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.

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.

Natural variation in gene expression and viral susceptibility revealed by neural progenitor cell villages.

Human genome variation contributes to diversity in neurodevelopmental outcomes and vulnerabilities; recognizing the underlying molecular and cellular mechanisms will require scalable approaches. Here, we describe a "cell village" experimental platform we used to analyze genetic, molecular, and phenotypic heterogeneity across neural progenitor cells from 44 human donors cultured in a shared in vitro environment using algorithms (Dropulation and Census-seq) to assign cells and phenotypes to individual donors. Through rapid induction of human stem cell-derived neural progenitor cells, measurements of natural genetic variation, and CRISPR-Cas9 genetic perturbations, we identified a common variant that regulates antiviral IFITM3 expression and explains most inter-individual variation in susceptibility to the Zika virus. We also detected expression QTLs corresponding to GWAS loci for brain traits and discovered novel disease-relevant regulators of progenitor proliferation and differentiation such as CACHD1. This approach provides scalable ways to elucidate the effects of genes and genetic variation on cellular phenotypes.

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.

Antagonistic effect of cyclin-dependent kinases and a calcium-dependent phosphatase on polyglutamine-expanded androgen receptor toxic gain of function.

Spinal and bulbar muscular atrophy is caused by polyglutamine (polyQ) expansions in androgen receptor (AR), generating gain-of-function toxicity that may involve phosphorylation. Using cellular and animal models, we investigated what kinases and phosphatases target polyQ-expanded AR, whether polyQ expansions modify AR phosphorylation, and how this contributes to neurodegeneration. Mass spectrometry showed that polyQ expansions preserve native phosphorylation and increase phosphorylation at conserved sites controlling AR stability and transactivation. In small-molecule screening, we identified that CDC25/CDK2 signaling could enhance AR phosphorylation, and the calcium-sensitive phosphatase calcineurin had opposite effects. Pharmacologic and genetic manipulation of these kinases and phosphatases modified polyQ-expanded AR function and toxicity in cells, flies, and mice. Ablation of CDK2 reduced AR phosphorylation in the brainstem and restored expression of Myc and other genes involved in DNA damage, senescence, and apoptosis, indicating that the cell cycle-regulated kinase plays more than a bystander role in SBMA-vulnerable postmitotic cells.

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.

The 22q11.2 region regulates presynaptic gene-products linked to schizophrenia.

It is unclear how the 22q11.2 deletion predisposes to psychiatric disease. To study this, we generated induced pluripotent stem cells from deletion carriers and controls and utilized CRISPR/Cas9 to introduce the heterozygous deletion into a control cell line. Here, we show that upon differentiation into neural progenitor cells, the deletion acted in trans to alter the abundance of transcripts associated with risk for neurodevelopmental disorders including autism. In excitatory neurons, altered transcripts encoded presynaptic factors and were associated with genetic risk for schizophrenia, including common and rare variants. To understand how the deletion contributed to these changes, we defined the minimal protein-protein interaction network that best explains gene expression alterations. We found that many genes in 22q11.2 interact in presynaptic, proteasome, and JUN/FOS transcriptional pathways. Our findings suggest that the 22q11.2 deletion impacts genes that may converge with psychiatric risk loci to influence disease manifestation in each deletion carrier.

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.

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.

FIN-Seq: transcriptional profiling of specific cell types from frozen archived tissue of the human central nervous system.

Thousands of frozen, archived tissue samples from the human central nervous system (CNS) are currently available in brain banks. As recent developments in RNA sequencing technologies are beginning to elucidate the cellular diversity present within the human CNS, it is becoming clear that an understanding of this diversity would greatly benefit from deeper transcriptional analyses. Single cell and single nucleus RNA profiling provide one avenue to decipher this heterogeneity. An alternative, complementary approach is to profile isolated, pre-defined cell types and use methods that can be applied to many archived human tissue samples that have been stored long-term. Here, we developed FIN-Seq (Frozen Immunolabeled Nuclei Sequencing), a method that accomplishes these goals. FIN-Seq uses immunohistochemical isolation of nuclei of specific cell types from frozen human tissue, followed by bulk RNA-Sequencing. We applied this method to frozen postmortem samples of human cerebral cortex and retina and were able to identify transcripts, including low abundance transcripts, in specific cell types.

