Inherited Motor Neuron Disease Research Articles

Experiences of predictive genetic testing in inherited motor neuron disease: Findings from a qualitative interview study

Experiences of predictive genetic testing in inherited motor neuron disease: Findings from a qualitative interview study

Patient Reported Impact of Symptoms in Spinal Bulbar Muscular Atrophy (PRISM-SBMA) (S33.007)

Patient Reported Impact of Symptoms in Spinal Bulbar Muscular Atrophy (PRISM-SBMA) (S33.007)

T38. Altered axonal excitability in spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease characterized with prominent fasciculations. Fasciculations suggest motor nerve hyperexcitability and potentially result in a burden to motor neuron. Axonal excitability and nerve conduction studies were performed in 31 patients with SBMA and 140 with amyotrophic lateral sclerosis (ALS). These results were compared with those of 16 healthy males (HM). In SBMA, correlations between neurophysiological studies and clinical profiles were examined. Patient backgrounds in SBMA were as follows; mean age; 58 years, mean disease duration; 152 months, mean ALS functional rating scale (ALSFRS-R); 41, mean CAG repeat expansion; 46, mean compound muscle action potential (CMAP) amplitude on abductor pollicis brevis muscle was 6.0 mV. Compared with HM, SBMA patients had longer strength-duration time constant (SDTC), greater threshold change in depolarizing threshold electrotonus (TEd) and greater supernormality (all p < 0.05). ALS patients had similar findings and smaller late subexcitability (all p < 0.05). In SBMA, TEd (90–100 ms) was related to ALSFRS-R (R = 0.42, p < 0.05), and CAG repeat was related to CMAP amplitude (R = −0.52, p < 0.05) and superexcitability (R = −0.46, p < 0.05). Patients were divided into two groups according to CMAP amplitude (over and under 5 mV). Compared with HM, SBMA patients with over 5 mV had greater TEd (10–20 ms) and greater superexcitability (all p < 0.05). SBMA patients with under 5 mV had longer SDTC, greater TEd and greater superexcitability (all p < 0.05). ALS patients with over 5 mV had greater TEd (90–100 ms) and greater superexcitability (all p < 0.05). ALS patients with under 5 mV had longer SDTC, greater TEd, greater superexcitability and smaller subexcitability (all p < 0.05). A previous study investigated axonal excitabilities in 7 patients with SBMA and revealed increased sodium currents. Our findings suggest that SBMA patients have increased sodium and decreased fast potassium currents. Similar findings and decreased slow potassium currents were found in ALS. This difference may suggest that ALS patients have more hyperexcitable motor nerve. Moreover, our findings suggest that decreased fast potassium currents are related to CAG repeat expansion in SBMA. It is already reported that SBMA patients have disruption of axonal transport. CAG repeat expansion may bring about axonal hyperexcitability and result in a burden to motor neuron.

Overexpression of hepatocyte growth factor in SBMA model mice has an additive effect on combination therapy with castration

Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine (polyQ)-encoding 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. Hepatocyte growth factor (HGF) is a polypeptide growth factor which has neuroprotective properties. To investigate whether HGF overexpression can affect disease progression in a mouse model of SBMA, we crossed SBMA transgenic model mice expressing an AR gene with an expanded CAG repeat with mice overexpressing HGF. Here, we report that high expression of HGF induces Akt phosphorylation and modestly ameliorated motor symptoms in an SBMA transgenic mouse model treated with or without castration. These findings suggest that HGF overexpression can provide a potential therapeutic avenue as a combination therapy with disease-modifying therapies in SBMA.

Dysfunction of spatacsin leads to axonal pathology in SPG11-linked hereditary spastic paraplegia.

