Mutant Androgen Receptor Research Articles

Male fertility in mice requires classical and nonclassical androgen signaling.

Molecular mechanisms by which androgens signal through the androgen receptor (AR) to maintain male fertility are poorly understood. Transgenic mice were produced expressing mutant ARs that can only (1) alter gene transcription through the classical response pathway (AR-C) or (2) activate kinase signaling cascades via the nonclassical pathway (AR-NC). AR-C is sufficient to produce sperm and fertility. Haploid germ cell production, the blood-testis barrier, and spermatid migration are supported by AR-NC. Gene expression essential for chromosome synapsis during meiosis requires AR-C. We identify targets of androgen signaling required for male fertility and provide a mechanistic explanation for meiotic germ cell arrest in the absence of androgen signaling. Prostate differentiation occurs with AR-C alone, but full development requires synergistic nonclassical signaling. Both AR signaling pathways are necessary for normal male reproductive tract development and function, validating our mouse models for studies of AR functions in other target tissues.

Functional Effects In Silico Prediction for Androgen Receptor Ligand-Binding Domain Novel I836S Mutation.

Over 1000 mutations are described in the androgen receptor (AR) gene. Of those, about 600 were found in androgen insensitivity syndrome (AIS) patients, among which 400 mutations affect the ligand-binding domain (LBD) of the AR protein. Recently, we reported a novel missense mutation c.2507T>G I836S (ClinVarID: 974911) in a patient with complete AIS (CAIS) phenotype. In the present study, we applied a set of computational approaches for the structural analysis of the ligand-binding domains in a wild-type and mutant AR to evaluate the functional impact of the novel I836S mutation. We revealed that the novel I836S substitution leads to a shorter existence time of the ligand’s gating tunnel and internal cavity, occurring only in the presence of S836 phosphorylation. Additionally, the analysis of phosphorylation of the 836 mutant residues explained the negative impact on AR homodimerization, since monomer surface changes indirectly impacted the binding site. Our analyses provide evidence that I836S causes disruptions of AR protein functionality and development of CAIS clinical features in patients.

Evaluation of Darolutamide (ODM201) Efficiency on Androgen Receptor Mutants Reported to Date in Prostate Cancer Patients.

Simple SummaryProstate cancer (PCa) is the most commonly diagnosed non-skin cancer in men and one of the leading causes of cancer-related death. The driver of PCa proliferation and growth is the androgen receptor (AR) and inhibiting this receptor is the standard of care for patients, following surgery or radiotherapy. Unfortunately, the effectiveness of current therapeutics is temporary, with the cancer eventually developing drug resistance. Among the mechanisms of resistance are the arising mutations in the AR that make the receptor promiscuously activated by drugs or non-specific ligands, thus promoting cancer progression. The aim of this study is to characterize the responses of 44 AR mutants, derived from PCa patients, to available steroids that activate the receptor as well as to various treatments currently used in the clinic. This work will help create a tool to guide the medical team in selecting the best personalized treatment option for each patient.Resistance to drug treatments is common in prostate cancer (PCa), and the gain-of-function mutations in human androgen receptor (AR) represent one of the most dominant drivers of progression to resistance to AR pathway inhibitors (ARPI). Previously, we evaluated the in vitro response of 24 AR mutations, identified in men with castration-resistant PCa, to five AR antagonists. In the current work, we evaluated 44 additional PCa-associated AR mutants, reported in the literature, and thus expanded the study of the effect of darolutamide to a total of 68 AR mutants. Unlike other AR antagonists, we demonstrate that darolutamide exhibits consistent efficiency against all characterized gain-of-function mutations in a full-length AR. Additionally, the response of the AR mutants to clinically used bicalutamide and enzalutamide, as well as to major endogenous steroids (DHT, estradiol, progesterone and hydrocortisone), was also investigated. As genomic profiling of PCa patients becomes increasingly feasible, the developed “AR functional encyclopedia” could provide decision-makers with a tool to guide the treatment choice for PCa patients based on their AR mutation status.

Analysis of the binding modes of the first- and second-generation antiandrogens with respect to F876L mutation.

