Survival motor neuron deficiency slows myoblast fusion through reduced myomaker and myomixer expression.

BACKGROUND: Spinal muscular atrophy is an inherited neurodegenerative disease caused by insufficient levels of the survival motor neuron (SMN) protein. Recently approved treatments aimed at increasing SMN protein levels have dramatically improved patient survival and have altered the disease landscape. While restoring SMN levels slows motor neuron loss, many patients continue to have smaller muscles and do not achieve normal motor milestones. While timing of treatment is important, it remains unclear why SMN restoration is insufficient to fully restore muscle size and function. We and others have shown that SMN-deficient muscle precursor cells fail to efficiently fuse into myotubes. However, the role of SMN in myoblast fusion is not known. METHODS: In this study, we show that SMN-deficient myoblasts readily fuse with wild-type myoblasts, demonstrating fusion competency. Conditioned media from wild type differentiating myoblasts do not rescue the fusion deficit of SMN-deficient cells, suggesting that compromised fusion may primarily be a result of altered membrane dynamics at the cell surface. Transcriptome profiling of skeletal muscle from SMN-deficient mice revealed altered expression of cell surface fusion molecules. Finally, using cell and mouse models, we investigate if myoblast fusion can be rescued in SMN-deficient myoblast and improve the muscle pathology in SMA mice. RESULTS: We found reduced expression of the muscle fusion proteins myomaker (P = 0.0060) and myomixer (P = 0.0051) in the muscle of SMA mice. Suppressing SMN expression in C2C12 myoblast cells reduces expression of myomaker (35% reduction; P < 0.0001) and myomixer, also known as myomerger and minion, (30% reduction; P < 0.0001) and restoring SMN levels only partially restores myomaker and myomixer expression. Ectopic expression of myomixer improves myofibre number (55% increase; P = 0.0006) and motor function (35% decrease in righting time; P = 0.0089) in SMA model mice and enhances motor function (82% decrease in righting time; P < 0.0001) and extends survival (28% increase; P < 0.01) when administered in combination with an antisense oligonucleotide that increases SMN protein levels. CONCLUSIONS: Here, we identified reduced expression of muscle fusion proteins as a key factor in the fusion deficits of SMN-deficient myoblasts. This discovery provides a novel target to improve SMA muscle pathology and motor function, which in combination with SMN increasing therapy could enhance clinical outcomes for SMA patients.

CNS uptake of bortezomib is enhanced by P-glycoprotein inhibition: implications for spinal muscular atrophy.

The development of therapeutics for neurological disorders is constrained by limited access to the central nervous system (CNS). ATP-binding cassette (ABC) transporters, particularly P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), are expressed on the luminal surface of capillaries in the CNS and transport drugs out of the endothelium back into the blood against the concentration gradient. Survival motor neuron (SMN) protein, which is deficient in spinal muscular atrophy (SMA), is a target of the ubiquitin proteasome system. Inhibiting the proteasome in a rodent model of SMA with bortezomib increases SMN protein levels in peripheral tissues but not the CNS, because bortezomib has poor CNS penetrance. We sought to determine if we could inhibit SMN degradation in the CNS of SMA mice with a combination of bortezomib and the ABC transporter inhibitor tariquidar. In cultured cells we show that bortezomib is a substrate of P-gp. Mass spectrometry analysis demonstrated that intraperitoneal co-administration of tariquidar increased the CNS penetrance of bortezomib, and reduced proteasome activity in the brain and spinal cord. This correlated with increased SMN protein levels and improved survival and motor function of SMA mice. These findings show that CNS penetrance of treatment for this neurological disorder can be improved by inhibiting drug efflux at the blood-brain barrier.

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.

Non-targeted identification of prions and amyloid-forming proteins from yeast and mammalian cells.

The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin.

The E3 ubiquitin ligase mind bomb 1 ubiquitinates and promotes the degradation of survival of motor neuron protein.

Spinal muscular atrophy is an inherited motor neuron disease that results from a deficiency of the survival of motor neuron (SMN) protein. SMN is ubiquitinated and degraded through the ubiquitin proteasome system (UPS). We have previously shown that proteasome inhibition increases SMN protein levels, improves motor function, and reduces spinal cord, muscle, and neuromuscular junction pathology of spinal muscular atrophy (SMA) mice. Specific targets in the UPS may be more efficacious and less toxic. In this study, we show that the E3 ubiquitin ligase, mind bomb 1 (Mib1), interacts with and ubiquitinates SMN and facilitates its degradation. Knocking down Mib1 levels increases SMN protein levels in cultured cells. Also, knocking down the Mib1 orthologue improves neuromuscular function in Caenorhabditis elegans deficient in SMN. These findings demonstrate that Mib1 ubiquitinates and catalyzes the degradation of SMN, and thus represents a novel therapeutic target for SMA.

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.

