CRISPR-Cas9 guided RNA-based model for the silencing of spinal bulbar muscular atrophy: A functional genetic disorder.

This study explores a novel therapeutic approach for spinal bulbar muscular atrophy (SBMA), a neurodegenerative disorder caused by a mutation in the Androgen Receptor (AR) gene. The aim is to investigate the potential of CRISPR-Cas9 technology in targeting the mutant AR gene to inhibit its production. The objectives include assessing the accuracy and efficacy of CRISPR-Cas9 guided RNAs in silencing the mutant gene and evaluating the feasibility of this approach as a treatment for SBMA. Computational and in-silico approaches are used to evaluate the feasibility of using CRISPR-Cas9 technology for treating SBMA. Computational analysis is used to design CRISPR-Cas9 guided RNAs targeting the mutant AR gene, assessing their on-target and off-target scores, GC content, and structural accuracy. In-silico simulations predict the potential therapeutic outcomes of the CRISPR-Cas9 approach in an artificial environment. Three guided RNA (gRNA) sequences were designed using the CHOPCHOP tool, targeting specific regions of the AR gene with high efficiency and 100% match. These gRNAs demonstrated effective targeting with minimal off-target scores and optimal GC content. Additionally, lentiCRISPR v2 plasmids were designed for the delivery of CRISPR materials, enabling high-efficiency multiplex genome editing of the AR gene. Thermodynamic ensemble predictions indicated favorable secondary structure stability of the designed gRNAs, further supporting their suitability for gene editing. The evaluation of designed gRNAs confirmed their strong binding ability to the target sequences, validating their potential as effective tools for genome editing. The study highlights the potential of CRISPR-Cas9 technology for targeting the Androgen Receptor gene associated with spinal bulbar muscular atrophy (SBMA). The findings support the feasibility of this approach for gene editing and suggest further exploration in preclinical and clinical settings. Recommendations include continued research to optimize CRISPR-Cas9 delivery methods and enhance specificity for therapeutic applications in SBMA.

Blocking the dimerization of polyglutamine-expanded androgen receptor protects cells from DHT-induced toxicity by increasing AR turnover.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular degenerative disease caused by a polyglutamine expansion in the androgen receptor (AR). This mutation causes AR to misfold and aggregate, contributing to toxicity in and degeneration of motor neurons and skeletal muscle. There is currently no effective treatment or cure for this disease. The role of an interdomain interaction between the amino- and carboxyl-termini of AR, termed the N/C interaction, has been previously identified as a component of androgen receptor-induced toxicity in cell and mouse models of SBMA. However, the mechanism by which this interaction contributes to disease pathology is unclear. This work seeks to investigate this mechanism by interrogating the role of AR homodimerization- a unique form of the N/C-interaction- in SBMA. We show that, although the AR N/C-interaction is reduced by polyglutamine-expansion, homodimers of 5α-dihydrotestosterone (DHT)-bound AR are increased. Additionally, blocking homodimerization results in decreased AR aggregation and toxicity in cell models. Blocking homodimerization results in the increased degradation of AR, which likely plays a role in the protective effects of this mutation. Overall, this work identifies a novel mechanism in SBMA pathology that may represent a novel target for the development of therapeutics for this disease.

Kennedy's disease.

A 57-year-old man developed worsening early morning headaches, muscle cramps and falls over 12 months. He had widespread fasciculation and was diagnosed with motor neurone disease, and treated with nocturnal hypoventilation. Based on this diagnosis, he made significant personal and financial decisions including retiring and selling his house. He subsequently developed a lump in his right breast and was found to have gynaecomastia. This triggered genetic testing for Kennedy's disease leading to the correct diagnosis. This case highlights an unusual presentation of a rare disease leading to misdiagnosis and major repercussions for the patient. Recent genetic analysis from the 100 000 genome project suggests Kennedy's disease may be four times more prevalent in the population than previously thought, highlighting the need to consider genetic testing, especially if there is a suggestion of multisystem disease.

271st ENMC international workshop: Towards a unifying effort to fight Kennedy's disease. 20-22 October 2023, Hoofddorp, Netherlands.