Androgen receptor (AR) is an important target for the treatment of prostate cancer, and mutations in the AR have an important impact on the resistance of existing drugs. In this work, we performed molecular dynamics simulations of the existing marketed antiandrogens flutamide, nilutamide, bicalutamide, enzalutamide, apalutamide, darolutamide, and its main metabolite ORM15341 in complex with the wild-type and F876L mutant AR. We calculated the residue-specific binding free energy contribution of the wild-type and mutant ARs with the AS-IE method and analyzed the hotspot residues and the binding free energy contributions of specific residues before and after the mutation. In addition, we analyzed the total binding obtained by adding residue binding energy contributions and compared the results with experimental values. The obtained residue-specific binding information should be very helpful in understanding the mechanism of drug resistance with respect to specific mutations and in the design of new generation drugs against possible new mutations.

Selective suppression of polyglutamine-expanded protein by lipid nanoparticle-delivered siRNA targeting CAG expansions in the mouse CNS

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by expansion of cytosine-adenine-guanine (CAG)-trinucleotide repeats in causative genes. These diseases include spinal and bulbar muscular atrophy (SBMA), Huntington’s disease, dentatorubral-pallidoluysian atrophy, and spinocerebellar ataxias. Targeting expanded CAG repeats is a common therapeutic approach to polyQ diseases, but concomitant silencing of genes with normal CAG repeats may lead to toxicity. Previous studies have shown that CAG repeat-targeting small interfering RNA duplexes (CAG-siRNAs) have the potential to selectively suppress mutant proteins in in vitro cell models of polyQ diseases. However, in vivo application of these siRNAs has not yet been investigated. In this study, we demonstrate that an unlocked nucleic acid (UNA)-modified CAG-siRNA shows high selectivity for polyQ-expanded androgen receptor (AR) inhibition in in vitro cell models and that lipid nanoparticle (LNP)-mediated delivery of the CAG-siRNA selectively suppresses mutant AR in the central nervous system of an SBMA mouse model. In addition, a subcutaneous injection of the LNP-delivered CAG-siRNA efficiently suppresses mutant AR in the skeletal muscle of the SBMA mouse model. These results support the therapeutic potential of LNP-delivered UNA-modified CAG-siRNAs for selective suppression of mutant proteins in SBMA and other polyQ diseases.

V-ATPase Inhibition Decreases Mutant Androgen Receptor Activity in Castrate-resistant Prostate Cancer.

Prostate cancer is critically dependent on androgen receptor (AR) signaling. Despite initial responsiveness to androgen deprivation, most patients with advanced prostate cancer subsequently progress to a clinically aggressive castrate-resistant prostate cancer (CRPC) phenotype, typically associated with expression of splice-variant or mutant AR forms. Although current evidence suggests that the vacuolar-ATPase (V-ATPase), a multiprotein complex that catalyzes proton transport across intracellular and plasma membranes, influences wild-type AR function, the effect of V-ATPase inhibition on variant AR function is unknown.Inhibition of V-ATPase reduced AR function in wild-type and mutant AR luciferase reporter models. In hormone-sensitive prostate cancer cell lines (LNCaP, DuCaP) and mutant AR CRPC cell lines (22Rv1, LNCaP-F877L/T878A), V-ATPase inhibition using bafilomycin-A1 and concanamycin-A reduced AR expression, and expression of AR target genes, at mRNA and protein levels. Furthermore, combining chemical V-ATPase inhibition with the AR antagonist enzalutamide resulted in a greater reduction in AR downstream target expression than enzalutamide alone in LNCaP cells. To investigate the role of individual subunit isoforms, siRNA and CRISPR-Cas9 were used to target the V1C1 subunit in 22Rv1 cells. Whereas transfection with ATP6V1C1-targeted siRNA significantly reduced AR protein levels and function, CRISPR-Cas9-mediated V1C1 knockout showed no substantial change in AR expression, but a compensatory increase in protein levels of the alternate V1C2 isoform.Overall, these results indicate that V-ATPase dysregulation is directly linked to both hormone-responsive prostate cancer and CRPC via impact on AR function. In particular, V-ATPase inhibition can reduce AR signaling regardless of mutant AR expression.

