Kentaro Sahashi

Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model.

Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality; it results from loss-of-function mutations in the survival motor neuron 1 (SMN1) gene. Humans have a paralogue, SMN2, whose exon 7 is predominantly skipped, but the limited amount of functional, full-length SMN protein expressed from SMN2 cannot fully compensate for a lack of SMN1. SMN is important for the biogenesis of spliceosomal small nuclear ribonucleoprotein particles, but downstream splicing targets involved in pathogenesis remain elusive. There is no effective SMA treatment, but SMN restoration in spinal cord motor neurons is thought to be necessary and sufficient. Non-central nervous system (CNS) pathologies, including cardiovascular defects, were recently reported in severe SMA mouse models and patients, reflecting autonomic dysfunction or direct effects in cardiac tissues. Here we compared systemic versus CNS restoration of SMN in a severe mouse model. We used an antisense oligonucleotide (ASO), ASO-10-27, that effectively corrects SMN2 splicing and restores SMN expression in motor neurons after intracerebroventricular injection. Systemic administration of ASO-10-27 to neonates robustly rescued severe SMA mice, much more effectively than intracerebroventricular administration; subcutaneous injections extended the median lifespan by 25 fold. Furthermore, neonatal SMA mice had decreased hepatic Igfals expression, leading to a pronounced reduction in circulating insulin-like growth factor 1 (IGF1), and ASO-10-27 treatment restored IGF1 to normal levels. These results suggest that the liver is important in SMA pathogenesis, underscoring the importance of SMN in peripheral tissues, and demonstrate the efficacy of a promising drug candidate.

Motor neuron cell-nonautonomous rescue of spinal muscular atrophy phenotypes in mild and severe transgenic mouse models

Survival of motor neuron (SMN) deficiency causes spinal muscular atrophy (SMA), but the pathogenesis mechanisms remain elusive. Restoring SMN in motor neurons only partially rescues SMA in mouse models, although it is thought to be therapeutically essential. Here, we address the relative importance of SMN restoration in the central nervous system (CNS) versus peripheral tissues in mouse models using a therapeutic splice-switching antisense oligonucleotide to restore SMN and a complementary decoy oligonucleotide to neutralize its effects in the CNS. Increasing SMN exclusively in peripheral tissues completely rescued necrosis in mild SMA mice and robustly extended survival in severe SMA mice, with significant improvements in vulnerable tissues and motor function. Our data demonstrate a critical role of peripheral pathology in the mortality of SMA mice and indicate that peripheral SMN restoration compensates for its deficiency in the CNS and preserves motor neurons. Thus, SMA is not a cell-autonomous defect of motor neurons in SMA mice.

In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5′ splice sites

We have found that two previously reported exonic mutations in the PINK1 and PARK7 genes affect pre-mRNA splicing. To develop an algorithm to predict underestimated splicing consequences of exonic mutations at the 5′ splice site, we constructed and analyzed 31 minigenes carrying exonic splicing mutations and their derivatives. We also examined 189 249 U2-dependent 5′ splice sites of the entire human genome and found that a new variable, the SD-Score, which represents a common logarithm of the frequency of a specific 5′ splice site, efficiently predicts the splicing consequences of these minigenes. We also employed the information contents (Ri) to improve the prediction accuracy. We validated our algorithm by analyzing 32 additional minigenes as well as 179 previously reported splicing mutations. The SD-Score algorithm predicted aberrant splicings in 198 of 204 sites (sensitivity = 97.1%) and normal splicings in 36 of 38 sites (specificity = 94.7%). Simulation of all possible exonic mutations at positions −3, −2 and −1 of the 189 249 sites predicts that 37.8, 88.8 and 96.8% of these mutations would affect pre-mRNA splicing, respectively. We propose that the SD-Score algorithm is a practical tool to predict splicing consequences of mutations affecting the 5′ splice site.

Pathological impact of SMN2 mis-splicing in adult SMA mice

Loss‐of‐function mutations in SMN1 cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. The related SMN2 gene expresses suboptimal levels of functional SMN protein, due to a splicing defect. Many SMA patients reach adulthood, and there is also adult‐onset (type IV) SMA. There is currently no animal model for adult‐onset SMA, and the tissue‐specific pathogenesis of post‐developmental SMN deficiency remains elusive. Here, we use an antisense oligonucleotide (ASO) to exacerbate SMN2 mis‐splicing. Intracerebroventricular ASO injection in adult SMN2‐transgenic mice phenocopies key aspects of adult‐onset SMA, including delayed‐onset motor dysfunction and relevant histopathological features. SMN2 mis‐splicing increases during late‐stage disease, likely accelerating disease progression. Systemic ASO injection in adult mice causes peripheral SMN2 mis‐splicing and affects prognosis, eliciting marked liver and heart pathologies, with decreased IGF1 levels. ASO dose–response and time‐course studies suggest that only moderate SMN levels are required in the adult central nervous system, and treatment with a splicing‐correcting ASO shows a broad therapeutic time window. We describe distinctive pathological features of adult‐onset and early‐onset SMA.

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.