The workshop held in the Netherlands from October 20-22, 2023, united 27 scientists from academia, healthcare, and industry representing 11 countries, alongside four patient and charity representatives. Focused on Kennedy's Disease (KD), also known as spinal and bulbar muscular atrophy (SBMA), the workshop aimed to consolidate knowledge, align on clinical trial designs, and promote participative medicine for effective treatments. Discussions emphasized KD's molecular mechanisms, highlighting its status as a neuromuscular disorder with motor neuron degeneration. Strategies for therapeutic intervention, including AR activity modulation and targeting post-translational modifications, were proposed. The need for diagnostic, prognostic, and target engagement biomarkers was stressed. Challenges in patient stratification and clinical trial recruitment were acknowledged, with the International KD/SBMA Registry praised for its role. The workshop concluded with a patient-focused session, underscoring challenges in KD diagnosis and the vital support provided by patient associations.

Differentially disrupted spinal cord and muscle energy metabolism in spinal and bulbar muscular atrophy.

Prior studies showed that polyglutamine-expanded androgen receptor (AR) is aberrantly acetylated and that deacetylation of the mutant AR by overexpression of nicotinamide adenine dinucleotide-dependent (NAD+-dependent) sirtuin 1 is protective in cell models of spinal and bulbar muscular atrophy (SBMA). Based on these observations and reduced NAD+ in muscles of SBMA mouse models, we tested the therapeutic potential of NAD+ restoration in vivo by treating postsymptomatic transgenic SBMA mice with the NAD+ precursor nicotinamide riboside (NR). NR supplementation failed to alter disease progression and had no effect on increasing NAD+ or ATP content in muscle, despite producing a modest increase of NAD+ in the spinal cords of SBMA mice. Metabolomic and proteomic profiles of SBMA quadriceps muscles indicated alterations in several important energy-related pathways that use NAD+, in addition to the NAD+ salvage pathway, which is critical for NAD+ regeneration for use in cellular energy production. We also observed decreased mRNA levels of nicotinamide riboside kinase 2 (Nmrk2), which encodes a key kinase responsible for NR phosphorylation, allowing its use by the NAD+ salvage pathway. Together, these data suggest a model in which NAD+ levels are significantly decreased in muscles of an SBMA mouse model and intransigent to NR supplementation because of decreased levels of Nmrk2.

A phenotypically robust model of spinal and bulbar muscular atrophy in Drosophila.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked disorder that affects males who inherit the androgen receptor (AR) gene with an abnormal CAG triplet repeat expansion. The resulting protein contains an elongated polyglutamine (polyQ) tract and causes motor neuron degeneration in an androgen-dependent manner. The precise molecular sequelae of SBMA are unclear. To assist with its investigation and the identification of therapeutic options, we report here a new model of SBMA in Drosophila melanogaster. We generated transgenic flies that express the full-length, human AR with a wild-type or pathogenic polyQ repeat. Each transgene is inserted into the same safe harbor site on the third chromosome of the fly as a single copy and in the same orientation. Expression of pathogenic AR, but not of its wild-type variant, in neurons or muscles leads to consistent, progressive defects in longevity and motility that are concomitant with polyQ-expanded AR protein aggregation and reduced complexity in neuromuscular junctions. Additional assays show adult fly eye abnormalities associated with the pathogenic AR species. The detrimental effects of pathogenic AR are accentuated by feeding flies the androgen, dihydrotestosterone. This new, robust SBMA model can be a valuable tool toward future investigations of this incurable disease.

Exercise attenuates polyglutamine-mediated neuromuscular degeneration in a mouse model of spinal and bulbar muscular atrophy.