Male Fertility Requires Classical and Nonclassical Androgen Signaling

The molecular mechanisms by which androgens act via the androgen receptor (AR) to maintain male fertility are poorly understood. Transgenic mice were produced expressing mutant ARs that can only (1) alter gene transcription through the classical response pathway (AR-C) or 2) activate kinase signaling cascades via the nonclassical pathway (AR-NC). AR-NC allowed completion of meiosis and the production of haploid germ cells. Expression of AR-C was sufficient to produce sperm and fertility. However, added AR-NC activity contributed to maintaining the blood-testis barrier, plus correct migration and orientation of maturing spermatids needed for fully efficient sperm production. AR-C activity is required for expression of cohesion and synaptonemal complex proteins that are essential for chromosome synapsis. AR-C supported partial development of prostate and coagulating glands, but full development required both pathways. Our results indicate that AR-C activity alone partially restores male fertility, but AR-NC is obligatory to maximize fertility.

Deubiquitinase USP7 contributes to the pathogenicity of spinal and bulbar muscular atrophy.

Polyglutamine (polyQ) diseases are devastating, slowly progressing neurodegenerative conditions caused by expansion of polyQ-encoding CAG repeats within the coding regions of distinct, unrelated genes. In spinal and bulbar muscular atrophy (SBMA), polyQ expansion within the androgen receptor (AR) causes progressive neuromuscular toxicity, the molecular basis of which is unclear. Using quantitative proteomics, we identified changes in the AR interactome caused by polyQ expansion. We found that the deubiquitinase USP7 preferentially interacts with polyQ-expanded AR and that lowering USP7 levels reduced mutant AR aggregation and cytotoxicity in cell models of SBMA. Moreover, USP7 knockdown suppressed disease phenotypes in SBMA and spinocerebellar ataxia type 3 (SCA3) fly models, and monoallelic knockout of Usp7 ameliorated several motor deficiencies in transgenic SBMA mice. USP7 overexpression resulted in reduced AR ubiquitination, indicating the direct action of USP7 on AR. Using quantitative proteomics, we identified the ubiquitinated lysine residues on mutant AR that are regulated by USP7. Finally, we found that USP7 also differentially interacts with mutant Huntingtin (HTT) protein in striatum and frontal cortex of a knockin mouse model of Huntington's disease. Taken together, our findings reveal a critical role for USP7 in the pathophysiology of SBMA and suggest a similar role in SCA3 and Huntington's disease.

Role of SPTSSB-Regulated de Novo Sphingolipid Synthesis in Prostate Cancer Depends on Androgen Receptor Signaling.

SummaryAnti-androgens are a common therapy in prostate cancer (PCa) targeting androgen receptor (AR) signaling. However, these therapies fail due to selection of highly aggressive AR-negative cancer cells that have no therapeutic options available. We demonstrate that elevating endogenous ceramide levels with administration of exogenous ceramide nanoliposomes (CNLs) was efficacious in AR-negative cell lines with limited efficacy in AR-positive cells. This effect is mediated through reduced de novo sphingolipid synthesis in AR-positive cells. We show that anti-androgens elevate de novo generation of sphingolipids via SPTSSB, a rate-limiting mediator of sphingolipid generation. Moreover, pharmacological inhibition of AR increases the efficacy of CNL in AR-positive cells through de novo synthesis, while SPTSSB knockdown limited CNL's efficacy in AR-negative cells. Alluding to clinical relevance, SPTSSB is upregulated in patients with advanced PCa after anti-androgens treatment. These findings emphasize the relevance of AR regulation upon sphingolipid metabolism and the potential of CNL as a PCa therapeutic.

Virtual Screening and Bioactivity Evaluation of Novel Androgen Receptor Antagonists From Anti-PCa Traditional Chinese Medicine Prescriptions.

