Genetic factors affecting survival in Japanese patients with sporadic amyotrophic lateral sclerosis: a genome-wide association study and verification in iPSC-derived motor neurons from patients.

BACKGROUND: Several genetic factors are associated with the pathogenesis of sporadic amyotrophic lateral sclerosis (ALS) and its phenotypes, such as disease progression. Here, in this study, we aimed to identify the genes that affect the survival of patients with sporadic ALS. METHODS: We enrolled 1076 Japanese patients with sporadic ALS with imputed genotype data of 7 908 526 variants. We used Cox proportional hazards regression analysis with an additive model adjusted for sex, age at onset and the first two principal components calculated from genotyped data to conduct a genome-wide association study. We further analysed messenger RNA (mRNA) and phenotype expression in motor neurons derived from induced pluripotent stem cells (iPSC-MNs) of patients with ALS. RESULTS: Three novel loci were significantly associated with the survival of patients with sporadic ALS-FGF1 at 5q31.3 (rs11738209, HR=2.36 (95% CI, 1.77 to 3.15), p=4.85×10-9), THSD7A at 7p21.3 (rs2354952, 1.38 (95% CI, 1.24 to 1.55), p=1.61×10-8) and LRP1 at 12q13.3 (rs60565245, 2.18 (95% CI, 1.66 to 2.86), p=2.35×10-8). FGF1 and THSD7A variants were associated with decreased mRNA expression of each gene in iPSC-MNs and reduced in vitro survival of iPSC-MNs obtained from patients with ALS. The iPSC-MN in vitro survival was reduced when the expression of FGF1 and THSD7A was partially disrupted. The rs60565245 was not associated with LRP1 mRNA expression. CONCLUSIONS: We identified three loci associated with the survival of patients with sporadic ALS, decreased mRNA expression of FGF1 and THSD7A and the viability of iPSC-MNs from patients. The iPSC-MN model reflects the association between patient prognosis and genotype and can contribute to target screening and validation for therapeutic intervention.

Monomerization of TDP-43 is a key determinant for inducing TDP-43 pathology in amyotrophic lateral sclerosis.

The cytoplasmic aggregation of TAR DNA binding protein-43 (TDP-43), also known as TDP-43 pathology, is the pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism underlying TDP-43 cytoplasmic mislocalization and subsequent aggregation remains unclear. Here, we show that TDP-43 dimerization/multimerization is impaired in the postmortem brains and spinal cords of patients with sporadic ALS and that N-terminal dimerization-deficient TDP-43 consists of pathological inclusion bodies in ALS motor neurons. Expression of N-terminal dimerization-deficient mutant TDP-43 in Neuro2a cells and induced pluripotent stem cell-derived motor neurons recapitulates TDP-43 pathology, such as Nxf1-dependent cytoplasmic mislocalization and aggregate formation, which induces seeding effects. Furthermore, TDP-DiLuc, a bimolecular luminescence complementation reporter assay, could detect decreased N-terminal dimerization of TDP-43 before TDP-43 pathological changes caused by the transcription inhibition linked to aberrant RNA metabolism in ALS. These findings identified TDP-43 monomerization as a critical determinant inducing TDP-43 pathology in ALS.

Simple and efficient differentiation of human iPSCs into contractible skeletal muscles for muscular disease modeling.

Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patient pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) differentiated into affected cell types can potentially recapitulate disease pathology more accurately than existing disease models. Such successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection, and clonal variations must be overcome. Moreover, their functionality should be carefully examined. Here, we demonstrated that bulk MYOD1-hiPSCs established with puromycin selection rather than G418 selection showed rapid and highly efficient differentiation. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties of clonally established MYOD1-hiPSCs, suggesting that it is possible to minimize clonal variations. Moreover, disease-specific hiPSCs of spinal bulbar muscular atrophy (SBMA) could be efficiently differentiated via this method into skeletal muscle that showed disease phenotypes, suggesting the applicability of this method for disease analysis. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical stimulation, indicating their functionality. Thus, our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and may facilitate the generation of muscular disease models.

Modulation of oxygen tension, acidosis, and cell density is crucial for neural differentiation of human induced pluripotent stem cells.

