Introduction to the Special Issue "Skeletal Muscle Atrophy: Mechanisms at a Cellular Level".

Skeletal muscle is the most abundant tissue in the body and requires high levels of energy to function properly. Skeletal muscle allows voluntary movement and body posture, which require different types of fiber, innervation, energy, and metabolism. Here, we summarize the contribution received at the time of publication of this Introductory Issue for the Special Issue dedicated to "Skeletal Muscle Atrophy: Mechanisms at a Cellular Level". The Special Issue is divided into three sections. The first is dedicated to skeletal muscle pathophysiology, the second to disease mechanisms, and the third to therapeutic development.

Skeletal Muscle Pathogenesis in Polyglutamine Diseases.

Polyglutamine diseases are characterized by selective dysfunction and degeneration of specific types of neurons in the central nervous system. In addition, nonneuronal cells can also be affected as a consequence of primary degeneration or due to neuronal dysfunction. Skeletal muscle is a primary site of toxicity of polyglutamine-expanded androgen receptor, but it is also affected in other polyglutamine diseases, more likely due to neuronal dysfunction and death. Nonetheless, pathological processes occurring in skeletal muscle atrophy impact the entire body metabolism, thus actively contributing to the inexorable progression towards the late and final stages of disease. Skeletal muscle atrophy is well recapitulated in animal models of polyglutamine disease. In this review, we discuss the impact and relevance of skeletal muscle in patients affected by polyglutamine diseases and we review evidence obtained in animal models and patient-derived cells modeling skeletal muscle.

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.

Motor Neuron Diseases and Neuroprotective Peptides: A Closer Look to Neurons.

Motor neurons (MNs) are specialized neurons responsible for muscle contraction that specifically degenerate in motor neuron diseases (MNDs), such as amyotrophic lateral sclerosis (ALS), spinal and bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA). Distinct classes of MNs degenerate at different rates in disease, with a particular class named fast-fatigable MNs (FF-MNs) degenerating first. The etiology behind the selective vulnerability of FF-MNs is still largely under investigation. Among the different strategies to target MNs, the administration of protective neuropeptides is one of the potential therapeutic interventions. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with beneficial effects in many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and more recently SBMA. Another neuropeptide that has a neurotrophic effect on MNs is insulin-like growth factor 1 (IGF-1), also known as somatomedin C. These two peptides are implicated in the activation of neuroprotective pathways exploitable in the amelioration of pathological outcomes related to MNDs.

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.

ClC-2-like Chloride Current Alterations in a Cell Model of Spinal and Bulbar Muscular Atrophy, a Polyglutamine Disease.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by expansions of a polyglutamine (polyQ) tract in the androgen receptor (AR) gene. SBMA is associated with the progressive loss of lower motor neurons, together with muscle weakness and atrophy. PolyQ-AR is converted to a toxic species upon binding to its natural ligands, testosterone, and dihydrotestosterone (DHT). Our previous patch-clamp studies on a motor neuron-derived cell model of SBMA showed alterations in voltage-gated ion currents. Here, we identified and characterized chloride currents most likely belonging to the chloride channel-2 (ClC-2) subfamily, which showed significantly increased amplitudes in the SBMA cells. The treatment with the pituitary adenylyl cyclase-activating polypeptide (PACAP), a neuropeptide with a proven protective effect in a mouse model of SBMA, recovered chloride channel current alterations in SBMA cells. These observations suggest that the CIC-2 currents are affected in SBMA, an alteration that may contribute and potentially determine the pathophysiology of the disease.

The E3 ubiquitin-protein ligase MDM2 is a novel interactor of the von Hippel-Lindau tumor suppressor.

Mutations of the von Hippel-Lindau (pVHL) tumor suppressor are causative of a familiar predisposition to develop different types of cancer. pVHL is mainly known for its role in regulating hypoxia-inducible factor 1 α (HIF-1α) degradation, thus modulating the hypoxia response. There are different pVHL isoforms, including pVHL30 and pVHL19. However, little is known about isoform-specific functions and protein-protein interactions. Integrating in silico predictions with in vitro and in vivo assays, we describe a novel interaction between pVHL and mouse double minute 2 homolog (MDM2). We found that pVHL30, and not pVHL19, forms a complex with MDM2, and that the N-terminal acidic tail of pVHL30 is required for its association with MDM2. Further, we demonstrate that an intrinsically disordered region upstream of the tetramerization domain of MDM2 is responsible for its isoform-specific association with pVHL30. This region is highly conserved in higher mammals, including primates, similarly to what has been already shown for the N-terminal tail of pVHL30. Finally, we show that overexpression of pVHL30 and MDM2 together reduces cell metabolic activity and necrosis, suggesting a synergistic effect of these E3 ubiquitin ligases. Collectively, our data show an isoform-specific interaction of pVHL with MDM2, suggesting an interplay between these two E3 ubiquitin ligases.