TSUNAMI: an antisense method to phenocopy splicing-associated diseases in animals

Antisense oligonucleotides (ASOs) are versatile molecules that can be designed to specifically alter splicing patterns of target pre-mRNAs. Here we exploit this feature to phenocopy a genetic disease. Spinal muscular atrophy (SMA) is a motor neuron disease caused by loss-of-function mutations in the SMN1 gene. The related SMN2 gene expresses suboptimal levels of functional SMN protein due to alternative splicing that skips exon 7; correcting this defect-e.g., with ASOs-is a promising therapeutic approach. We describe the use of ASOs that exacerbate SMN2 missplicing and phenocopy SMA in a dose-dependent manner when administered to transgenic Smn(-/-) mice. Intracerebroventricular ASO injection in neonatal mice recapitulates SMA-like progressive motor dysfunction, growth impairment, and shortened life span, with α-motor neuron loss and abnormal neuromuscular junctions. These SMA-like phenotypes are prevented by a therapeutic ASO that restores correct SMN2 splicing. We uncovered starvation-induced splicing changes, particularly in SMN2, which likely accelerate disease progression. These results constitute proof of principle that ASOs designed to cause sustained splicing defects can be used to induce pathogenesis and rapidly and accurately model splicing-associated diseases in animals. This approach allows the dissection of pathogenesis mechanisms, including spatial and temporal features of disease onset and progression, as well as testing of candidate therapeutics.

Functional characterisation of mitochondrial tRNA(Tyr) mutation (5877-->GA) associated with familial chronic progressive external ophthalmoplegia.

Editor—Chronic progressive external ophthalmoplegia (CPEO) is a common clinical manifestation of mitochondrial cytopathies characterised by ophthalmoplegia and ptosis.1 Approximately two-thirds of CPEO patients harbour a large, heteroplasmic, mitochondrial DNA (mtDNA) deletion.2 Some other CPEO patients carry a point mutation in the mitochondrial tRNA genes. Twelve point mutations in six mitochondrial tRNA genes have been reported to date in association with CPEO (Mitomap at http://www.gen.emory.edu/mitomap.html). Among the 12 mutations, 5703C→T in the tRNAAsn gene has been functionally characterised.3 Here we report functional analysis of 5877G→A in tRNATyr identified in a patient with CPEO4 using ρ cells that lack mtDNA. A 45 year old woman had moderate degrees of ptosis, external ophthalmoplegia, and proximal muscle weakness from the age of 28. She had no sensorineural hearing loss, ataxia, pigmentary retinopathy, hypogonadism, or mental retardation. She had episodic diarrhoea of unknown aetiology. An ECG showed atrioventricular conduction block, while EEG, brain CT, and brain MRI showed no abnormalities. An exercise loading test of 15 watts for 15 minutes on a bicycle ergometer5 raised her serum lactate from 6.9 mg/dl to 24.0 mg/dl (normal, less than 18.0 mg/dl), and her serum pyruvate from 0.6 mg/dl to 1.4 mg/dl (normal, less than 1.3 mg/dl), thereby increasing the lactate to pyruvate ratio from 10.7 to 17.1 (normal, less than 13.8). A biopsy specimen obtained from the biceps brachii showed 4.0% ragged red fibres and 0.7% cytochrome …

HMGCR antibody-associated myopathy as a paraneoplastic manifestation of esophageal carcinoma

Immune-mediated myopathies with antibodies may be triggered by statin exposure, but some patients with these disorders are statin-naive; thus, there are probably other etiologies.1 Several malignant tumors, including esophageal squamous cell carcinoma (ESCC), overexpress 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) protein.2 To date, the relationship between anti-HMGCR antibody–associated myopathy (HMGCR-M) and malignancy remains unclear.3,4 Here, we report a case of HMGCR-M in a patient with ESCC and paraneoplastic syndrome.

BIIB021, a synthetic Hsp90 inhibitor, induces mutant ataxin-1 degradation through the activation of heat shock factor 1

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by the expansion of a polyglutamine (polyQ) tract in ataxin-1 (ATXN1). The pathological hallmarks of SCA1 are the loss of cerebellar Purkinje cells and neurons in the brainstem and the presence of nuclear aggregates containing the polyQ-expanded ATXN1 protein. Heat shock protein 90 (Hsp90) inhibitors have been shown to reduce polyQ-induced toxicity. This study was designed to examine the therapeutic effects of BIIB021, a purine-scaffold Hsp90 inhibitor, on the protein homeostasis of polyQ-expanded mutant ATXN1 in a cell culture model of SCA1. Our results demonstrated that BIIB021 activated heat shock factor 1 (HSF1) and suppressed the abnormal accumulation of ATXN1 and its toxicity. The pharmacological degradation of mutant ATXN1 via activated HSF1 was dependent on both the proteasome and autophagy systems. These findings indicate that HSF1 is a key molecule in the regulation of the protein homeostasis of the polyQ-expanded mutant ATXN1 and that Hsp90 has potential as a novel therapeutic target in patients with SCA1.

Correction of RNA Splicing with Antisense Oligonucleotides as a Therapeutic Strategy for a Neurodegenerative Disease

Spinal muscular atrophy (SMA) is a severe genetic disease inherited in autosomal recessive fashion. It is the leading genetic cause of infant mortality. SMA is a neuromuscular disease, characterized by progressive degeneration and loss of α-motor neurons in the anterior horn of the spinal cord, which in turn leads to muscle weakness and atrophy, resulting in gradual paralysis. SMA is classified into four types on the basis of severity and time of onset: childhood-onset SMA ranges from type I, which is the most severe, to type III, which is considerably milder, with type II having intermediate severity [1–4]; adult-onset SMA is classified as type IV. There is no effective therapy for SMA.