BACKGROUND: Spinal and bulbar muscular atrophy (SBMA) is a hereditary neuromuscular disorder caused by the expansion of trinucleotide cytosine-adenine-guanine (CAG) repeats, which encodes a polyglutamine (polyQ) tract in the androgen receptor (AR) gene. Recent evidence suggests that, in addition to motor neuron degeneration, defective skeletal muscles are also the primary contributors to the pathogenesis in SBMA. While benefits of physical exercise have been suggested in SBMA, underlying mechanism remains elusive. METHODS: We investigated the effect of running exercise in a transgenic mouse model of SBMA carrying human AR with 97 expanded CAGs (AR97Q). We assigned AR97Q mice to exercise and sedentary control groups, and mice in the exercise group received 1-h forced running wheel (5 m/min) 5 days a week for 4 weeks during the early stage of the disease. Motor function (grip strength and rotarod performance) and survival of each group were analysed, and histopathological and biological features in skeletal muscles and motor neurons were evaluated. RESULTS: AR97Q mice in the exercise group showed improvement in motor function (~40% and ~50% increase in grip strength and rotarod performance, respectively, P < 0.05) and survival (median survival 23.6 vs. 16.7 weeks, P < 0.05) with amelioration of neuronal and muscular histopathology (~1.4-fold and ~2.8-fold increase in motor neuron and muscle fibre size, respectively, P < 0.001) compared to those in the sedentary group. Nuclear accumulation of polyQ-expanded AR in skeletal muscles and motor neurons was suppressed in the mice with exercise compared to the sedentary mice (~50% and ~30% reduction in 1C2-positive cells in skeletal muscles and motor neurons, respectively, P < 0.05). We found that the exercise activated 5'-adenosine monophosphate-activated protein kinase (AMPK) signalling and inhibited mammalian target of rapamycin pathway that regulates protein synthesis in skeletal muscles of SBMA mice. Pharmacological activation of AMPK inhibited protein synthesis and reduced polyQ-expanded AR proteins in C2C12 muscle cells. CONCLUSIONS: Our findings suggest the therapeutic potential of exercise-induced effect via AMPK activation in SBMA.

Spinal and bulbar muscular atrophy: From molecular pathogenesis to pharmacological intervention targeting skeletal muscle.

The clinical characteristics of SBMA, also known as Kennedy's disease (OMIM 313200), were initially documented by Dr. H Kawahara in the 18th century and a hundred years later by Dr. W. Kennedy. SBMA is a neuromuscular disease caused by expansions of a CAG microsatellite tandem repeat in exon 1 of the androgen receptor (AR) gene located on the X chromosome. These expansions result in the production of AR with an aberrantly expanded polyglutamine (polyQ) tract. In this review, we explore recent advancements in the significance of gene expression changes in skeletal muscle and discuss how pharmacological interventions targeting this aspect of disease pathogenesis can potentially be translated into therapies for SBMA patients.

Incidence and prevalence of Spinal and bulbar muscular atrophy in South Korea: a nationwide population-based study.

Spinal and bulbar muscular atrophy (SBMA), or Kennedy's disease, is a rare X-linked neuromuscular disorder predominantly affecting males and caused by a mutation in the androgen receptor gene. The epidemiology and comorbidities associated with SBMA in different ethnicities remain poorly understood. This study investigated the prevalence, incidence, and comorbidities associated with SBMA in the South Korean population using the Health Insurance Review and Assessment Service (HIRA) database. We retrospectively reviewed diagnosed cases of SBMA (G12.25 code of the Korean Classification of Diseases-7th edition) registered from January 1, 2016, to December 31, 2019, to calculate incidence and prevalence rates and concomitant comorbidities. Additionally, we surveyed SBMA patients (questionnaire group) visiting our clinic in 2022 to compare comorbidities with the HIRA data. The mean incidence rate of SBMA in the Korean population was 0.36/100,000 males from 2018 to 2019, while the prevalence rate was approximately 0.46/100,000 males from 2016 to 2019. The most common comorbidities identified in HIRA were gastritis and duodenitis (99.7%), gastroesophageal reflux (90.5%), hyperlipidemia (88.4%), and liver disorders (75.2%), which showed similar results in the questionnaire group. Additionally, gastric cancer was the most common type of cancer reported in SBMA in South Korea; although indeterminate, age-related factors may contribute to the development of cancer in these patients. Our findings provide valuable insights into the epidemiology and associated comorbidities of SBMA within the Korean population, which could inform clinical practice and future clinical research.

Genotype and clinical phenotype analysis of a Family with Kennedy disease.