Hereditary spastic paraplegias are a group of inherited motor neuron diseases characterized by progressive paraparesis and spasticity. Mutations in the spastic paraplegia gene SPG11, encoding spatacsin, cause an autosomal-recessive disease trait; however, the precise knowledge about the role of spatacsin in neurons is very limited. We for the first time analyzed the expression and function of spatacsin in human forebrain neurons derived from human pluripotent stem cells including lines from two SPG11 patients and two controls. SPG11 patients'-derived neurons exhibited downregulation of specific axonal-related genes, decreased neurite complexity and accumulation of membranous bodies within axonal processes. Altogether, these data point towards axonal pathologies in human neurons with SPG11 mutations. To further corroborate spatacsin function, we investigated human pluripotent stem cell-derived neurons and mouse cortical neurons. In these cells, spatacsin was located in axons and dendrites. It colocalized with cytoskeletal and synaptic vesicle (SV) markers and was present in synaptosomes. Knockdown of spatacsin in mouse cortical neurons evidenced that the loss of function of spatacsin leads to axonal instability by downregulation of acetylated tubulin. Finally, time-lapse assays performed in SPG11 patients'-derived neurons and spatacsin-silenced mouse neurons highlighted a reduction in the anterograde vesicle trafficking indicative of impaired axonal transport. By employing SPG11 patient-derived forebrain neurons and mouse cortical neurons, this study provides the first evidence that SPG11 is implicated in axonal maintenance and cargo trafficking. Understanding the cellular functions of spatacsin will allow deciphering mechanisms of motor cortex dysfunction in autosomal-recessive hereditary spastic paraplegia.

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.

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.

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.

Widespread aggregation of mutant VAPB associated with ALS does not cause motor neuron degeneration or modulate mutant SOD1 aggregation and toxicity in mice

BackgroundA proline-to-serine substitution at position-56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) causes a form of dominantly inherited motor neuron disease (MND), including typical and atypical amyotrophic lateral sclerosis (ALS) and a mild late-onset spinal muscular atrophy (SMA). VAPB is an integral endoplasmic reticulum (ER) protein and has been implicated in various cellular processes, including ER stress, the unfolded protein response (UPR) and Ca2+ homeostasis. However, it is unclear how the P56S mutation leads to neurodegeneration and muscle atrophy in patients. The formation of abnormal VAPB-positive inclusions by mutant VAPB suggests a possible toxic gain of function as an underlying mechanism. Furthermore, the amount of VAPB protein is reported to be reduced in sporadic ALS patients and mutant SOD1G93A mice, leading to the hypothesis that wild type VAPB plays a role in the pathogenesis of ALS without VAPB mutations.ResultsTo investigate the pathogenic mechanism in vivo, we generated human wild type (wtVAPB) and mutant VAPB (muVAPB) transgenic mice that expressed the transgenes broadly in the CNS. We observed robust VAPB-positive aggregates in the spinal cord of muVAPB transgenic mice. However, we failed to find an impairment of motor function and motor neuron degeneration. We also did not detect any change in the endogenous VAPB level or evidence for induction of the unfolded protein response (UPR) and coaggregation of VAPA with muVAPB. Furthermore, we crossed these VAPB transgenic mice with mice that express mutant SOD1G93A and develop motor neuron degeneration. Overexpression of neither wtVAPB nor muVAPB modulated the protein aggregation and disease progression in the SOD1G93A mice.ConclusionOverexpression of VAPBP56S mutant to approximately two-fold of the endogenous VAPB in mouse spinal cord produced abundant VAPB aggregates but was not sufficient to cause motor dysfunction or motor neuron degeneration. Furthermore, overexpression of either muVAPB or wtVAPB does not modulate the course of ALS in SOD1G93A mice. These results suggest that changes in wild type VAPB do not play a significant role in ALS cases that are not caused by VAPB mutations. Furthermore, these results suggest that muVAPB aggregates are innocuous and do not cause motor neuron degeneration by a gain-of-toxicity, and therefore, a loss of function may be the underlying mechanism.

Spinal muscular atrophy, John Griffin, and mentorship

Hereditary canine spinal muscular atrophy is an inherited motor neuron disease that occurs in Brittany Spaniels and has remarkable similarities with human spinal muscular atrophy. Both disorders are characterized by proximal limb and truncal muscle weakness of variable severity. Detailed pathological studies indicate that there is early dysfunction of motor neuron synapses, particularly the neuromuscular junction synapse, prior to motor neuron death. This period of synaptic dysfunction may define a critical window of opportunity for disease reversibility in motor neuron disease.

