Naohide Kondo

Viral delivery of miR-196a ameliorates the SBMA phenotype via the silencing of CELF2

Spinal and bulbar muscular atrophy (SBMA) is an inherited neurodegenerative disorder caused by the expansion of the polyglutamine (polyQ) tract of the androgen receptor (AR-polyQ). Characteristics of SBMA include proximal muscular atrophy, weakness, contraction fasciculation and bulbar involvement. MicroRNAs (miRNAs) are a diverse class of highly conserved small RNA molecules that function as crucial regulators of gene expression in animals and plants. Recent functional studies have shown the potent activity of specific miRNAs as disease modifiers both in vitro and in vivo. Thus, potential therapeutic approaches that target the miRNA processing pathway have recently attracted attention. Here we describe a novel therapeutic approach using the adeno-associated virus (AAV) vector–mediated delivery of a specific miRNA for SBMA. We found that miR-196a enhanced the decay of the AR mRNA by silencing CUGBP, Elav-like family member 2 (CELF2). CELF2 directly acted on AR mRNA and enhanced the stability of AR mRNA. Furthermore, we found that the early intervention of miR-196a delivered by an AAV vector ameliorated the SBMA phenotypes in a mouse model. Our results establish the proof of principle that disease-specific miRNA delivery could be useful in neurodegenerative diseases.

Disrupted Transforming Growth Factor-β Signaling in Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA) is a late-onset lower motor neuron disease caused by the expansion of a trinucleotide CAG repeat, which encodes a polyglutamine tract in androgen receptor (AR). Although it is commonly held that the pathogenic polyglutamine proteins accumulate in neurons and thereby induce transcriptional dysregulation, the downstream molecular events have remained elusive. Here, we examined whether TGF-β signaling is dysregulated in SBMA. Nuclear translocation of phosphorylated Smad2/3, a key step in TGF-β signaling, is suppressed in the spinal motor neurons of male transgenic mice carrying the mutant human AR. A similar finding was also observed in the motor neurons, but not in Purkinje cells, of SBMA patients. The pathogenic AR, the causative protein of SBMA, inhibits the transcription of TGF-β receptor type II (TβRII) via abnormal interactions with NF-Y and p300/CBP-associated factor. Furthermore, overexpression of TβRII dampens polyglutamine-induced cytotoxicity in a neuroblastoma cell line expressing the pathogenic AR. The present study thus indicates that disruption of TGF-β due to the transcriptional dysregulation of TβRII is associated with polyglutamine-induced motor neuron damage in SBMA.

p62/SQSTM1 Differentially Removes the Toxic Mutant Androgen Receptor via Autophagy and Inclusion Formation in a Spinal and Bulbar Muscular Atrophy Mouse Model

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders that are caused by the expansion of trinucleotide CAG repeats in the causative genes. Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease that is caused by the expansion of a polyQ tract within the androgen receptor (AR). p62 is a ubiquitin- and light-chain 3-binding protein that is known to regulate the degradation of targeted proteins via autophagy and inclusion formation. In this study, we examined the effects of p62 depletion and overexpression on cultured cells and in a transgenic mouse model that overexpressed the mutant AR. Here, we demonstrate that depletion of p62 significantly exacerbated motor phenotypes and the neuropathological outcome, whereas overexpression of p62 protected against mutant AR toxicity in SBMA mice. Depletion of p62 significantly increased the levels of monomeric mutant AR and mutant AR protein complexes in an SBMA mouse model via the impairment of autophagic degradation. In addition, p62 overexpression improved SBMA mouse phenotypes by inducing cytoprotective inclusion formation. Our results demonstrate that p62 provides two different therapeutic targets in SBMA pathogenesis: (1) autophagy-dependent degradation and (2) benevolent inclusion formation of the mutant AR.

Heat shock factor-1 influences pathological lesion distribution of polyglutamine-induced neurodegeneration

A crucial feature of adult-onset neurodegenerative diseases is accumulation of abnormal protein in specific brain regions, although the mechanism underlying this pathological selectivity remains unclear. Heat shock factor-1 is a transcriptional regulator of heat shock proteins, molecular chaperones that abrogate neurodegeneration by refolding and solubilizing pathogenic proteins. Here we show that heat shock factor-1 expression levels are associated with the accumulation of pathogenic androgen receptor in spinal and bulbar muscular atrophy, a polyglutamine-induced neurodegenerative disease. In heterozygous heat shock factor-1-knockout spinal and bulbar muscular atrophy mice, abnormal androgen receptor accumulates in the cerebral visual cortex, liver and pituitary, which are not affected in their genetically unmodified counterparts. The depletion of heat shock factor-1 also expands the distribution of pathogenic androgen receptor accumulation in other neuronal regions. Furthermore, lentiviral-mediated delivery of heat shock factor-1 into the brain of spinal and bulbar muscular atrophy mice topically suppresses the pathogenic androgen receptor accumulation and neuronal atrophy. These results suggest that heat shock factor-1 influences the pathological lesion selectivity in spinal and bulbar muscular atrophy.

Naratriptan mitigates CGRP1-associated motor neuron degeneration caused by an expanded polyglutamine repeat tract

Spinal and bulbar muscular atrophy (SBMA) is a motor neuron disease caused by the expansion of the CAG triplet repeat within the androgen receptor (AR) gene. Here, we demonstrated that pathogenic AR upregulates the gene encoding calcitonin gene-related peptide α (CGRP1). In neuronal cells, overexpression of CGRP1 induced cellular damage via the activation of the c-Jun N-terminal kinase (JNK) pathway, whereas pharmacological suppression of CGRP1 or JNK attenuated the neurotoxic effects of pathogenic AR. The depletion of CGRP1 inactivated JNK and suppressed neurodegeneration in a mouse model of SBMA. Naratriptan, a serotonin 1B/1D (5-hydroxytryptamine 1B/1D, or 5-HT1B/1D) receptor agonist, decreased CGRP1 expression via the induction of dual-specificity protein phosphatase 1 (DUSP1), attenuated JNK activity and mitigated pathogenic AR-mediated neuronal damage in cellular and mouse SBMA models. These observations suggest that pharmacological activation of the 5-HT1B/1D receptor may be used therapeutically to treat SBMA and other polyglutamine-related neurodegenerative diseases.

