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.

Rif2 interaction with Rad50 counteracts Tel1 functions in checkpoint signalling and DNA tethering by releasing Tel1 from MRX binding.

The yeast Rif2 protein is known to inhibit Mre11 nuclease and the activation of Tel1 kinase through a short motif termed MIN, which binds the Rad50 subunit and simulates its ATPase activity in vitro. The mechanism by which Rif2 restrains Tel1 activation and the consequences of this inhibition at DNA double-strand breaks (DSBs) are poorly understood. In this study, we employed AlphaFold-Multimer modelling to pinpoint and validate the interaction surface between Rif2 MIN and Rad50. We also engineered the rif2-S6E mutation that enhances the inhibitory effect of Rif2 by increasing Rif2-Rad50 interaction. Unlike rif2Δ, the rif2-S6E mutation impairs hairpin cleavage. Furthermore, it diminishes Tel1 activation by inhibiting Tel1 binding to DSBs while leaving MRX association unchanged, indicating that Rif2 can directly inhibit Tel1 recruitment to DSBs. Additionally, Rif2S6E reduces Tel1-MRX interaction and increases stimulation of ATPase by Rad50, indicating that Rif2 binding to Rad50 induces an ADP-bound MRX conformation that is not suitable for Tel1 binding. The decreased Tel1 recruitment to DSBs in rif2-S6E cells impairs DSB end-tethering and this bridging defect is suppressed by expressing a Tel1 mutant variant that increases Tel1 persistence at DSBs, suggesting a direct role for Tel1 in the bridging of DSB ends.

The multistep path to replicative senescence onset: zooming on triggering and inhibitory events at telomeric DNA.

Replicative senescence is an essential cellular process playing important physiological functions, but it is better known for its implications in aging, cancer, and other pathologies. One of the main triggers of replicative senescence is telomere shortening and/or its dysfunction and, therefore, a deep understanding of the molecular determinants is crucial. However, replicative senescence is a heterogeneous and hard to study process, especially in mammalian cells, and some important questions still need an answer. These questions concern i) the exact molecular causes triggering replicative senescence, ii) the role of DNA repair mechanisms and iii) the importance of R-loops at telomeres in regulating senescence onset, and iv) the mechanisms underlying the bypass of replicative senescence. In this review, we will report and discuss recent findings about these mechanisms both in mammalian cells and in the model organism Saccharomyces cerevisiae.

The DNA damage checkpoint: A tale from budding yeast.

Studies performed in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have led the way in defining the DNA damage checkpoint and in identifying most of the proteins involved in this regulatory network, which turned out to have structural and functional equivalents in humans. Subsequent experiments revealed that the checkpoint is an elaborate signal transduction pathway that has the ability to sense and signal the presence of damaged DNA and transduce this information to influence a multifaceted cellular response that is essential for cancer avoidance. This review focuses on the work that was done in Saccharomyces cerevisiae to articulate the checkpoint concept, to identify its players and the mechanisms of activation and deactivation.

To Fix or Not to Fix: Maintenance of Chromosome Ends Versus Repair of DNA Double-Strand Breaks.

Early work by Muller and McClintock discovered that the physical ends of linear chromosomes, named telomeres, possess an inherent ability to escape unwarranted fusions. Since then, extensive research has shown that this special feature relies on specialized proteins and structural properties that confer identity to the chromosome ends, thus allowing cells to distinguish them from intrachromosomal DNA double-strand breaks. Due to the inability of conventional DNA replication to fully replicate the chromosome ends and the downregulation of telomerase in most somatic human tissues, telomeres shorten as cells divide and lose this protective capacity. Telomere attrition causes the activation of the DNA damage checkpoint that leads to a cell-cycle arrest and the entering of cells into a nondividing state, called replicative senescence, that acts as a barrier against tumorigenesis. However, downregulation of the checkpoint overcomes this barrier and leads to further genomic instability that, if coupled with re-stabilization of telomeres, can drive tumorigenesis. This review focuses on the key experiments that have been performed in the model organism Saccharomyces cerevisiae to uncover the mechanisms that protect the chromosome ends from eliciting a DNA damage response, the conservation of these pathways in mammals, as well as the consequences of their loss in human cancer.

AR cooperates with SMAD4 to maintain skeletal muscle homeostasis.

