media

About

  • About the Journal
  • Editorial Board
  • Privacy Policy
  • Contact
  • Feedback
(eNeuro logo)
(SfN logo)

Copyright © 2021 by the Society for Neuroscience.
eNeuro eISSN: 2373-2822

The ideas and opinions expressed in eNeuro do not necessarily reflect those of SfN or the eNeuro Editorial Board. Publication of an advertisement or other product mention in eNeuro should not be construed as an endorsement of the manufacturer’s claims. SfN does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of any material contained in eNeuro.