Novel mutation in VCP gene causes atypical amyotrophic lateral sclerosis

To identify the genetic variant that causes autosomal dominantly inherited motor neuron disease in a 4-generation Israeli-Arab family using genetic linkage and whole exome sequencing. Genetic linkage analysis was performed in this family using Illumina single nucleotide polymorphism chips. Whole exome sequencing was then undertaken on DNA samples from 2 affected family members using an Illumina 2000 HiSeq platform in pursuit of potentially pathogenic genetic variants that comigrate with the disease in this pedigree. Variants meeting these criteria were then screened in all affected individuals. A novel mutation (p.R191G) in the valosin-containing protein (VCP) gene was identified in the index family. Direct sequencing of the VCP gene in a panel of DNA from 274 unrelated individuals with familial amyotrophic lateral sclerosis (FALS) revealed 5 additional mutations. Among them, 2 were previously identified in pedigrees with a constellation of inclusion body myopathy with Paget disease of the bone and frontotemporal dementia (IBMPFD) and in FALS, and 2 other mutations (p.R159C and p.R155C) in IBMPFD alone. We did not detect VCP gene mutations in DNA from 178 cases of sporadic amyotrophic lateral sclerosis. We report a novel VCP mutation identified in an amyotrophic lateral sclerosis family (p.R191G) with atypical clinical features. In our experience, VCP mutations arise in approximately 1.5% of FALS cases. Our study supports the view that motor neuron disease is part of the clinical spectrum of VCP-associated disease.

Motor terminal degeneration unaffected by activity changes in SOD1G93A mice; a possible role for glycolysis

This study examined whether activity is a contributing factor to motor terminal degeneration in mice that overexpress the G93A mutation of the SOD1 enzyme found in humans with inherited motor neuron disease. Previously, we showed that overload of muscles accomplished by synergist denervation accelerated motor terminal degeneration in dogs with hereditary canine spinal muscular atrophy (HCSMA). In the present study, we found that SOD1 plantaris muscles overloaded for 2months showed no differences of neuromuscular junction innervation status when compared with normally loaded, contralateral plantaris muscles. Complete elimination of motor terminal activity using blockade of sciatic nerve conduction with tetrodotoxin cuffs for 1month also produced no change of plantaris innervation status. To assess possible effects of activity on motor terminal function, we examined the synaptic properties of SOD1 soleus neuromuscular junctions at a time when significant denervation of close synergists had occurred as a result of natural disease progression. When examined in glucose media, SOD1 soleus synaptic properties were similar to wildtype. When glycolysis was inhibited and ATP production limited to mitochondria, however, blocking of evoked synaptic transmission occurred and a large increase in the frequency of spontaneous mEPCs was observed. Similar effects were observed at neuromuscular junctions in muscle from dogs with inherited motor neuron disease (HCSMA), although significant defects of synaptic transmission exist at these neuromuscular junctions when examined in glucose media, as reported previously. These results suggest that glycolysis compensates for mitochondrial dysfunction at motor terminals of SOD1 mice and HCSMA dogs. This compensatory mechanism may help to support resting and activity-related metabolism in the presence of dysfunctional mitochondria and prolong the survival of SOD1 motor terminals.

Survival Motor Neuron Protein in Motor Neurons Determines Synaptic Integrity in Spinal Muscular Atrophy

The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein and results in severe muscle weakness. In SMA mice, synaptic dysfunction of both neuromuscular junctions (NMJs) and central sensorimotor synapses precedes motor neuron cell death. To address whether this synaptic dysfunction is due to SMN deficiency in motor neurons, muscle, or both, we generated three lines of conditional SMA mice with tissue-specific increases in SMN expression. All three lines of mice showed increased survival, weights, and improved motor behavior. While increased SMN expression in motor neurons prevented synaptic dysfunction at the NMJ and restored motor neuron somal synapses, increased SMN expression in muscle did not affect synaptic function although it did improve myofiber size. Together these data indicate that both peripheral and central synaptic integrity are dependent on motor neurons in SMA, but SMN may have variable roles in the maintenance of these different synapses. At the NMJ, it functions at the presynaptic terminal in a cell-autonomous fashion, but may be necessary for retrograde trophic signaling to presynaptic inputs onto motor neurons. Importantly, SMN also appears to function in muscle growth and/or maintenance independent of motor neurons. Our data suggest that SMN plays distinct roles in muscle, NMJs, and motor neuron somal synapses and that restored function of SMN at all three sites will be necessary for full recovery of muscle power.