Silencing neuronal mutant androgen receptor in a mouse model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA), an adult-onset neurodegenerative disease that affects males, results from a CAG triplet repeat/polyglutamine expansions in the androgen receptor (AR) gene. Patients develop progressive muscular weakness and atrophy, and no effective therapy is currently available. The tissue-specific pathogenesis, especially relative pathological contributions between degenerative motor neurons and muscles, remains inconclusive. Though peripheral pathology in skeletal muscle caused by toxic AR protein has been recently reported to play a pivotal role in the pathogenesis of SBMA using mouse models, the role of motor neuron degeneration in SBMA has not been rigorously investigated. Here, we exploited synthetic antisense oligonucleotides to inhibit the RNA levels of mutant AR in the central nervous system (CNS) and explore its therapeutic effects in our SBMA mouse model that harbors a mutant AR gene with 97 CAG expansions and characteristic SBMA-like neurogenic phenotypes. A single intracerebroventricular administration of the antisense oligonucleotides in the presymptomatic phase efficiently suppressed the mutant gene expression in the CNS, and delayed the onset and progression of motor dysfunction, improved body weight gain and survival with the amelioration of neuronal histopathology in motor units such as spinal motor neurons, neuromuscular junctions and skeletal muscle. These findings highlight the importance of the neurotoxicity of mutant AR protein in motor neurons as a therapeutic target.

Paeoniflorin eliminates a mutant AR via NF-YA-dependent proteolysis in spinal and bulbar muscular atrophy

The accumulation of abnormal proteins is a common characteristic of neurodegenerative diseases. This accumulation reflects a severe disturbance of cellular homeostasis in pathogenic protein clearance. Here, we demonstrated that the activation of the two major proteolytic machineries, the molecular chaperone-ubiquitin proteasome system (UPS) and the autophagy system, were simultaneously enhanced by paeoniflorin (PF), a major component of Paeonia plants, and exerted therapeutic effects in models of spinal and bulbar muscular atrophy (SBMA). PF significantly increased the expression of nuclear factor-YA (NF-YA), which strongly upregulated the molecules involved in the proteolytic machinery [molecular chaperones, carboxyl terminus of Hsc70-interacting protein and transcription factor EB], which thus mitigated the behavioral and pathological impairments in an SBMA mouse model through the upregulation of pathogenic androgen receptor protein clearance in motor neurons and muscles. These findings demonstrated that PF is able to enhance both the UPS and autophagy systems by upregulating the expression of NF-YA, which promotes therapeutic effects in an SBMA model.

IgM MGUS anti‐MAG neuropathy with predominant muscle weakness and extensive muscle atrophy

We report a patient with anti–myelin‐associated glycoprotein (MAG) neuropathy, predominantly exhibiting severe motor symptoms, accompanied by extensive muscle atrophy mimicking Charcot–Marie–Tooth disease. Nerve conduction studies revealed mild retardation of motor conduction velocities and significant prolongation of distal latency. Sural nerve biopsy revealed widely spaced myelin and positive staining of myelinated fibers with an IgM antibody. Predominant motor symptoms with muscle atrophy can be one of the clinical manifestations of anti‐MAG neuropathy. Muscle Nerve, 2010

Pioglitazone suppresses neuronal and muscular degeneration caused by polyglutamine-expanded androgen receptors

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by the expansion of a CAG repeat in the androgen receptor (AR) gene. Mutant AR has been postulated to alter the expression of genes important for mitochondrial function and induce mitochondrial dysfunction. Here, we show that the expression levels of peroxisome proliferator-activated receptor-γ (PPARγ), a key regulator of mitochondrial biogenesis, were decreased in mouse and cellular models of SBMA. Treatment with pioglitazone (PG), an activator of PPARγ, improved the viability of the cellular model of SBMA. The oral administration of PG also improved the behavioral and histopathological phenotypes of the transgenic mice. Furthermore, immunohistochemical and biochemical analyses demonstrated that the administration of PG suppressed oxidative stress, nuclear factor-κB (NFκB) signal activation and inflammation both in the spinal cords and skeletal muscles of the SBMA mice. These findings suggest that PG is a promising candidate for the treatment of SBMA.

Genistein, a natural product derived from soybeans, ameliorates polyglutamine‐mediated motor neuron disease

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine (polyQ) tract within the androgen receptor (AR) gene. The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem, and diffuse nuclear accumulation and nuclear inclusions of mutant AR in residual motor neurons and certain visceral organs. AR‐associated coregulator 70 (ARA70) was the first coregulator of AR to be identified, and it has been shown to interact with AR and increase its protein stability. Here, we report that genistein, an isoflavone found in soy, disrupts the interaction between AR and ARA70 and promotes the degradation of mutant AR in neuronal cells and transgenic mouse models of SBMA. We also demonstrate that dietary genistein ameliorates behavioral abnormalities, improves spinal cord and muscle pathology, and decreases the amounts of monomeric AR and high‐molecular‐weight mutant AR protein aggregates in SBMA transgenic mice. Thus, genistein treatment may be a potential therapeutic approach for alleviating the symptoms of SBMA by disrupting the interactions between AR and ARA70.