The pVHL neglected functions, a tale of hypoxia-dependent and -independent regulations in cancer.

The von Hippel-Lindau protein (pVHL) is a tumour suppressor mainly known for its role as master regulator of hypoxia-inducible factor (HIF) activity. Functional inactivation of pVHL is causative of the von Hippel-Lindau disease, an inherited predisposition to develop different cancers. Due to its impact on human health, pVHL has been widely studied in the last few decades. However, investigations mostly focus on its role in degrading HIFs, whereas alternative pVHL protein-protein interactions and functions are insistently surfacing in the literature. In this review, we analyse these almost neglected functions by dissecting specific conditions in which pVHL is proposed to have differential roles in promoting cancer. We reviewed its role in regulating phosphorylation as a number of works suggest pVHL to act as an inhibitor by either degrading or promoting downregulation of specific kinases. Further, we summarize hypoxia-dependent and -independent pVHL interactions with multiple protein partners and discuss their implications in tumorigenesis.

MEF2 impairment underlies skeletal muscle atrophy in polyglutamine disease.

Polyglutamine (polyQ) tract expansion leads to proteotoxic misfolding and drives a family of nine diseases. We study spinal and bulbar muscular atrophy (SBMA), a progressive degenerative disorder of the neuromuscular system caused by the polyQ androgen receptor (AR). Using a knock-in mouse model of SBMA, AR113Q mice, we show that E3 ubiquitin ligases which are a hallmark of the canonical muscle atrophy machinery are not induced in AR113Q muscle. Similarly, we find no evidence to suggest dysfunction of signaling pathways that trigger muscle hypertrophy or impairment of the muscle stem cell niche. Instead, we find that skeletal muscle atrophy is characterized by diminished function of the transcriptional regulator Myocyte Enhancer Factor 2 (MEF2), a regulator of myofiber homeostasis. Decreased expression of MEF2 target genes is age- and glutamine tract length-dependent, occurs due to polyQ AR proteotoxicity, and is associated with sequestration of MEF2 into intranuclear inclusions in muscle. Skeletal muscle from R6/2 mice, a model of Huntington disease which develops progressive atrophy, also sequesters MEF2 into inclusions and displays age-dependent loss of MEF2 target genes. Similarly, SBMA patient muscle shows loss of MEF2 target gene expression, and restoring MEF2 activity in AR113Q muscle rescues fiber size and MEF2-regulated gene expression. This work establishes MEF2 impairment as a novel mechanism of skeletal muscle atrophy downstream of toxic polyglutamine proteins and as a therapeutic target for muscle atrophy in these disorders.

Insulin-like growth factor 1 signaling in motor neuron and polyglutamine diseases: From molecular pathogenesis to therapeutic perspectives.

The pleiotropic peptide insulin-like growth factor 1 (IGF-I) regulates human body homeostasis and cell growth. IGF-I activates two major signaling pathways, namely phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB/Akt) and Ras/extracellular signal-regulated kinase (ERK), which contribute to brain development, metabolism and function as well as to neuronal maintenance and survival. In this review, we discuss the general and tissue-specific effects of the IGF-I pathways. In addition, we present a comprehensive overview examining the role of IGF-I in neurodegenerative diseases, such as spinal and muscular atrophy, amyotrophic lateral sclerosis, and polyglutamine diseases. In each disease, we analyze the disturbances of the IGF-I pathway, the modification of the disease protein by IGF-I signaling, and the therapeutic strategies based on the use of IGF-I developed to date. Lastly, we highlight present and future considerations in the use of IGF-I for the treatment of these disorders.

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.

NURR1 and ERR1 Modulate the Expression of Genes of a DRD2 Coexpression Network Enriched for Schizophrenia Risk.