Human induced pluripotent stem cell (hiPSC)-derived neural cells provide valuable disease models for pathophysiological analysis and drug discovery for intractable neurodegenerative diseases. However, neural differentiation of hiPSCs requires a complex and long culture procedure, which has been a bottleneck for analysis. We previously demonstrated rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells (hPSCs). Although optimization of the microenvironment for the differentiation of hPSCs has been considered to achieve more efficient differentiation, it has never been investigated in detail. Here, we demonstrated that three microenvironmental modifiers, oxygen (O2) tension, pH, and cell density, critically affect neural differentiation of hiPSCs. Hypoxia is known to be involved in neural development in vivo and to promote neural differentiation of PSCs. However, in this study, it caused significant cell death in aggregation culture of human embryoid bodies (hEBs) and negatively affected neural differentiation. Modulation of pH by optimized carbon dioxide (CO2) tension improved neural differentiation of hiPSCs, but mild acidosis caused by increased CO2 tension suppressed neural differentiation without cell death. Moreover, high-cell density culture resulted in prominent acidosis and cell death under hypoxic conditions, which synergistically suppressed neural differentiation of hiPSCs. These results suggest that optimization of the microenvironment via O2 tension, pH, and cell density enables more efficient neural differentiation of hiPSCs for the analysis of neurological diseases.

Selective suppression of polyglutamine-expanded protein by lipid nanoparticle-delivered siRNA targeting CAG expansions in the mouse CNS.

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by expansion of cytosine-adenine-guanine (CAG)-trinucleotide repeats in causative genes. These diseases include spinal and bulbar muscular atrophy (SBMA), Huntington's disease, dentatorubral-pallidoluysian atrophy, and spinocerebellar ataxias. Targeting expanded CAG repeats is a common therapeutic approach to polyQ diseases, but concomitant silencing of genes with normal CAG repeats may lead to toxicity. Previous studies have shown that CAG repeat-targeting small interfering RNA duplexes (CAG-siRNAs) have the potential to selectively suppress mutant proteins in in vitro cell models of polyQ diseases. However, in vivo application of these siRNAs has not yet been investigated. In this study, we demonstrate that an unlocked nucleic acid (UNA)-modified CAG-siRNA shows high selectivity for polyQ-expanded androgen receptor (AR) inhibition in in vitro cell models and that lipid nanoparticle (LNP)-mediated delivery of the CAG-siRNA selectively suppresses mutant AR in the central nervous system of an SBMA mouse model. In addition, a subcutaneous injection of the LNP-delivered CAG-siRNA efficiently suppresses mutant AR in the skeletal muscle of the SBMA mouse model. These results support the therapeutic potential of LNP-delivered UNA-modified CAG-siRNAs for selective suppression of mutant proteins in SBMA and other polyQ diseases.

Unveiling synapse pathology in spinal bulbar muscular atrophy by genome-wide transcriptome analysis of purified motor neurons derived from disease specific iPSCs.

Spinal bulbar muscular atrophy (SBMA) is an adult-onset, slowly progressive motor neuron disease caused by abnormal CAG repeat expansion in the androgen receptor (AR) gene. Although ligand (testosterone)-dependent mutant AR aggregation has been shown to play important roles in motor neuronal degeneration by the analyses of transgenic mice models and in vitro cell culture models, the underlying disease mechanisms remain to be fully elucidated because of the discrepancy between model mice and SBMA patients. Thus, novel human disease models that recapitulate SBMA patients' pathology more accurately are required for more precise pathophysiological analysis and the development of novel therapeutics. Here, we established disease specific iPSCs from four SBMA patients, and differentiated them into spinal motor neurons. To investigate motor neuron specific pathology, we purified iPSC-derived motor neurons using flow cytometry and cell sorting based on the motor neuron specific reporter, HB9e438::Venus, and proceeded to the genome-wide transcriptome analysis by RNA sequences. The results revealed the involvement of the pathology associated with synapses, epigenetics, and endoplasmic reticulum (ER) in SBMA. Notably, we demonstrated the involvement of the neuromuscular synapse via significant upregulation of Synaptotagmin, R-Spondin2 (RSPO2), and WNT ligands in motor neurons derived from SBMA patients, which are known to be associated with neuromuscular junction (NMJ) formation and acetylcholine receptor (AChR) clustering. These aberrant gene expression in neuromuscular synapses might represent a novel therapeutic target for SBMA.