Motor Unit Pathology in SMA Patients (P03.175)

Objective: To examine the pathology of the motor unit in spinal muscular atrophy (SMA). Background SMA is an inherited motor neuron disease, which often causes early mortality. Although there is currently no disease-modify treatment to offer patients, several drugs show promising effects in SMA mouse models in which the pathology of the motor unit has been well defined. In mice, there are early abnormalities of motor neuron (MN) terminals prior to MN loss with differential susceptibility of certain MN groups. Limited prior studies of human SMA pathology have not characterized early changes of MNs, their axons, or terminals. Design/Methods: At autopsy, spinal cord, nerve roots, phrenic nerve, and skeletal muscle were collected from 14 SMA cases and analyzed with confocal and electron microscopy methods. Results: Vast numbers of small, round myofibers were evident in the psoas, paraspinal, and intercostal muscles, but myofiber size was relatively preserved in diaphragm muscle. Phrenic nerve showed an average of 1830±177 axons/nerve in SMA cases, while ventral roots exhibited a severe reduction in axonal density. Active axonal degeneration was present in ventral roots and phrenic nerve where 3.7±0.45% of axons showed a compacted myelin appearance. Neuromuscular junctions (NMJs) in the diaphragm were often well innervated, but many axon terminals displayed neurofilament aggregation and others were very thin. EM analysis confirmed neurofilament aggregation and reduced density of synaptic vesicles in SMA diaphragm NMJs. Conclusions: These studies show that motor units are differentially affected in human SMA, with phrenic nerve motor neurons innervating the diaphragm less affected than those innervating the paraspinal, intercostal, or proximal limb muscles. The diaphragm may therefore provide important insights into early SMA disease pathology. In this muscle, most NMJs were innervated but structurally abnormal. These studies raise the possibility that the distal motor neuron terminal and axon are the earliest sites of disease pathogenesis in human SMA. Supported by: The SMA Foundation and by R01NS062869 (CJS) from NINDS and by R01-HD054599 (KJS) from NICHD. Disclosure: Dr. Crowder has nothing to disclose. Dr. Polley has nothing to disclose. Dr. Kong has nothing to disclose. Dr. Van Meerbeke has nothing to disclose. Dr. Murphy has nothing to disclose. Dr. Swoboda has received research support from BioMarin Pharmaceuticals and Orphamed, Dr. Crawford has nothing to disclose. Dr. Sumner has nothing to disclose.

The Therapeutic Effects of RG3039 in Severe Spinal Muscular Atrophy Mice and Normal Human Volunteers (SC01.002)

