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.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.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.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.
Both amyloid plaques and neurofibrillary tangles are pathological hallmarks in the brains of patients with Alzheimer's disease (AD). However, the constituents of these hallmarks, amyloid beta (Aβ) 40, Aβ42, and total Tau (t-Tau), have been detected in the blood of cognitively normal subjects by using an immunomagnetic reduction (IMR) assay. Whether these levels are age dependent is not known, and their interrelation remains undefined. We determined the levels of these biomarkers in cognitively normal subjects of different age groups. A total of 391 cognitively normal subjects aged 23 to 91 were enrolled from hospitals in Asia, Europe, and North America. Healthy cognition was evaluated by NIA-AA guidelines to exclude subjects with mild cognitive impairment (MCI) and AD and by cognitive assessment using the Mini Mental State Examination and Clinical Dementia Rating. We examined the effect of age on plasma levels of Aβ40, Aβ42, and t-Tau and the relationship between these biomarkers during aging. Additionally, we explored age-related reference intervals for each biomarker. Plasma t-Tau and Aβ42 levels had modest but significant correlations with chronological age (r = 0.127, p = 0.0120 for t-Tau; r = -0.126, p = 0.0128 for Aβ42), ranging from ages 23 to 91. Significant positive correlations were detected between Aβ42 and t-Tau in the groups aged 50 years and older, with Rho values ranging from 0.249 to 0.474. Significant negative correlations were detected between Aβ40 and t-Tau from age 40 to 91 (r ranged from -0.293 to -0.582) and between Aβ40 and Aβ42 in the age groups of 30-39 (r = -0.562, p = 0.0235), 50-59 (r = -0.261, p = 0.0142), 60-69 (r = -0.303, p = 0.0004), and 80-91 (r = 0.459, p = 0.0083). We also provided age-related reference intervals for each biomarker. In this multicenter study, age had weak but significant effects on the levels of Aβ42 and t-Tau in plasma. However, the age group defined by decade revealed the emergence of a relationship between Aβ40, Aβ42, and t-Tau in the 6th and 7th decades. Validation of our findings in a large-scale and longitudinal study is warranted.
A previous study reported that a massive cerebral infarct in the territory of the middle cerebral artery (MCA) may be associated with development of neurofibrillary tangles (NFTs) in the ipsilateral basal nucleus of Meynert (BNM). We analyzed 19 cases of an MCA territory infarct and 12 with a putaminal hemorrhage (mean age 82.5 years; female/male ratio 8/23; mean time from stroke onset to autopsy 4182 days). In both groups, 74–100% had a significantly higher rate of phosphorylated tau immunoreactive or Gallyas Braak silver stain-positive neurons on the BNM-affected side than on the BNM-unaffected side. These NFTs were immunoreactive for anti-RD3 and anti-RD4 antibodies, and a triple-band pattern was observed by immunoblot analysis with anti-tau antibody. Most NFTs might be formed within the 5–10 years after stroke onset. There were significantly more TAR DNA-binding protein 43 (TDP43) immunoreactive structures on the BNM-affected side than on the BNM-unaffected side. We showed that many NFTs with TDP43-immunoreactive structures were observed in the ipsilateral BNM associated with a massive cerebral infarct in the MCA territory or a putaminal hemorrhage.
Objective: We are starting randomized clinical trial testing ropinirole hydrochloride in ALS patients in December, 2018. Background: No effective treatment has been established for patients with amyotrophic lateral sclerosis (ALS). It may imply a limitation of animal model (e.g. transgenic animal models with SOD-1 mutations) for drug development. Thus, we performed drug screening using motor neurons (MNs) derived from disease-specific-induced pluripotent stem cells (iPSC) for ALS and we found that ropinirole hydrochloride inhibited reactive oxygen species (ROS) and the abnormal aggregation of TDP-43 or FUS, improved mitochondrial function, and prevented motor neuron death (Fujimori K, Okano H, et al. Nat Med 2018). Now, we are planning a randomized clinical trial testing ropinirole hydrochloride in ALS patients. Design/Methods: This is a phase I/IIa randomized, double-blind, placebo-controlled, single-center (Keio University, Japan), open-label continuation clinical trial. The primary aim is to assess the safety and tolerability of ropinirole hydrochloride in patients with ALS. Secondary aims include the following effectiveness evaluations: ALS Functional Rating Scale-Revised, quantitative muscle strength by hand-held dynamometer, muscle volume by muscle CT scan, forced vital capacity, physical activity by activity tracker, survival, quality of life for ALS patients (ALSAQ40 scale), and Zarit Caregiver Burden Interview. Moreover, we will performed an efficacy evaluation using subjects-derived iPSCs/MNs and assess plasma/CSF biomarkers (TDP-43, and ALS-related RNA/micro RNA) as exploratory research. Results: Ropinirole hydrochloride potentially targets multiple mechanisms in ALS (i.e., oxidative stress, mitochondrial dysfunction, and abnormal aggregation of TDP-43/FUS protein, which is representative of the ALS phenotype), with promising preclinical study results based on iPSCs research. This drug has been approved for the treatment of Parkinson disease for many years; the availability of the drug suggests that rapid translation to daily clinical use might be possible. Conclusions: Our trial will provide reliable and important data for further potential trials. The results will appear in March, 2021. Disclosure: Dr. Takahashi has nothing to disclose. Dr. Morimoto has nothing to disclose. Dr. Saya has nothing to disclose. Dr. Okano has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with K Pharma Inc., and SanBio Co. Ltd. Dr. Okano has received compensation for serving on the Board of Directors of SanBio Co.Ltd. Dr. Aoki has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Eisai Inc., Mitsubishi Tanabe Pharma Corporation, Astellas Pharma Inc., Takeda Pharmaceutical Company Ltd, Sanofi K.K. and Dainippon Sumitomo Pharma Co. Ltd.. Dr. Suzuki has nothing to disclose. Dr. Nakahara has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Alexion, Biogen, Daiichi-Sankyo, Eisai, Fujimoto Pharma, JB, Mitsubishi-Tanabe, Novartis, and Takeda. Dr. Nakahara has received research support from Astellas, Biogen, Boehringer-Ingelheim, Daiichi-Sankyo, Eisai, Kyowa-Kirin, Mitsubishi-Tanabe, Otsuka, Pfizer, Shionogi, Sumitomo Dainippon, and UCB..