Src inhibition attenuates polyglutamine-mediated neuromuscular degeneration in spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by an expanded CAG repeat in the androgen receptor (AR) gene. Here, we perform a comprehensive analysis of signaling pathways in a mouse model of SBMA (AR-97Q mice) utilizing a phosphoprotein assay. We measure the levels of 17 phosphorylated proteins in spinal cord and skeletal muscle of AR-97Q mice at three stages. The level of phosphorylated Src (p-Src) is markedly increased in the spinal cords and skeletal muscles of AR-97Q mice prior to the onset. Intraperitoneal administration of a Src kinase inhibitor improves the behavioral and histopathological phenotypes of the transgenic mice. We identify p130Cas as an effector molecule of Src and show that the phosphorylated p130Cas is elevated in murine and cellular models of SBMA. These results suggest that Src kinase inhibition is a potential therapy for SBMA.

Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells.

BACKGROUND: Human pluripotent stem cells (hPSCs) are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders. In particular, neural cells derived from disease-specific human induced pluripotent stem cells (hiPSCs) established from patients with neurological disorders have been used as in vitro disease models to recapitulate in vivo pathogenesis because neural cells cannot be usually obtained from patients themselves. RESULTS: In this study, we established a rapid, efficient, and simple method for efficiently deriving motor neurons from hPSCs that is useful for pathophysiological analysis and the development of drugs to treat motor neuron diseases. Treatment with GSK3ß inhibitors during the initial phase of differentiation in combination with dual SMAD inhibition was sufficient to induce PAX6 (+) and SOX1 (+) neural progenitors within 1 week, and subsequent treatment with retinoic acid (RA) and purmorphamine, which activates sonic hedgehog (SHH) signaling, resulted in the highly efficient induction of HB9(+) and ISL-1(+) motor neurons within 2 weeks. After 4 weeks of monolayer differentiation in motor neuron maturation medium, hPSC-derived motor neurons were shown to mature, displaying larger somas and clearer staining for the mature motor neuron marker choline acetyltransferase (ChAT). Moreover, hPSC-derived motor neurons were able to form neuromuscular junctions with human myotubes in vitro and induced acetylcholine receptor (AChR) clustering, as detected by Alexa 555-conjugated α-Bungarotoxin (α-BTX), suggesting that these hPSC-derived motor neurons formed functional contacts with skeletal muscles. This differentiation system is simple and is reproducible in several hiPSC clones, thereby minimizing clonal variation among hPSC clones. We also established a system for visualizing motor neurons with a lentiviral reporter for HB9 (HB9 (e438) ::Venus). The specificity of this reporter was confirmed through immunocytochemistry and quantitative RT-PCR analysis of high-positive fractions obtained via fluorescence-activated cell sorting (FACS), suggesting its applicability for motor neuron-specific analysis. CONCLUSIONS: Our motor neuron differentiation system and lentivirus-based reporter system for motor neurons facilitate the analysis of disease-specific hiPSCs for motor neuron diseases.

[Bilateral internal carotid artery occlusion and severe basilar artery stenosis in a patient with fibromuscular dysplasia: a case report].

Fibromuscular dysplasia (FMD) is a non-atherosclerotic, non-inflammatory arterial disease of unknown etiology. We report a 26-year-old woman who presented with transient ischemic attack (TIA) due to bilateral internal carotid artery (ICA) occlusion and severe basilar artery stenosis, as FMD was diagnosed by a biopsy specimen of right ICA. Imaging investigations included magnetic resonance angiography and catheter angiogram without characteristic "string of beads" pattern, before reaching a definitive diagnosis by pathologist. Anti-platelet therapy and bypass surgery of superficial temporal artery-middle cerebral artery revealed no more clinical symptoms. This case of intra- and extra-cranial FMD gives a consideration of such rare disease in the differential diagnosis of TIA or stroke in healthy young patients. The literature of FMD is reviewed including pathological findings.

A case of consciousness disturbance resulting from severe hypothyroidism due to chronic thyroiditis and excess iodine absorption.

An 82-year-old Japanese man had consciousness disturbance due to severe hypothyroidism triggered by percutaneous absorption of iodine from an iodine-containing ointment used in diabetic gangrene treatment. Laboratory data revealed extremely high urinary iodine concentrations, and chronic thyroiditis-induced hypothyroidism. Excess iodine intake can also cause hypothyroidism. It was unlikely that iodine intoxication or Hashimoto's encephalopathy had caused the consciousness disturbance. The patient regained consciousness after discontinuing the use of the ointment and commencing thyroid hormone therapy. We conclude that consciousness disturbance resulted from severe hypothyroidism caused by chronic thyroiditis and excess iodine absorption.