Androgens and androgen-related molecules exert a plethora of functions across different tissues, mainly through binding to the transcription factor androgen receptor (AR). Despite widespread therapeutic use and misuse of androgens as potent anabolic agents, the molecular mechanisms of this effect on skeletal muscle are currently unknown. Muscle mass in adulthood is mainly regulated by the bone morphogenetic protein (BMP) axis of the transforming growth factor (TGF)-ß pathway via recruitment of mothers against decapentaplegic homolog 4 (SMAD4) protein. Here we show that, upon activation, AR forms a transcriptional complex with SMAD4 to orchestrate a muscle hypertrophy programme by modulating SMAD4 chromatin binding dynamics and enhancing its transactivation activity. We challenged this mechanism of action using spinal and bulbar muscular atrophy (SBMA) as a model of study. This adult-onset neuromuscular disease is caused by a polyglutamine expansion (polyQ) in AR and is characterized by progressive muscle weakness and atrophy secondary to a combination of lower motor neuron degeneration and primary muscle atrophy. Here we found that the presence of an elongated polyQ tract impairs AR cooperativity with SMAD4, leading to an inability to mount an effective anti-atrophy gene expression programme in skeletal muscle in response to denervation. Furthermore, adeno-associated virus, serotype 9 (AAV9)-mediated muscle-restricted delivery of BMP7 is able to rescue the muscle atrophy in SBMA mice, supporting the development of treatments able to fine-tune AR-SMAD4 transcriptional cooperativity as a promising target for SBMA and other conditions associated with muscle loss.

Gene therapy with AR isoform 2 rescues spinal and bulbar muscular atrophy phenotype by modulating AR transcriptional activity.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset neuromuscular condition caused by an abnormal polyglutamine (polyQ) tract expansion in androgen receptor (AR) protein. SBMA is a disease with high unmet clinical need. Recent studies have shown that mutant AR-altered transcriptional activity is key to disease pathogenesis. Restoring the transcriptional dysregulation without affecting other AR critical functions holds great promise for the treatment of SBMA and other AR-related conditions; however, how this targeted approach can be achieved and translated into a clinical application remains to be understood. Here, we characterized the role of AR isoform 2, a naturally occurring variant encoding a truncated AR lacking the polyQ-harboring domain, as a regulatory switch of AR genomic functions in androgen-responsive tissues. Delivery of this isoform using a recombinant adeno-associated virus vector type 9 resulted in amelioration of the disease phenotype in SBMA mice by restoring polyQ AR-dysregulated transcriptional activity.

Modelling Neuromuscular Diseases in the Age of Precision Medicine.

Advances in knowledge resulting from the sequencing of the human genome, coupled with technological developments and a deeper understanding of disease mechanisms of pathogenesis are paving the way for a growing role of precision medicine in the treatment of a number of human conditions. The goal of precision medicine is to identify and deliver effective therapeutic approaches based on patients' genetic, environmental, and lifestyle factors. With the exception of cancer, neurological diseases provide the most promising opportunity to achieve treatment personalisation, mainly because of accelerated progress in gene discovery, deep clinical phenotyping, and biomarker availability. Developing reproducible, predictable and reliable disease models will be key to the rapid delivery of the anticipated benefits of precision medicine. Here we summarize the current state of the art of preclinical models for neuromuscular diseases, with particular focus on their use and limitations to predict safety and efficacy treatment outcomes in clinical trials.

Polyglutamine-Expanded Androgen Receptor Alteration of Skeletal Muscle Homeostasis and Myonuclear Aggregation Are Affected by Sex, Age and Muscle Metabolism.