Prostate cancer (PCa), a type of malignancy that arises in the prostate gland, is the most commonly diagnosed neoplasm and the second leading cause of cancer-related deaths in men. Acquisition of resistance to conventional therapy is a major problem for PCa patient treatment. Androgen receptor (AR) signaling pathway is necessary in the pathogenesis of prostate cancer, and there is a heightened interest in finding novel AR antagonists that target AR and its regulatory pathways. In our search for novel androgen receptor antagonists, we focus on the Traditional Chinese Medicine (TCM), which has been used for thousands of years to prove effective in the treatment of cancer. In this study, we collected 653 traditional Chinese medicine prescriptions that have certain therapeutic effect to prostate cancer, including the prescriptions and even the folk prescriptions. After summarizing the frequency of herbs and gathering the natural products contained in these prescriptions, we built a natural products database to do computer-aided virtual screening and drug-like evaluation to find potential AR antagonists. Totally 25 compounds were submitted to experimental biological activity tests. Through the MTT cell proliferation experiment, 5 chemicals were found to inhibit the proliferation of LNCaP cells in a concentration-dependent manner. Especially, MoL_11 was found to have good antagonistic activity and significantly inhibit fluorescence enzyme activity by the AR reporter gene experiment. Finally, the molecular dynamics simulation method was used to study the interaction between the most active compound MoL_11 and the wild-type and F876L mutant androgen receptor (WT/F876L AR), and it was found that F876L AR could not cause resistance to MoL_11.

Darolutamide as a Second-Generation Androgen Receptor Inhibitor in the Treatment of Prostate Cancer.

Prostate cancer (PC) is known as the most frequent cancer among men in the world. Androgen Deprivation Therapy (ADT) is one of the initial treatment approaches in the PC therapy and various drugs can be used in routine Hormonal therapy for PC therapy. Nevertheless, PC cells can survive and continue their growth via different mechanisms which lead to their resistance to common treatments i.e., Enzalutamide. olutamide (ODM-201) is a second-generation androgen receptor (AR) inhibitor with a new chemical structure and has a high affinity to the AR. Darolutamide does not cross the blood-brain barrier and for this reason, reduces the possibility of seizures. Darolutamide can also inhibit the transcriptional activity of several AR mutant variants (F877L, F877L/T878A, and H875Y/T878A), which are Enzalutamide resistant. In this review, we reviewed the results of different studies: in vitro, animal model and phase 1, 2 and 3 clinical trials (ARADES, ARAFOR and ARAMIS). We shall discuss worldwide phase 2 and 3 clinical trials (ARASENS and ODENZA) that are in progress, in order to demonstrate the advantages of Darolutamide consumption in different groups of patients. Darolutamide has shown high potential in inhibiting the growth of MR49F (Enzalutamide resistant PC cells) and VCaP (Castration-resistant PC cells) cell lines and transcriptional activities of AR. Fewer doses of Darolutamide are needed compared to Enzalutamide. The drug had significant anti-tumor activity and no effect on serum testosterone levels in animal models. Darolutamide demonstrates its safety and efficacy in different studies and was well tolerated nearly in all of the patients.

Molecular pathogenesis of spinal bulbar muscular atrophy (Kennedy's disease) and avenues for treatment.

The aim of this study was to illustrate the current understanding and avenues for developing treatment in spinal and bulbar muscular atrophy (SBMA), an inherited neuromuscular disorder caused by a CAG trinucleotide repeat expansion in the androgen receptor (AR) gene. Important advances have been made in characterizing the molecular mechanism of the disease, including the disruption of protein homeostasis, intracellular trafficking and signalling pathways. Biomarkers such as MRI quantification of muscle volume and fat fraction have been used to track disease progression, and will be useful in future clinical studies. Therapies tested and under development have been based on diverse strategies, including targeting mutant AR gene expression, stability and activity, and pathways that mitigate disease toxicity. We provide an overview of the recent advances in understanding the SBMA disease mechanism and highlight efforts to translate these insights into well tolerated and effective therapy.

Deep Learning Modeling of Androgen Receptor Responses to Prostate Cancer Therapies.