To investigate the clinical phenotype-genotype correlations of a family with Kennedy disease (KD) and improve our understanding of the disease. KD was confirmed after clinical phenotypic analyses, laboratory tests, polymerase chain reaction assays for cytosine-adenine-guanine (CAG) repeats, and neuro-electrophysiological tests. The disease was assessed using the KD1234 scale and the spinal and bulbar muscular atrophy functional rating scale. The average age of disease onset was 30.8 ± 2.85 years. Clinically diagnosed members had 48 CAG repeats (≥35 is abnormal) in the androgen receptor gene. The patients exhibited gynecomastia and testicular dysfunction. The lesions mainly involved the medulla oblongata and spinal cord. Progesterone and serum creatine kinase levels were significantly high. Electromyography showed chronic neurogenic damage and abnormal sensory and motor conduction in family members who did not participate in sports, exercise, or physical hobbies. Our study showed that this family had a stable inheritance of CAG repeats, and the genotype was consistent with the clinical phenotype. Gynecomastia was the first symptom, with progressive androgen resistance resulting in testicular atrophy, infertility, and sexual dysfunction. Changes in serum creatine kinase may indicate the progression or relief of symptoms, and rehabilitation may delay the progression of muscle atrophy.

LSD1/PRMT6-targeting gene therapy to attenuate androgen receptor toxic gain-of-function ameliorates spinobulbar muscular atrophy phenotypes in flies and mice.

Spinobulbar muscular atrophy (SBMA) is caused by CAG expansions in the androgen receptor gene. Androgen binding to polyQ-expanded androgen receptor triggers SBMA through a combination of toxic gain-of-function and loss-of-function mechanisms. Leveraging cell lines, mice, and patient-derived specimens, we show that androgen receptor co-regulators lysine-specific demethylase 1 (LSD1) and protein arginine methyltransferase 6 (PRMT6) are overexpressed in an androgen-dependent manner specifically in the skeletal muscle of SBMA patients and mice. LSD1 and PRMT6 cooperatively and synergistically transactivate androgen receptor, and their effect is enhanced by expanded polyQ. Pharmacological and genetic silencing of LSD1 and PRMT6 attenuates polyQ-expanded androgen receptor transactivation in SBMA cells and suppresses toxicity in SBMA flies, and a preclinical approach based on miRNA-mediated silencing of LSD1 and PRMT6 attenuates disease manifestations in SBMA mice. These observations suggest that targeting overexpressed co-regulators can attenuate androgen receptor toxic gain-of-function without exacerbating loss-of-function, highlighting a potential therapeutic strategy for patients with SBMA.

Defective excitation-contraction coupling and mitochondrial respiration precede mitochondrial Ca2+ accumulation in spinobulbar muscular atrophy skeletal muscle.

Polyglutamine expansion in the androgen receptor (AR) causes spinobulbar muscular atrophy (SBMA). Skeletal muscle is a primary site of toxicity; however, the current understanding of the early pathological processes that occur and how they unfold during disease progression remains limited. Using transgenic and knock-in mice and patient-derived muscle biopsies, we show that SBMA mice in the presymptomatic stage develop a respiratory defect matching defective expression of genes involved in excitation-contraction coupling (ECC), altered contraction dynamics, and increased fatigue. These processes are followed by stimulus-dependent accumulation of calcium into mitochondria and structural disorganization of the muscle triads. Deregulation of expression of ECC genes is concomitant with sexual maturity and androgen raise in the serum. Consistent with the androgen-dependent nature of these alterations, surgical castration and AR silencing alleviate the early and late pathological processes. These observations show that ECC deregulation and defective mitochondrial respiration are early but reversible events followed by altered muscle force, calcium dyshomeostasis, and dismantling of triad structure.

Bicalutamide and Trehalose Ameliorate Spinal and Bulbar Muscular Atrophy Pathology in Mice.