Mitochondrial abnormalities in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a motor neuron disease caused by polyglutamine expansion mutation in the androgen receptor (AR). We investigated whether the mutant protein alters mitochondrial function. We found that constitutive and doxycycline-induced expression of the mutant AR in MN-1 and PC12 cells, respectively, are associated with depolarization of the mitochondrial membrane. This was mitigated by cyclosporine A, which inhibits opening of the mitochondrial permeability transition pore. We also found that the expression of the mutant protein in the presence of ligand results in an elevated level of reactive oxygen species, which is blocked by the treatment with the antioxidants co-enzyme Q10 and idebenone. The mutant protein in MN-1 cells also resulted in increased Bax, caspase 9 and caspase 3. We assessed the effects of mutant AR on the transcription of mitochondrial proteins and found altered expression of the peroxisome proliferator-activated receptor gamma coactivator 1 and the mitochondrial specific antioxidant superoxide dismutase-2 in affected tissues of SBMA knock-in mice. In addition, we found that the AR associates with mitochondria in cultured cells. This study thus provides evidence for mitochondrial dysfunction in SBMA cell and animal models, either through indirect effects on the transcription of nuclear-encoded mitochondrial genes or through direct effects of the mutant protein on mitochondria or both. These findings indicate possible benefit from mitochondrial therapy for SBMA.

Expression of expanded polyglutamine targets profilin for degradation and alters actin dynamics.

Huntington's disease is caused by polyglutamine expansion in the huntingtin protein. Huntingtin directly interacts with profilin, a major actin monomer sequestering protein and a key integrator of signals leading to actin polymerization. We observed a progressive loss of profilin in the cerebral cortex of Huntington's disease patients, and in cell culture and Drosophila models of polyglutamine disease. This loss of profilin is likely due to increased degradation through the ubiquitin proteasome system. Profilin loss reduces the F/G actin ratio, indicating a shift in actin polymerization. Overexpression of profilin abolishes mutant huntingtin toxicity in cells and partially ameliorates the morphological and functional eye phenotype and extends lifespan in a transgenic polyglutamine Drosophila model. These results indicate a link between huntingtin and profilin and implicate profilin in Huntington's disease pathogenesis.

Akt blocks ligand binding and protects against expanded polyglutamine androgen receptor toxicity.

Spinal and bulbar muscular atrophy (SBMA) is a progressive neurodegenerative disease caused by an expansion of the polyglutamine tract in the androgen receptor (AR). Here, we investigated the regulation of AR phosphorylation in order to understand factors that may modify SBMA disease progression. We show that expanded polyglutamine AR is phosphorylated by Akt. Substitution of the AR at two Akt consensus sites, S215 and S792, with aspartate, which mimics phosphorylation, reduces ligand binding, ligand-dependent nuclear translocation, transcriptional activation and toxicity of expanded polyglutamine AR. Co-expression of constitutively active Akt and the AR has similar consequences, which are blocked by alanine substitutions at residues 215 and 792. Furthermore, in motor neuron-derived MN-1 cells toxicity associated with polyglutamine-expanded AR is rescued by co-expression with Akt. Insulin-like growth factor-1 (IGF-1) stimulation, which activates several cell survival promoting pathways, also reduces toxicity of the expanded polyglutamine AR in MN-1 cells, in a manner dependent upon phospho-inositol-3-kinase. IGF-1 rescue of AR toxicity is diminished by alanine substitutions at the Akt consensus sites. These results highlight potential targets for therapeutic intervention in SBMA.

MicroRNA pathways modulate polyglutamine-induced neurodegeneration.

Nine human neurodegenerative diseases are due to expansion of a CAG repeat- encoding glutamine within the open reading frame of the respective genes. Polyglutamine (polyQ) expansion confers dominant toxicity, resulting in neuronal degeneration. MicroRNAs (miRNAs) have been shown to modulate programmed cell death during development. To address whether miRNA pathways play a role in neurodegeneration, we tested whether genes critical for miRNA processing modulated toxicity induced by the spinocerebellar ataxia type 3 (SCA3) protein. These studies revealed a striking enhancement of polyQ toxicity upon reduction of miRNA processing in Drosophila and human cells. In parallel genetic screens, we identified the miRNA bantam (ban) as a potent modulator of both polyQ and tau toxicity in flies. Our studies suggest that ban functions downstream of toxicity of the SCA3 protein, to prevent degeneration. These findings indicate that miRNA pathways dramatically modulate polyQ- and tau-induced neurodegeneration, providing the foundation for new insight into therapeutics.

The polyglutamine neurodegenerative protein ataxin 3 regulates aggresome formation.

The polyglutamine-containing neurodegenerative protein ataxin 3 (AT3) has deubiquitylating activity and binds ubiquitin chains with a preference for chains of four or more ubiquitins. Here we characterize the deubiquitylating activity of AT3 in vitro and show it trims/edits K48-linked ubiquitin chains. AT3 also edits polyubiquitylated (125)I-lysozyme and decreases its degradation by proteasomes. Cellular studies show that endogenous AT3 colocalizes with aggresomes and preaggresome particles of the misfolded cystic fibrosis transmembrane regulator (CFTR) mutant CFTRDeltaF508 and associates with histone deacetylase 6 and dynein, proteins required for aggresome formation and transport of misfolded protein. Small interfering RNA knockdown of AT3 greatly reduces aggresomes formed by CFTRDeltaF508, demonstrating a critical role of AT3 in this process. Wild-type AT3 restores aggresome formation; however, AT3 with mutations in the active site or ubiquitin interacting motifs cannot restore aggresome formation in AT3 knockdown cells. These same mutations decrease the association of AT3 and dynein. These data indicate that the deubiquitylating activity of AT3 and its ubiquitin interacting motifs as well play essential roles in CFTRDeltaF508 aggresome formation.