Multiple schizophrenia (SCZ) risk loci may be involved in gene co-regulation mechanisms, and analysis of coexpressed gene networks may help to clarify SCZ molecular basis. We have previously identified a dopamine D2 receptor (DRD2) coexpression module enriched for SCZ risk genes and associated with cognitive and neuroimaging phenotypes of SCZ, as well as with response to treatment with antipsychotics. Here we aimed to identify regulatory factors modulating this coexpression module and their relevance to SCZ. We performed motif enrichment analysis to identify transcription factor (TF) binding sites in human promoters of genes coexpressed with DRD2. Then, we measured transcript levels of a group of these genes in primary mouse cortical neurons in basal conditions and upon overexpression and knockdown of predicted TFs. Finally, we analyzed expression levels of these TFs in dorsolateral prefrontal cortex (DLPFC) of SCZ patients. Our in silico analysis revealed enrichment for NURR1 and ERR1 binding sites. In neuronal cultures, the expression of genes either relevant to SCZ risk (Drd2, Gatad2a, Slc28a1, Cnr1) or indexing coexpression in our module (Btg4, Chit1, Osr1, Gpld1) was significantly modified by gain and loss of Nurr1 and Err1. Postmortem DLPFC expression data analysis showed decreased expression levels of NURR1 and ERR1 in patients with SCZ. For NURR1 such decreased expression is associated with treatment with antipsychotics. Our results show that NURR1 and ERR1 modulate the transcription of DRD2 coexpression partners and support the hypothesis that NURR1 is involved in the response to SCZ treatment.SIGNIFICANCE STATEMENT In the present study, we provide in silico and experimental evidence for a role of the TFs NURR1 and ERR1 in modulating the expression pattern of genes coexpressed with DRD2 in human DLPFC. Notably, genetic variations in these genes is associated with SCZ risk and behavioral and neuroimaging phenotypes of the disease, as well as with response to treatment. Furthermore, this study presents novel findings on a possible interplay between D2 receptor-mediated dopamine signaling involved in treatment with antipsychotics and the transcriptional regulation mechanisms exerted by NURR1. Our results suggest that coexpression and co-regulation mechanisms may help to explain some of the complex biology of genetic associations with SCZ.

Muscleblind acts as a modifier of FUS toxicity by modulating stress granule dynamics and SMN localization.

Mutations in fused in sarcoma (FUS) lead to amyotrophic lateral sclerosis (ALS) with varying ages of onset, progression and severity. This suggests that unknown genetic factors contribute to disease pathogenesis. Here we show the identification of muscleblind as a novel modifier of FUS-mediated neurodegeneration in vivo. Muscleblind regulates cytoplasmic mislocalization of mutant FUS and subsequent accumulation in stress granules, dendritic morphology and toxicity in mammalian neuronal and human iPSC-derived neurons. Interestingly, genetic modulation of endogenous muscleblind was sufficient to restore survival motor neuron (SMN) protein localization in neurons expressing pathogenic mutations in FUS, suggesting a potential mode of suppression of FUS toxicity. Upregulation of SMN suppressed FUS toxicity in Drosophila and primary cortical neurons, indicating a link between FUS and SMN. Our data provide in vivo evidence that muscleblind is a dominant modifier of FUS-mediated neurodegeneration by regulating FUS-mediated ALS pathogenesis.

Pituitary Adenylyl Cyclase Activating Polypeptide (PACAP) Signaling and the Cell Cycle Machinery in Neurodegenerative Diseases.

Pituitary adenylyl cyclase activating polypeptide (PACAP) is a neuropeptide with great neuroprotective effects and remarkable therapeutic potential. PACAP activates several cellular pathways to exert its protective effects. Emerging evidence shows that PACAP can modify the levels and activity of cell cycle components involved in neurodegeneration to protect neurons from death. Cell cycle is a highly regulated process that controls the balance between proliferation, differentiation and death of every cell in the body. Aberrant expression and function of components of the cell cycle machinery have been linked to neurodegenerative diseases, in which different types of neuronal cells become dysfunctional and die in response to toxic insults. Since neurons are postmitotic cells, re-entry into the cell cycle has been shown to be pathological and contributes to the process of neurodegeneration. Moreover, an increasing number of studies highlight the importance of the role of cell cycle components outside the cell cycle and their involvement in neurodegenerative disorders. Here, we discuss the pleiotropic effects of PACAP on cell cycle machinery and the implication for the treatment of neurodegenerative diseases.

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.

Altered ionic currents and amelioration by IGF-1 and PACAP in motoneuron-derived cells modelling SBMA.

Spinal and bulbar muscular atrophy (SBMA), also known as Kennedy's disease, is a motor neuron disease caused by the expansion of a polymorphic CAG tandem repeat encoding a polyglutamine (polyQ) tract in the androgen receptor (AR) gene. SBMA is triggered by the binding of mutant AR to its natural ligands, testosterone and dihydrotestosterone (DHT). To investigate the neuronal alterations of motor neuron cell models of SBMA, we applied patch-clamp methods to verify how polyQ expansions in the AR alter cell ionic currents. We used mouse motoneuron-derived MN-1 cells expressing normal AR (MN24Q) and mutant AR (MN100Q treated cells with vehicle EtOH and DHT). We observed a reduction of the current flux mainly at depolarizing potentials in the DHT-treated cells, while the dissection of macroscopic currents showed single different cationic currents belonging to voltage-gated channels. Also, we treated the cells with IGF-1 and PACAP, which have previously been shown to protect MN-1 cells from the toxicity of mutant AR, and we found an amelioration of the altered currents. Our results suggest that the electrophysiological correlate of SBMA is a suitable reference point for the identification of disease symptoms and for future therapeutic targets.