Objective: To investigate the therapeutic effects of RG3039 in spinal muscular atrophy (SMA). Background Spinal muscular atrophy (SMA) is an autosomal recessively inherited motor neuron disease that frequently results in early mortality. SMA is caused by homozygous mutation of the SMN1 gene and retention of SMN2, a highly similar gene that expresses insufficient levels of SMN protein. Currently there is no disease modifying treatment to offer patients. SMN2 copy number inversely correlates with disease severity in humans and robust increases in SMN expression prevents SMA in mice. RG3039, a quinazoline derivative, increases SMN2 promoter activity (Jarecki et al., 2005) and inhibits the scavenger decapping enzyme DcpS (Singh et al., 2008). Design/Methods: Severe SMA mice were treated with 0, 3, or 10 mg/kg of RG3039 daily beginning on the day of birth (P1). Behavioral, biochemical, and histological outcomes were assessed. A Phase I safety study of RG3039 in healthy human volunteers is ongoing. Results: RG3039 improves median survival by 29%, maximum body weight achieved by 15%, and motor behavioral outcomes in severe SMA mice. These effects are associated with increased SMN2 transcript levels, improved neuromuscular junction (NMJ) morphology and physiology, increased muscle and myofiber area, and increased number of vGluT1 synapses onto lumbar motor neurons. Interim phase I clinical results in healthy volunteers indicate no adverse effects at drug exposure that inhibits DcpS enzyme completely in circulating PBMC. Conclusions: RG3039 improves survival, motor behavior, and motor unit structure and function in severe SMA mice. Dosing in humans has produced no adverse effects to date. Phase I trials in SMA patients will be initiated in early 2012 and will represent the first clinical trial of a compound developed solely for the treatment of SMA. Supported by: Funding from the Repligen Corporation and grant support from the MDA, Families of Spinal Muscular Atrophy to CK and the National Institutes of Health (R01NS062869 to CJS). Disclosure: Dr. Van Meerbeke has nothing to disclose. Dr. Gibbs has nothing to disclose. Dr. Plasterer has received personal compensation for activities with Repligen Corporation as an employee. Dr. Feng has nothing to disclose. Dr. Lin has nothing to disclose. Dr. Wee has nothing to disclose. Dr. Xia has received personal compensation for activities with Repligen Corporation as an employee. Dr. Jacques has received personal compensation for activities with Repligen Corporation as an employee. Dr. Rusche has received personal compensation for activities with Repligen Corporation as an employee. Dr. Ko has nothing to disclose. Dr. Sumner has nothing to disclose.

A Role for SMN Exon 7 Splicing in the Selective Vulnerability of Motor Neurons in Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by homozygous loss of the Survival Motor Neuron 1 (SMN1) gene. In the absence of SMN1, inefficient inclusion of exon 7 in transcripts from the nearly identical SMN2 gene results in ubiquitous SMN decrease but selective motor neuron degeneration. Here we investigated whether cell type-specific differences in the efficiency of exon 7 splicing contribute to the vulnerability of SMA motor neurons. We show that normal motor neurons express markedly lower levels of full-length SMN mRNA from SMN2 than do other cells in the spinal cord. This is due to inefficient exon 7 splicing that is intrinsic to motor neurons under normal conditions. We also find that SMN depletion in mammalian cells decreases exon 7 inclusion through a negative feedback loop affecting the splicing of its own mRNA. This mechanism is active in vivo and further decreases the efficiency of exon 7 inclusion specifically in motor neurons of severe-SMA mice. Consistent with expression of lower levels of full-length SMN, we find that SMN-dependent downstream molecular defects are exacerbated in SMA motor neurons. These findings suggest a mechanism to explain the selective vulnerability of motor neurons to loss of SMN1.

Increased IGF-1 in muscle modulates the phenotype of severe SMA mice

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by the mutation of the survival motor neuron 1 (SMN1) gene and deficiency of the SMN protein. Severe SMA mice have abnormal motor function and small, immature myofibers early in development suggesting that SMN protein deficiency results in retarded muscle growth. Insulin-like growth factor 1 (IGF-1) stimulates myoblast proliferation, induces myogenic differentiation and generates myocyte hypertrophy in vitro and in vivo. We hypothesized that increased expression of IGF-1 specifically in skeletal muscle would attenuate disease features of SMAΔ7 mice. SMAΔ7 mice overexpressing a local isoform of IGF-1 (mIGF-1) in muscle showed enlarged myofibers and a 40% increase in median survival compared with mIGF-1-negative SMA littermates (median survival = 14 versus 10 days, respectively, log-rank P = 0.025). Surprisingly, this was not associated with a significant improvement in motor behavior. Treatment of both mIGF-1(NEG) and mIGF-1(POS) SMA mice with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in a further extension of survival and improved motor behavior, but the combination of mIGF-1 and TSA treatment was not synergistic. These results show that increased mIGF-1 expression restricted to muscle can modulate the phenotype of SMA mice indicating that therapeutics targeted to muscle alone should not be discounted as potential disease-modifying therapies in SMA. IGF-1 may warrant further investigation in mild SMA animal models and perhaps SMA patients.