Polyglutamine (polyQ) expansions in the androgen receptor (AR) gene cause spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease characterized by lower motor neuron (MN) loss and skeletal muscle atrophy, with an unknown mechanism. We generated new mouse models of SBMA for constitutive and inducible expression of mutant AR and performed biochemical, histological and functional analyses of phenotype. We show that polyQ-expanded AR causes motor dysfunction, premature death, IIb-to-IIa/IIx fiber-type change, glycolytic-to-oxidative fiber-type switching, upregulation of atrogenes and autophagy genes and mitochondrial dysfunction in skeletal muscle, together with signs of muscle denervation at late stage of disease. PolyQ expansions in the AR resulted in nuclear enrichment. Within the nucleus, mutant AR formed 2% sodium dodecyl sulfate (SDS)-resistant aggregates and inclusion bodies in myofibers, but not spinal cord and brainstem, in a process exacerbated by age and sex. Finally, we found that two-week induction of expression of polyQ-expanded AR in adult mice was sufficient to cause premature death, body weight loss and muscle atrophy, but not aggregation, metabolic alterations, motor coordination and fiber-type switch, indicating that expression of the disease protein in the adulthood is sufficient to recapitulate several, but not all SBMA manifestations in mice. These results imply that chronic expression of polyQ-expanded AR, i.e. during development and prepuberty, is key to induce the full SBMA muscle pathology observed in patients. Our data support a model whereby chronic expression of polyQ-expanded AR triggers muscle atrophy through toxic (neomorphic) gain of function mechanisms distinct from normal (hypermorphic) gain of function mechanisms.

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents.

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

From gene to therapy in spinal and bulbar muscular atrophy: Are we there yet?

Abnormal polyglutamine expansions in the androgen receptor (AR) cause a muscular condition, known as Kennedy's disease or spinal and bulbar muscular atrophy (SBMA). The disease is transmitted in an X-linked fashion and is clinically characterized by weakness, atrophy and fasciculations of the limb and bulbar muscles as a result of a toxic gain-of-function of the mutant protein. Notably, affected males also show signs of androgen insensitivity, such as gynaecomastia and reduced fertility. The characterization of the natural history of the disease, the increasing understanding of the mechanism of pathogenesis and the elucidation of the functions of normal and mutant AR have offered a momentum for developing a rational therapeutic strategy for this disease. In this special issue on androgens and AR functions, we will review the molecular, biochemical, and cellular mechanisms underlying the pathogenesis of SBMA. We will discuss recent advances on therapeutic approaches and opportunities for this yet incurable disease, ranging from androgen deprivation, to gene silencing, to an expanding repertoire of peripheral targets, including muscle. With the advancement of these strategies into the clinic, it can be reasonably anticipated that the landscape of treatment options for SBMA and other neuromuscular conditions will change rapidly in the near future.

A small-molecule Nrf1 and Nrf2 activator mitigates polyglutamine toxicity in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA, also known as Kennedy's disease) is one of nine neurodegenerative disorders that are caused by expansion of polyglutamine-encoding CAG repeats. Intracellular accumulation of abnormal proteins in these diseases, a pathological hallmark, is associated with defects in protein homeostasis. Enhancement of the cellular proteostasis capacity with small molecules has therefore emerged as a promising approach to treatment. Here, we characterize a novel curcumin analog, ASC-JM17, as an activator of central pathways controlling protein folding, degradation and oxidative stress resistance. ASC-JM17 acts on Nrf1, Nrf2 and Hsf1 to increase the expression of proteasome subunits, antioxidant enzymes and molecular chaperones. We show that ASC-JM17 ameliorates toxicity of the mutant androgen receptor (AR) responsible for SBMA in cell, fly and mouse models. Knockdown of the Drosophila Nrf1 and Nrf2 ortholog cap 'n' collar isoform-C, but not Hsf1, blocks the protective effect of ASC-JM17 on mutant AR-induced eye degeneration in flies. Our observations indicate that activation of the Nrf1/Nrf2 pathway is a viable option for pharmacological intervention in SBMA and potentially other polyglutamine diseases.

MiR-298 Counteracts Mutant Androgen Receptor Toxicity in Spinal and Bulbar Muscular Atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a currently untreatable adult-onset neuromuscular disease caused by expansion of a polyglutamine repeat in the androgen receptor (AR). In SBMA, as in other polyglutamine diseases, a toxic gain of function in the mutant protein is an important factor in the disease mechanism; therefore, reducing the mutant protein holds promise as an effective treatment strategy. In this work, we evaluated a microRNA (miRNA) to reduce AR expression. From a list of predicted miRNAs that target human AR, we selected microRNA-298 (miR-298) for its ability to downregulate AR mRNA and protein levels when transfected in cells overexpressing wild-type and mutant AR and in SBMA patient-derived fibroblasts. We showed that miR-298 directly binds to the 3'-untranslated region of the human AR transcript, and counteracts AR toxicity in vitro. Intravenous delivery of miR-298 with adeno-associated virus serotype 9 vector resulted in efficient transduction of muscle and spinal cord and amelioration of the disease phenotype in SBMA mice. Our findings support the development of miRNAs as a therapeutic strategy for SBMA and other neurodegenerative disorders caused by toxic proteins.