Gain-of-function mutations in human androgen receptor (AR) are among the major causes of drug resistance in prostate cancer (PCa). Identifying mutations that cause resistant phenotype is of critical importance for guiding treatment protocols, as well as for designing drugs that do not elicit adverse responses. However, experimental characterization of these mutations is time consuming and costly; thus, predictive models are needed to anticipate resistant mutations and to guide the drug discovery process. In this work, we leverage experimental data collected on 68 AR mutants, either observed in the clinic or described in the literature, to train a deep neural network (DNN) that predicts the response of these mutants to currently used and experimental anti-androgens and testosterone. We demonstrate that the use of this DNN, with general 2D descriptors, provides a more accurate prediction of the biological outcome (inhibition, activation, no-response, mixed-response) in AR mutant-drug pairs compared to other machine learning approaches. Finally, the developed approach was used to make predictions of AR mutant response to the latest AR inhibitor darolutamide, which were then validated by in-vitro experiments.

Computational analysis of androgen receptor (AR) variants to decipher the relationship between protein stability and related-diseases

Although more than 1,000 androgen receptor (AR) mutations have been identified and these mutants are pathologically important, few theoretical studies have investigated the role of AR protein folding stability in disease and its relationship with the phenotype of the patients. Here, we extracted AR variant data from four databases: ARDB, HGMD, Cosmic, and 1,000 genome. 905 androgen insensitivity syndrome (AIS)-associated loss-of-function mutants and 168 prostate cancer-associated gain-of-function mutants in AR were found. We analyzed the effect of single-residue variation on the folding stability of AR by FoldX and guanidine hydrochloride denaturation experiment, and found that genetic disease-associated mutations tend to have a significantly greater effect on protein stability than gene polymorphisms. Moreover, AR mutants in complete androgen insensitivity syndrome (CAIS) tend to have a greater effect on protein stability than in partial androgen insensitive syndrome (PAIS). This study, by linking disease phenotypes to changes in AR stability, demonstrates the importance of protein stability in the pathogenesis of hereditary disease.

Enhanced Clearance of Neurotoxic Misfolded Proteins by the Natural Compound Berberine and Its Derivatives.

Background: Accumulation of misfolded proteins is a common hallmark of several neurodegenerative disorders (NDs) which results from a failure or an impairment of the protein quality control (PQC) system. The PQC system is composed by chaperones and the degradative systems (proteasome and autophagy). Mutant proteins that misfold are potentially neurotoxic, thus strategies aimed at preventing their aggregation or at enhancing their clearance are emerging as interesting therapeutic targets for NDs. Methods: We tested the natural alkaloid berberine (BBR) and some derivatives for their capability to enhance misfolded protein clearance in cell models of NDs, evaluating which degradative pathway mediates their action. Results: We found that both BBR and its semisynthetic derivatives promote degradation of mutant androgen receptor (ARpolyQ) causative of spinal and bulbar muscular atrophy, acting mainly via proteasome and preventing ARpolyQ aggregation. Overlapping effects were observed on other misfolded proteins causative of amyotrophic lateral sclerosis, frontotemporal-lobar degeneration or Huntington disease, but with selective and specific action against each different mutant protein. Conclusions: BBR and its analogues induce the clearance of misfolded proteins responsible for NDs, representing potential therapeutic tools to counteract these fatal disorders.

SUN-028 Concomitant Mutations in the POR and AR Genes in a Boy Presenting with Micropenis and Premature Adrenarche