Cardiac defects contribute to the pathology of spinal muscular atrophy models

Spinal muscular atrophy (SMA) is an autosomal recessive disorder, which is the leading genetic cause of infantile death. SMA is the most common inherited motor neuron disease and occurs in approximately 1:6000 live births. The gene responsible for SMA is called Survival Motor Neuron-1 (SMN1). Interestingly, a human-specific copy gene is present on the same region of chromosome 5q, called SMN2. Motor neurons are the primary tissue affected in SMA. Although it is clear that SMA is a neurodegenerative disease, there are clinical reports that suggest that other tissues contribute to the overall phenotype, especially in the most severe forms of the disease. In severe SMA cases, a growing number of congenital heart defects have been identified upon autopsy. The most common defect is a developmental defect referred to as hypoplastic left heart. The purpose of this report is to determine whether cardiac tissue is altered in SMA models and whether this could contribute to SMA pathogenesis. Here we identified early-stage developmental defects in a severe model of SMA. Additionally, pathological responses including fibrosis and oxidative stress markers were observed shortly after birth in a less severe model of disease. Similarly, functional differences were detected between wild-type and early-stage SMA animals. Collectively, this work demonstrates the importance of cardiac development and function in these severe models of SMA.

Abnormal interaction of motor neuropathy-associated mutant HspB8 (Hsp22) forms with the RNA helicase Ddx20 (gemin3)

A number of missense mutations in the two related small heat shock proteins HspB8 (Hsp22) and HspB1 (Hsp27) have been associated with the inherited motor neuron diseases (MND) distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 and HspB1 interact with each other, suggesting that these two etiologic factors may act through a common biochemical mechanism. However, their role in neuron biology and in MND is not understood. In a yeast two-hybrid screen, we identified the DEAD box protein Ddx20 (gemin3, DP103) as interacting partner of HspB8. Using co-immunoprecipitation, chemical cross-linking, and in vivo quantitative fluorescence resonance energy transfer, we confirmed this interaction. We also show that the two disease-associated mutant HspB8 forms have abnormally increased binding to Ddx20. Ddx20 itself binds to the survival-of-motor-neurons protein (SMN protein), and mutations in the SMN1 gene cause spinal muscular atrophy, another MND and one of the most prevalent genetic causes of infant mortality. Thus, these protein interaction data have linked the three etiologic factors HspB8, HspB1, and SMN protein, and mutations in any of their genes cause the various forms of MND. Ddx20 and SMN protein are involved in spliceosome assembly and pre-mRNA processing. RNase treatment affected the interaction of the mutant HspB8 with Ddx20 suggesting RNA involvement in this interaction and a potential role of HspB8 in ribonucleoprotein processing.

Inhibition of myostatin does not ameliorate disease features of severe spinal muscular atrophy mice

There is currently no treatment for the inherited motor neuron disease, spinal muscular atrophy (SMA). Severe SMA causes lower motor neuron loss, impaired myofiber development, profound muscle weakness and early mortality. Myostatin is a transforming growth factor-beta family member that inhibits muscle growth. Loss or blockade of myostatin signaling increases muscle mass and improves muscle strength in mouse models of primary muscle disease and in the motor neuron disease, amyotrophic lateral sclerosis. In this study, we evaluated the effects of blocking myostatin signaling in severe SMA mice (hSMN2/delta7SMN/mSmn(-/-)) by two independent strategies: (i) transgenic overexpression of the myostatin inhibitor follistatin and (ii) post-natal administration of a soluble activin receptor IIB (ActRIIB-Fc). SMA mice overexpressing follistatin showed little increase in muscle mass and no improvement in motor function or survival. SMA mice treated with ActRIIB-Fc showed minimal improvement in motor function, and no extension of survival compared with vehicle-treated mice. Together these results suggest that inhibition of myostatin may not be a promising therapeutic strategy in severe forms of SMA.