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.

Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission.

Transcription factor programming of pluripotent stem cells (PSCs) has emerged as an approach to generate human neurons for disease modeling. However, programming schemes produce a variety of cell types, and those neurons that are made often retain an immature phenotype, which limits their utility in modeling neuronal processes, including synaptic transmission. We report that combining NGN2 programming with SMAD and WNT inhibition generates human patterned induced neurons (hpiNs). Single-cell analyses showed that hpiN cultures contained cells along a developmental continuum, ranging from poorly differentiated neuronal progenitors to well-differentiated, excitatory glutamatergic neurons. The most differentiated neurons could be identified using a CAMK2A::GFP reporter gene and exhibited greater functionality, including NMDAR-mediated synaptic transmission. We conclude that utilizing single-cell and reporter gene approaches for selecting successfully programmed cells for study will greatly enhance the utility of hpiNs and other programmed neuronal populations in the modeling of nervous system disorders.

Beyond motor neurons: expanding the clinical spectrum in Kennedy's disease.

Kennedy's disease, or spinal and bulbar muscular atrophy (SBMA), is an X-linked neuromuscular condition clinically characterised by weakness, atrophy and fasciculations of the limb and bulbar muscles, as a result of lower motor neuron degeneration. The disease is caused by an abnormally expanded triplet repeat expansions in the ubiquitously expressed androgen receptor gene, through mechanisms which are not entirely elucidated. Over the years studies from both humans and animal models have highlighted the involvement of cell populations other than motor neurons in SBMA, widening the disease phenotype. The most compelling aspect of these findings is their potential for therapeutic impact: muscle, for example, which is primarily affected in the disease, has been recently shown to represent a valid alternative target for therapy to motor neurons. In this review, we discuss the emerging study of the extra-motor neuron involvement in SBMA, which, besides increasingly pointing towards a multidisciplinary approach for affected patients, deepens our understanding of the pathogenic mechanisms and holds potential for providing new therapeutic targets for this disease.

Gene co-regulation by Fezf2 selects neurotransmitter identity and connectivity of corticospinal neurons.

The neocortex contains an unparalleled diversity of neuronal subtypes, each defined by distinct traits that are developmentally acquired under the control of subtype-specific and pan-neuronal genes. The regulatory logic that orchestrates the expression of these unique combinations of genes is unknown for any class of cortical neuron. Here, we report that Fezf2 is a selector gene able to regulate the expression of gene sets that collectively define mouse corticospinal motor neurons (CSMN). We find that Fezf2 directly induces the glutamatergic identity of CSMN via activation of Vglut1 (Slc17a7) and inhibits a GABAergic fate by repressing transcription of Gad1. In addition, we identify the axon guidance receptor EphB1 as a target of Fezf2 necessary to execute the ipsilateral extension of the corticospinal tract. Our data indicate that co-regulated expression of neuron subtype-specific and pan-neuronal gene batteries by a single transcription factor is one component of the regulatory logic responsible for the establishment of CSMN identity.

Polarized expression of p75(NTR) specifies axons during development and adult neurogenesis.

VIDEO ABSTRACT: Newly generated neurons initiate polarizing signals that specify a single axon and multiple dendrites, a process critical for patterning neuronal circuits in vivo. Here, we report that the pan-neurotrophin receptor p75(NTR) is a polarity regulator that localizes asymmetrically in differentiating neurons in response to neurotrophins and is required for specification of the future axon. In cultured hippocampal neurons, local exposure to neurotrophins causes early accumulation of p75(NTR) into one undifferentiated neurite to specify axon fate. Moreover, knockout or knockdown of p75(NTR) results in failure to initiate an axon in newborn neurons upon cell-cycle exit in vitro and in the developing cortex, as well as during adult hippocampal neurogenesis in vivo. Hence, p75(NTR) governs neuronal polarity, determining pattern and assembly of neuronal circuits in adult hippocampus and cortical development.

TrkB signaling directs the incorporation of newly generated periglomerular cells in the adult olfactory bulb.