Spinal and bulbar muscular atrophy (SBMA) is characterized by motor neuron (MN) degeneration that leads to slowly progressive muscle weakness. It is considered a neuromuscular disease since muscle has a primary role in disease onset and progression. SBMA is caused by a CAG triplet repeat expansion in the androgen receptor (AR) gene. The translated poly-glutamine (polyQ) tract confers a toxic gain of function to the mutant AR altering its folding, causing its aggregation into intracellular inclusions, and impairing the autophagic flux. In an in vitro SBMA neuronal model, we previously showed that the antiandrogen bicalutamide and trehalose, a natural disaccharide stimulating autophagy, block ARpolyQ activation, reduce its nuclear translocation and toxicity and facilitate the autophagic degradation of cytoplasmic AR aggregates. Here, in a knock-in SBMA mouse model (KI AR113Q), we show that bicalutamide and trehalose ameliorated SBMA pathology. Bicalutamide reversed the formation of the AR insoluble forms in KI AR113Q muscle, preventing autophagic flux blockage. We demonstrated that apoptosis is activated in KI AR113Q muscle, and that both compounds prevented its activation. We detected a decrease of mtDNA and an increase of OXPHOS enzymes, already at early symptomatic stages; these alterations were reverted by trehalose. Overall, bicalutamide and/or trehalose led to a partial recovery of muscle morphology and function, and improved SBMA mouse motor behavior, inducing an extension of their survival. Thus, bicalutamide and trehalose, by counteracting ARpolyQ toxicity in skeletal muscle, are valuable candidates for future clinical trials in SBMA patients.

Characteristics of spinal and bulbar muscular atrophy in South Korea: a cross-sectional study of 157 patients.

Spinal and bulbar muscular atrophy, namely Kennedy disease, is a rare progressive neurodegenerative disorder caused by the expansion of a CAG repeat in the first exon of the androgen receptor gene on the X chromosome. We assessed the clinical history, laboratory findings, functional scales and electrophysiological data, as well as the levels of luteinizing hormone, follicle-stimulating hormone and testosterone, in 157 Korean patients with genetically confirmed spinal and bulbar muscular atrophy (mean age at data collection = 56.9 years; range = 33-83 years). Hand tremor was the first symptom noticed by patients at a median age of 35 years, followed by gynaecomastia, orofacial fasciculation, cramps and fatigability in ascending order. Clinical symptoms such as paraesthesia and dysphagia appeared during the later stages of the disease. Cane use during ambulation began at a median age of 62 years. There were statistically significant differences between patients and controls in the results of sensory nerve studies, motor conduction velocity, and distal latencies. Furthermore, among the hormone markers analysed, the level of luteinizing hormone exhibited a negative correlation with the spinal and bulbar muscular atrophy functional rating scale, Korean version. However, among the patients with a disease duration of ≤5 years, the levels of luteinizing hormone showed a significant correlation with assessments using the amyotrophic lateral sclerosis functional rating scale-revised, spinal and bulbar muscular atrophy functional rating scale, Korean version and the 6-minute walk test. In conclusion, our findings provide clinical information from a substantial number of patients with spinal and bulbar muscular atrophy in Korea that accorded with that of patients with this disease worldwide but with updated clinical features.

Kennedy's disease presented with mastication fatigue combined with positive titin antibody: a case report.

BACKGROUND: Spinal and bulbar muscular atrophy (SBMA) is an X-linked recessive hereditary neuromuscular disorder caused by the expanded trinucleotide repeat in the androgen receptors gene. The major clinical manifestations of SBMA consist of weakness in the bulbar and limb muscles, fasciculations, tremors, cramps, sensory impairment, and gynecomastia. However, atypical SBMA cases may lead to misdiagnosis. Muscular fatigue and decremental responses to repetitive nerve stimulation (RNS), despite being observed in some SBMA patients, are usually occurred in MG patients, and patient with the symptom of mastication fatigue was rarely reported. In addition, cardiological investigations have been performed in SBMA patients and several ECG alterations were identified. Here we report an SBMA patient presenting with a rare onset symptom of mastication fatigue, who has been detected with a positive titin antibody in the serum and showed a WPW pattern electrocardiogram. CASE PRESENTATION: The patient showed mildly progressive fatigue in the muscles of mastication over 3 years. Neurological examination showed facial muscle weakness and a wasting tongue with fasciculations, but the weakness, wasting, or fasciculations were not obvious in the limbs. 3-Hz RNS showed a decremental response in bilateral orbicularis oculi. The test of titin antibody was positive in the serum, and the electrocardiogram showed a WPW pattern ECG. Genetic analysis revealed an increased number (39 repeats) of tandem CAG repeats in the AR gene, which confirmed the diagnosis of SBMA. The fatigue symptom was significantly improved after oral pyridostigmine bromide treatment. CONCLUSION: This case calls for more attention to muscular fatigue as the onset symptoms of Kennedy's disease. ECG screening is of importance in SBMA patients and further studies are needed to investigate the titin antibody in SBMA patients as well as other neurogenic disorders.