Background: Micropenis is caused either by a defect in androgen synthesis, conversion to Dihydrotestosterone, or action due to mutant androgen receptor. Premature adrenarche is caused by excess of adrenal androgens. Clinical case: A 6,5-year-old boy presented to our pediatric endocrinology clinic due to increased body weight and hyperthyreotropinemia. Physical examination revealed premature adrenarche with testicular volume 1,5 ml, micropenis (4cm, <-2 SDS) and acanthosis nigricans. His linear growth presented an acceleration the last 2 years from +0.85 to +1.81 SD and his bone age was advanced by 2 years with a statural height at -0.5 SD, not in accordance with the mean parental height (+0.90 SD). At the same time, his BMI was increased from +1,36 to +3,64 SDS.Laboratory investigations for premature adrenarche with synacthen test revealed mild elevation of compound-S 15,4 ng/dl at 60’, which could be theoretically attributed to partial 11-β-hydroxylase deficiency (CYP11B1). DHEA was also elevated 2,1 ng/ml with an increased DHEA/Δ4 ratio at 20 (normal < 10) which could be explained in the case of partial 3-β-Hydroxysteroid dehydrogenase deficiency. Method: Whole exome sequencing was preformed targeted to a gene panel related to premature adrenarche. Results: A heterozygous mutation c.[1174C>T]+[1174C>T]; p.[Pro329Ser]+[Pro329Ser] in exon 1 in Androgen Receptor (AR) gene (X-linked) was found to the patient and his mother. The mutation, according to Human Gene Mutation Database, can cause disorder of sex development (DSD), partial androgen insensitivity syndrome (PAIS), infertility and hypospadias. Additionally, sequencing of the coding region of the P450 oxyreductase (POR) gene revealed a heterozygous mutation c.[642-5C>G] in exon 7, which in homozygosity can cause steroidogenesis disorder due to oxyreductase deficiency. This heterozygous POR mutation was found to the patient and his father. Discussion: Our patient caries mutations in the AR and in POR genes. The AR mutation is obviously responsible for micropenis in our patient. CYP11B1 and 3β-Hydroxysteroid dehydrogenase genes were normal. POR deficiency is a disorder of steroidogenesis with phenotypic spectrum ranging from PCOS to ambiguous genitalia and glycocorticosteroid deficiency or even to classic Antley-Bixler syndrome (1). Conclusion: The genetic and hormonal results in our case imply a role for POR in the CYP11B1 gene expression, which is reported for the first time to our knowledge.1.Concomitant Mutations in the P450 Oxidoreductase and Androgen Receptor Genes Presenting with 46,XY Disordered Sex Development and Androgenization at Adrenarche. J Clin Endocrinol Metab. 2010 Jul; 95(7): 3418-3427

SAT-712 Computational Study of the Effect of Androgen Receptor BF 3 Site Mutations on DDE Binding

Exposure to endocrine disrupting chemicals (EDCs) affects the function of the androgen receptor (AR) causing reproductive system problems such as reduced sperm counts, increased infertility, testicular dysgenesis syndrome, and testicular and prostate cancers, as well as reduced bone mass and diabetes mellitus in males. Experimental results have shown that the presence of EDCs such as the diphenyl compound DDT and its analogue DDE, allosterically cause the release of the stably bound dihydrotestosterone (DHT) from the steroid binding site of the AR ligand binding domain. It was hypothesized that EDCs mediate this effect via binding to the Binding Function 3 (BF 3) surface binding site. Five mutations of three BF 3 amino acids (F673K, F673W, G724R, G724M, and L830D) showed that the ability of DDE to inhibit AR activity was reduced, suggesting that DDE binds to the BF 3 site and allosterically regulates AR activity. In this study, the Induced Fit Docking protocol of the Schrodinger software was used to dock DDE into the BF 3 site of the wild type AR ligand binding domain as well as the five mutant BF 3 sites. The docking poses generated for each receptor were clustered and representative structures were selected. The receptor-ligand complexes were energy minimized using the Schrodinger module Macromodel. Finally, the energy of interaction between DDE and the BF 3 site amino acids was evaluated for each of the selected docks of the wild type and mutant receptors. The relationship between the energies of interaction and the experimental results for DDE inhibition of the mutant AR activities will be discussed.

SUN-LB138 Dynamic Structural Model of Testosterone Entry Into the Unliganded Androgen Receptor