The Role of the Protein Quality Control System in SBMA.

Spinal and bulbar muscular atrophy (SBMA) or Kennedy's disease is an X-linked disease associated with the expansion of the CAG triplet repeat present in exon 1 of the androgen receptor (AR) gene. This results in the production of a mutant AR containing an elongated polyglutamine tract (polyQ) in its N-terminus. Interestingly, the ARpolyQ becomes toxic only after its activation by the natural androgenic ligands, possibly because of aberrant androgen-induced conformational changes of the ARpolyQ, which generate misfolded species. These misfolded ARpolyQ species must be cleared from motoneurons and muscle cells, and this process is mediated by the protein quality control (PQC) system. Experimental evidence suggested that failure of the PQC pathways occurs in disease, leading to ARpolyQ accumulation and toxicity in the target cells. In this review, we summarized the overall impact of mutant and misfolded ARpolyQ on the PQC system and described how molecular chaperones and the degradative pathways (ubiquitin-proteasome system (UPS), the autophagy-lysosome pathway (ALP), and the unfolded protein response (UPR), which activates the endoplasmic reticulum-associated degradation (ERAD)) are differentially affected in SBMA. We also extensively and critically reviewed several molecular and pharmacological approaches proposed to restore a global intracellular activity of the PQC system. Collectively, these data suggest that the fine and delicate equilibrium existing among the different players of the PQC system could be restored in a therapeutic perspective by the synergic/additive activities of compounds designed to tackle sequential or alternative steps of the intracellular defense mechanisms triggered against proteotoxic misfolded species.

Muscle matters in Kennedy's disease.

Polyglutamine expansion in the androgen receptor causes Kennedy's disease. Two recent reports, Cortes et al. (2014) in this issue of Neuron and Lieberman et al. (2014) in Cell Reports, raise the possibility that targeting expression of the mutant protein in skeletal muscle, instead of the nervous system, may mitigate manifestations of this disorder.

Insulinlike growth factor (IGF)-1 administration ameliorates disease manifestations in a mouse model of spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy is an X-linked motor neuron disease caused by polyglutamine expansion in the androgen receptor. Patients develop slowly progressive proximal muscle weakness, muscle atrophy and fasciculations. Affected individuals often show gynecomastia, testicular atrophy and reduced fertility as a result of mild androgen insensitivity. No effective disease-modifying therapy is currently available for this disease. Our recent studies have demonstrated that insulinlike growth factor (IGF)-1 reduces the mutant androgen receptor toxicity through activation of Akt in vitro, and spinal and bulbar muscular atrophy transgenic mice that also overexpress a noncirculating muscle isoform of IGF-1 have a less severe phenotype. Here we sought to establish the efficacy of daily intraperitoneal injections of mecasermin rinfabate, recombinant human IGF-1 and IGF-1 binding protein 3, in a transgenic mouse model expressing the mutant androgen receptor with an expanded 97 glutamine tract. The study was done in a controlled, randomized, blinded fashion, and, to reflect the clinical settings, the injections were started after the onset of disease manifestations. The treatment resulted in increased Akt phosphorylation and reduced mutant androgen receptor aggregation in muscle. In comparison to vehicle-treated controls, IGF-1-treated transgenic mice showed improved motor performance, attenuated weight loss and increased survival. Our results suggest that peripheral tissue can be targeted to improve the spinal and bulbar muscular atrophy phenotype and indicate that IGF-1 warrants further investigation in clinical trials as a potential treatment for this disease.

A candidate gene for autoimmune myasthenia gravis.