In the adult rodent brain, the olfactory bulb (OB) is continuously supplied with new neurons which survival critically depends on their successful integration into pre-existing networks. Yet, the extracellular signals that determine the selection which neurons will be ultimately incorporated into these circuits are largely unknown. Here, we show that immature neurons express the catalytic form of the brain-derived neurotrophic factor receptor TrkB [full-length TrkB (TrkB-FL)] only after their arrival in the OB, at the time when integration commences. To unravel the role of TrkB signaling in newborn neurons, we conditionally ablated TrkB-FL in mice via Cre expression in adult neural stem and progenitor cells. TrkB-deficient neurons displayed a marked impairment in dendritic arborization and spine growth. By selectively manipulating the signaling pathways initiated by TrkB in vivo, we identified the transducers Shc/PI3K to be required for dendritic growth, whereas the activation of phospholipase C-γ was found to be responsible for spine formation. Furthermore, long-term genetic fate mapping revealed that TrkB deletion severely compromised the survival of new dopaminergic neurons, leading to a substantial reduction in the overall number of adult-generated periglomerular cells (PGCs), but not of granule cells (GCs). Surprisingly, this loss of dopaminergic PGCs was mirrored by a corresponding increase in the number of calretinin+ PGCs, suggesting that distinct subsets of adult-born PGCs may respond differentially to common extracellular signals. Thus, our results identify TrkB signaling to be essential for balancing the incorporation of defined classes of adult-born PGCs and not GCs, reflecting their different mode of integration in the OB.

The quest for myelin in the adult brain.

Although myelination largely occurs during early postnatal life, myelinating oligodendrocytes are still generated in the adult brain. Myelin turnover in the adult is necessary for proper neuronal function and is gravely compromised in myelin disorders. The lineage relationship between adult neural stem cells and adult-born oligodendrocytes has been clarified, highlighting molecular pathways that could potentially be targeted to favour de novo myelination in pathological situations.

TI-VAMP/VAMP7 is the SNARE of secretory lysosomes contributing to ATP secretion from astrocytes.

BACKGROUND INFORMATION: ATP is the main transmitter stored and released from astrocytes under physiological and pathological conditions. Morphological and functional evidence suggest that besides secretory granules, secretory lysosomes release ATP. However, the molecular mechanisms involved in astrocytic lysosome fusion remain still unknown. RESULTS: In the present study, we identify tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP, also called VAMP7) as the vesicular SNARE which mediates secretory lysosome exocytosis, contributing to release of both ATP and cathepsin B from glial cells. We also demonstrate that fusion of secretory lysosomes is triggered by slow and locally restricted calcium elevations, distinct from calcium spikes which induce the fusion of glutamate-containing clear vesicles. Downregulation of TI-VAMP/VAMP7 expression inhibited the fusion of ATP-storing vesicles and ATP-mediated calcium wave propagation. TI-VAMP/VAMP7 downregulation also significantly reduced secretion of cathepsin B from glioma. CONCLUSIONS: Given that sustained ATP release from glia upon injury greatly contributes to secondary brain damage and cathepsin B plays a critical role in glioma dissemination, TI-VAMP silencing can represent a novel strategy to control lysosome fusion in pathological conditions.

Essential role of Rac1 and Rac3 GTPases in neuronal development.

Rac GTPases are members of the Rho family regulating the actin cytoskeleton and implicated in neuronal development. Ubiquitous Rac1 and neuron-specific Rac3 GTPases are coexpressed in the developing mammalian brain. We used Cre-mediated conditional deletion of Rac1 in neurons combined with knockout of neuron-specific Rac3 to study the role of these GTPases in neural development. We found that lack of both genes causes motor behavioral defects, epilepsy, and premature death of mice. Deletion of either GTPase does not produce evident phenotypes. Double-knockout mice show specific defects in the development of the hippocampus. Selective impairment of the dorsal hilus of double-knockout animals is associated with alteration in the formation of the hippocampal circuitry. Axonal pathways to and from the dorsal hilus are affected because of the deficit of hilar mossy cells. Moreover, analysis of Rac function in hippocampal cultures shows that spine formation is strongly hampered only in neurons lacking both Rac proteins. These findings show for the first time that both Rac1 and Rac3 are important for the development of the nervous system, wherein they play complementary roles during late stages of neuronal and brain development.