Background: Crystallographic structures of nuclear receptor ligand binding domains provide a static model of a receptor stably wrapped around an internalized ligand. Understanding the dynamics of a receptor at different stages of ligand binding has been hampered by the paucity of crystal structures for unliganded nuclear receptors. Molecular dynamic models have been constructed for some nuclear receptors to fill that void. Methods: The molecular simulation docking program MORDOR (MOlecular Recognition with a Driven dynamics OptimizeR)(1) was used to study the structural dynamics of the androgen receptor ligand binding domain (AR LBD) modeled from the static structure of the AR LBD bound to testosterone (T) (PDB ID: 2AM9). The goals of the study were to understand a) the dynamic interaction of the T in its binding pocket, b) AR LBD structural flexibilities that permit T entry/exit from the binding pocket and c) a model of the unliganded AR LBD. Results: Modeling AR LBD structure flexibility over time revealed possible alternative dynamic structures, including those without ligand, overlaid against the canonical nuclear receptor structure. The model dynamically tracks the structural changes as a ligand enters into the ligand binding domain and nestles into the ligand binding pocket. The model predicted the appearance of alpha helices within the AR LBD that transiently fold/unfold during the ligand entry phases. Once in the pocket, the ligand itself remains very dynamic in a still flexible pocket. The model predicted also AR LBD amino acids that sequentially interact with the ligand during its dynamic entry into the AR LBD. Intriguingly, those AR amino acids include those mutated in castration-resistant prostate tumors that continue to grow during androgen suppression therapy. Functional studies showed those mutant ARs had a primary consequence of enhancing response to lower level T, and other androgens, consistent with their role in creating a higher affinity AR that can scavenge low-level androgens in an androgen-suppressed patient. Conclusions: The molecular model of T binding to the AR LBD suggests a degree of structural dynamism not evident in the crystallographic structures commonly associated with nuclear receptors. Some AR mutations activating prostate tumor growth may do so by impacting androgen entry/exit, rather than by altering androgen fit into the ligand binding pocket. Reference: (1) Guilbert C, James TL (2008) J Chem Inf Model. 2008 48(6): 1257-1268. doi: 10.1021/ci8000327

THE PHYSIOPATHOLOGICAL ROLES OF ANDROGENS IN MOTONEURONS

The androgen receptor has been purified in the ‘70s and cloned in the ‘80s. It is a member of the steroid receptor superfamily and mediated the most important effects of androgen in androgen dependent or sensitive tissues. Several physiological function of the brain are differentially controlled in the two sexes and androgens play specific role in the processes of sexual differentiation and it is involved in the maintenance of male sex behaviour in adulthood. When mutated, the androgen receptor may impact on many of these androgen-regulated activities because of a loss of androgenic function in target cells. However, in the case of a peculiar type of mutation, the elongation of the polyglutamine tract normally present in its N-terminus, the androgen receptor becomes neurotoxic and induces cells death of a number of motoneurons in the spinal cord, which express very high level of this protein. Here, we will briefly discuss the most important actions of androgen receptor-mediated androgen activity in the brain and the mechanisms by which the mutant androgen receptor may lead to neurodegeneration in Spinal and Bulbar Muscular Atrophy (SBMA).

Unveiling synapse pathology in spinal bulbar muscular atrophy by genome-wide transcriptome analysis of purified motor neurons derived from disease specific iPSCs

Spinal bulbar muscular atrophy (SBMA) is an adult-onset, slowly progressive motor neuron disease caused by abnormal CAG repeat expansion in the androgen receptor (AR) gene. Although ligand (testosterone)-dependent mutant AR aggregation has been shown to play important roles in motor neuronal degeneration by the analyses of transgenic mice models and in vitro cell culture models, the underlying disease mechanisms remain to be fully elucidated because of the discrepancy between model mice and SBMA patients. Thus, novel human disease models that recapitulate SBMA patients’ pathology more accurately are required for more precise pathophysiological analysis and the development of novel therapeutics. Here, we established disease specific iPSCs from four SBMA patients, and differentiated them into spinal motor neurons. To investigate motor neuron specific pathology, we purified iPSC-derived motor neurons using flow cytometry and cell sorting based on the motor neuron specific reporter, HB9e438::Venus, and proceeded to the genome-wide transcriptome analysis by RNA sequences. The results revealed the involvement of the pathology associated with synapses, epigenetics, and endoplasmic reticulum (ER) in SBMA. Notably, we demonstrated the involvement of the neuromuscular synapse via significant upregulation of Synaptotagmin, R-Spondin2 (RSPO2), and WNT ligands in motor neurons derived from SBMA patients, which are known to be associated with neuromuscular junction (NMJ) formation and acetylcholine receptor (AChR) clustering. These aberrant gene expression in neuromuscular synapses might represent a novel therapeutic target for SBMA.