OBJECTIVE: We sought to identify a causative mutation in a previously reported kindred with parental consanguinity and 5 of 10 siblings with adult-onset autoimmune myasthenia gravis. METHODS: We performed genome-wide homozygosity mapping, and sequenced all known genes in the one region of extended homozygosity. Quantitative and allele-specific reverse transcriptase PCR (RT-PCR) were performed on a candidate gene to determine the RNA expression level in affected siblings and controls and the relative abundance of the wild-type and mutant alleles in a heterozygote. RESULTS: A region of shared homozygosity at chromosome 13q13.3-13q14.11 was found in 4 affected siblings and 1 unaffected sibling. A homozygous single nucleotide variant was found in the 3'-untranslated region of the ecto-NADH oxidase 1 gene (ENOX1). No other variants likely to be pathogenic were found in genes in this region or elsewhere. The ENOX1 sequence variant was not found in 764 controls. Quantitative RT-PCR showed that expression of ENOX1 decreased to about 20% of normal levels in lymphoblastoid cells from individuals homozygous for the variant and to about 50% in 2 unaffected heterozygotes. Allele-specific RT-PCR showed a 55%-60% reduction in the level of the variant transcript in heterozygous cells due to reduced mRNA stability. CONCLUSION: These results indicate that this sequence variant in ENOX1 may contribute to the familial autoimmune myasthenia in these patients.

Benign hereditary chorea: clinical and neuroimaging features in an Italian family.

Benign hereditary chorea is an autosomal dominant disorder characterized by early onset nonprogressive chorea, caused by mutations of the thyroid transcription factor-1 (TITF-1) gene. Clinical heterogeneity has been reported and thyroid and respiratory abnormalities may be present. We describe 3 patients of an Italian family carrying the S145X mutation in the TITF-1 gene with mild motor delay, childhood onset dyskinesias, and subtle cognitive impairment. A child in the third generation presented with congenital hypothyroidism and neonatal respiratory distress. Imaging studies in 2 patients showed mild ventricular enlargement and empty sella at magnetic resonance imaging and hypometabolism of basal ganglia and cortex at 18-Fluoro-2-deoxy-glucose positron emission tomography.

Blood vessel remodeling in pig ovarian follicles during the periovulatory period: an immunohistochemistry and SEM-corrosion casting study.

BACKGROUND: The present research aims to describe the process of vascular readjustment occurring in pig ovary during the periovulatory phase (from LH surge to ovulation) that drives the transformation of the follicle, a limited blood supplied structure, into the corpus luteum, a highly vascularised endocrine gland required to maintain high levels of progesterone in pregnancy. The swine model was chosen because it is characterized by a long periovulatory window (about 40-44 hrs-similar to human) that permits to recover follicles at a precise endocrinological timing. METHODS: By validated hormonal protocol (eCG+hCG), able to mimic the physiologic gonadotropin stimulation, preovulatory follicles (PreOFs, 60 h-eCG), follicles in the middle (early periovulatory follicles, EPerOFs, 18 h-hCG) or late (LPerOFs, 36 h-hCG) periovulatory phase were isolated from prepubertal gilts. To understand the angiogenic process, morphological/morphometrical analyses were performed by combining immunohistochemistry (IHC) and SEM of vascular corrosion casts (VCC) techniques. RESULTS: PreOFs showed a vascular plexus with proliferating endothelial cells (EPI). This plexus was characterized by a dense inner capillary network, with angiogenic figures, connected to the outer network by anastomotic vessels (arterioles and venules of the middle network). EPerOFs decreased their EPI, blood vessel extension in the outer network, and evidenced a reduced compactness of blood vessels. In LPerOFs, a rapid neovascularization was associated to an intensive tissue remodeling: the follicle acquired an undulated aspect presenting arterioles/venules near the basal membrane, increased vascular extension by EPI, sprouting and non-sprouting angiogenesis.The analysis of vascular geometric relations and branching angles evidenced similar values at all stages. CONCLUSION: These data allow us to hypothesize that EPerOFs are in a quiescent status. LPerOFs represent the "metamorphic" follicles that rapidly turn-on angiogenesis to sustain a successful corpus luteum formation. Particularly, it is interesting to underlie that the non-sprouting angiogenesis, typical of structures in rapid neovascularization, occurred only in the LPerOFs. Moreover, vascular geometric relations showed as blood vessel remodeling occurs with the "maximum output and the minimum energetic expense".This knowledge will allow to better understand the mechanisms regulating the reproductive success and to clarify the complex physiological angiogenic process in adult tissues.