Increased mitophagy in the skeletal muscle of spinal and bulbar muscular atrophy patients.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disorder caused by polyglutamine expansion in the androgen receptor (AR) and characterized by the loss of lower motor neurons. Here we investigated pathological processes occurring in muscle biopsy specimens derived from SBMA patients and, as controls, age-matched healthy subjects and patients suffering from amyotrophic lateral sclerosis (ALS) and neurogenic atrophy. We detected atrophic fibers in the muscle of SBMA, ALS and neurogenic atrophy patients. In addition, SBMA muscle was characterized by the presence of a large number of hypertrophic fibers, with oxidative fibers having a larger size compared with glycolytic fibers. Polyglutamine-expanded AR expression was decreased in whole muscle, yet enriched in the nucleus, and localized to mitochondria. Ultrastructural analysis revealed myofibrillar disorganization and streaming in zones lacking mitochondria and degenerating mitochondria. Using molecular (mtDNA copy number), biochemical (citrate synthase and respiratory chain enzymes) and morphological (dark blue area in nicotinamide adenine dinucleotide-stained muscle cross-sections) analyses, we found a depletion of the mitochondria associated with enhanced mitophagy. Mass spectrometry analysis revealed an increase of phosphatidylethanolamines and phosphatidylserines in mitochondria isolated from SBMA muscles, as well as a 50% depletion of cardiolipin associated with decreased expression of the cardiolipin synthase gene. These observations suggest a causative link between nuclear polyglutamine-expanded AR accumulation, depletion of mitochondrial mass, increased mitophagy and altered mitochondrial membrane composition in SBMA muscle patients. Given the central role of mitochondria in cell bioenergetics, therapeutic approaches toward improving the mitochondrial network are worth considering to support SBMA patients.

Beta-agonist stimulation ameliorates the phenotype of spinal and bulbar muscular atrophy mice and patient-derived myotubes.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease characterized by the loss of lower motor neurons. SBMA is caused by expansions of a polyglutamine tract in the gene coding for androgen receptor (AR). Expression of polyglutamine-expanded AR causes damage to motor neurons and skeletal muscle cells. Here we investigated the effect of ß-agonist stimulation in SBMA myotube cells derived from mice and patients, and in knock-in mice. We show that treatment of myotubes expressing polyglutamine-expanded AR with the ß-agonist clenbuterol increases their size. Clenbuterol activated the phosphatidylinositol-3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway and decreased the accumulation of polyglutamine-expanded AR. Treatment of SBMA knock-in mice with clenbuterol, which was started at disease onset, ameliorated motor function and extended survival. Clenbuterol improved muscle pathology, attenuated the glycolytic-to-oxidative metabolic alterations occurring in SBMA muscles and induced hypertrophy of both glycolytic and oxidative fibers. These results indicate that ß-agonist stimulation is a novel therapeutic strategy for SBMA.

Adenylyl cyclase activating polypeptide reduces phosphorylation and toxicity of the polyglutamine-expanded androgen receptor in spinobulbar muscular atrophy.

Spinobulbar muscular atrophy (SBMA) is an X-linked neuromuscular disease caused by polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. SBMA belongs to the family of polyQ diseases, which are fatal neurodegenerative disorders mainly caused by protein-mediated toxic gain-of-function mechanisms and characterized by deposition of misfolded proteins in the form of aggregates. The neurotoxicity of the polyQ proteins can be modified by phosphorylation at specific sites, thereby providing the rationale for the development of disease-specific treatments. We sought to identify signaling pathways that modulate polyQ-AR phosphorylation for therapy development. We report that cyclin-dependent kinase 2 (CDK2) phosphorylates polyQ-AR specifically at Ser96 Phosphorylation of polyQ-AR by CDK2 increased protein stabilization and toxicity and is negatively regulated by the adenylyl cyclase (AC)/protein kinase A (PKA) signaling pathway. To translate these findings into therapy, we developed an analog of pituitary adenylyl cyclase activating polypeptide (PACAP), a potent activator of the AC/PKA pathway. Chronic intranasal administration of the PACAP analog to knock-in SBMA mice reduced Ser96 phosphorylation, promoted polyQ-AR degradation, and ameliorated disease outcome. These results provide proof of principle that noninvasive therapy based on the use of PACAP analogs is a therapeutic option for SBMA.