Development of a functional composite for the evaluation of spinal and bulbar muscular atrophy.

This study aimed to develop a functional measurement that combines quantitative motor evaluation index of various body regions in patients with spinal and bulbar muscular atrophy (SBMA). We assessed subjects with SBMA and healthy controls with quantitative muscle strength measurements and functional scales. We selected tongue pressure, grip power, % peak expiratory flow (%PEF), timed walking test, and % forced vital capacity (%FVC) as components. By combining these values with Z-score, we created a functional composite (SBMA functional composite: SBMAFC). We also calculated the standardized response mean to compare the sensitivity of SBMAFC with that of existing measurements. A total of 97 genetically confirmed patients with SBMA and 36 age- and sex-matched healthy controls were enrolled. In the longitudinal analysis, the standardized response mean of SBMAFC was larger than that of existing rating scales. Receiver operating characteristic (ROC) analysis demonstrated that the SBMAFC is capable of distinguishing between subjects with early-stage SBMA and healthy controls. SBMAFC is more sensitive to disease progression than existing functional rating scales and is a potential outcome measure in clinical trials of SBMA.

Identification of UBA1 as the causative gene of an X-linked non-Kennedy spinal-bulbar muscular atrophy.

BACKGROUND AND PURPOSE: Spinal-bulbar muscular atrophy (SBMA) (Kennedy's disease) is a motor neuron disease. Kennedy's disease is nearly exclusively caused by mutations in the androgen receptor encoding gene (AR). The results of studies aimed at identification of the genetic cause of a disease that best approximates SBMA in a pedigree (four patients) without mutations in AR are reported. METHODS: Clinical investigations included thorough neurological and non-neurological examinations and testing. Genetic analysis was performed by exome sequencing using standard protocols. UBA1 mutations were modeled on the crystal structure of UBA1. RESULTS: The clinical features of the patients are described in detail. A missense mutation in UBA1 (c.T1499C; p.Ile500Thr) was identified as the probable cause of the non-Kennedy SBMA in the pedigree. Like AR, UBA1 is positioned on chromosome X. UBA1 is a highly conserved gene. It encodes ubiquitin-like modifier activating enzyme 1 (UBA1) which is the major E1 enzyme of the ubiquitin-proteasome system. Interestingly, UBA1 mutations can also cause infantile-onset X-linked spinal muscular atrophy (XL-SMA). The mutation identified here and the XL-SMA causative mutations were shown to affect amino acids positioned in the vicinity of UBA1's ATP binding site and to cause structural changes. CONCLUSION: UBA1 was identified as a novel SBMA causative gene. The gene affects protein homeostasis which is one of most important components of the pathology of neurodegeneration. The contribution of this same gene to the etiology of XL-SMA is discussed.

C9orf72 hexanucleotide repeat expansion found in suspected spinobulbar muscular atrophy (SBMA).

INTRODUCTION: The expansion of a hexanucleotide GGGGCC repeat (G4C2) in the C9orf72 locus is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In addition, C9orf72 expansion has also been detected in patients with a clinical manifestation of Parkinson's Disease (PD), Alzheimer's Disease (AD), Huntington's Disease (HD), and ataxic disorders. MATERIAL AND METHODS: A total of 1,387 patients with clinically suspected ALS, HD or spinal and bulbar muscular atrophy (SBMA) were enrolled, and the prevalence of C9orf72 expansions was estimated. RESULTS: The hexanucleotide expansion accounted for 3.7% of the ALS patients, 0.2% of the HD suspected patients with excluded HTT mutation, and 1.3% of the suspected SBMA patients with excluded mutation in AR gene. CONCLUSIONS: This is the first report revealing the presence of C9orf72 expansion in patients with a suspected SBMA diagnosis. Consequently, we advise testing for C9orf72 expansion in patients presenting with the SBMA phenotype and a genetically unsolved diagnosis.