Amyotrophic lateral sclerosis (ALS) is a major neurodegenerative disease for which there is currently no curative treatment. The blood-brain barrier (BBB), multiple physiological functions formed by mainly specialized brain microvascular endothelial cells (BMECs), serves as a gatekeeper to protect the central nervous system (CNS) from harmful molecules in the blood and aberrant immune cell infiltration. The accumulation of evidence indicating that alterations in the peripheral milieu can contribute to neurodegeneration within the CNS suggests that the BBB may be a previously overlooked factor in the pathogenesis of ALS. Animal models suggest BBB breakdown may precede neurodegeneration and link BBB alteration to the disease progression or even onset. However, the lack of a useful patient-derived model hampers understanding the pathomechanisms of BBB dysfunction and the development of BBB-targeted therapies. In this study, we differentiated BMEC-like cells from human induced pluripotent stem cells (hiPSCs) derived from ALS patients to investigate BMEC functions in ALS patients.
Our laboratory previously established spinal motor neurons (MN) from induced-pluripotent stem cells (iPSCs) prepared from both sporadic and familial ALS patients, and successfully recapitulated disease-specific pathophysiological processes. We next searched for effective drugs capable of slowing the progression of ALS using a drug library of 1232 existing compounds and discovered that ropinirole hydrochloride prevented MN death. In December 2018, we started an investigator-initiated clinical trial testing ropinirole hydrochloride extended-release tablets in ALS patients. This is an on-going phase I/IIa randomized, double-blind, placebo-controlled, single-center, open-label continuation clinical trial (UMIN000034954). The primary aim is to assess the safety and tolerability of ropinirole hydrochloride in patients with ALS. We will also perform an efficacy evaluation using patient-derived iPSCs/MN. Major inclusion criteria were as follows: 1) 'clinically possible and laboratory-supported ALS', 'clinically probable ALS' or 'clinically definite ALS', according to the criteria for the diagnosis of ALS (El Escorial revised) and within 60 months after disease onset; 2) change in ALSFRS-R score of -2 to -5 points during the 12-week run-in period. Finally, 15 patients will be assigned to the active drug and 5 patients to the placebo. Our trial will be a touchstone trial for iPSC-based drug development strategies.
iPSC-based drug discovery is a promising technology for developing novel therapeutics for neurodegenerative diseases lacking useful disease models, such as amyotrophic lateral sclerosis (ALS).Ropinirole, retigabine, and bosutinib were identified as candidate therapeutic agents for ALS by the combination of iPSC-based drug discovery and drug repositioning.The potential anti-ALS mechanism of ropinirole is independent of antioxidant activity, rescue of mitochondria, reduction of stress granules, and abnormal proteins such as phosphorylated TDP-43 and FUS, and dopamine D2 receptor (D2R) agonism.Retigabine inhibits the hyperexcitability of motor neurons in ALS and bosutinib prompts autophagy and reduces abnormal proteins such as SOD-1 and phosphorylated TDP-43 via the Src/c-Abl pathway in motor neurons in ALS.Stratification of ALS, personalized medicine strategies, and the identification of common mechanisms with other neurodegenerative diseases are key aspects in the development of ALS therapies. Induced pluripotent stem cells (iPSCs) are increasingly used in the study of disease mechanisms and the development of effective disease-modifying therapies for neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Recently, three candidate anti-ALS drugs – ropinirole (ROPI), retigabine, and bosutinib – have been identified in iPSC-based drug screens and are now being evaluated in clinical trials for safety and effectiveness. We review the preclinical data, clinical research design, and rationale for ROPI as an anti-ALS drug candidate compared with those of the other two drugs. We also discuss the use of iPSCs for understanding and monitoring treatment response as well as for new insights into the development of new drugs and therapeutic interventions for major neurodegenerative diseases. Induced pluripotent stem cells (iPSCs) are increasingly used in the study of disease mechanisms and the development of effective disease-modifying therapies for neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Recently, three candidate anti-ALS drugs – ropinirole (ROPI), retigabine, and bosutinib – have been identified in iPSC-based drug screens and are now being evaluated in clinical trials for safety and effectiveness. We review the preclinical data, clinical research design, and rationale for ROPI as an anti-ALS drug candidate compared with those of the other two drugs. We also discuss the use of iPSCs for understanding and monitoring treatment response as well as for new insights into the development of new drugs and therapeutic interventions for major neurodegenerative diseases.
iPSC-based drug discovery led to a phase 1/2a trial of ropinirole in ALS. 20 participants with sporadic ALS received ropinirole or placebo for 24 weeks in the double-blind period to evaluate safety, tolerability, and therapeutic effects. Adverse events were similar in both groups. During the double-blind period, muscle strength and daily activity were maintained, but a decline in the ALSFRS-R, which assesses the functional status of ALS patients, was not different from that in the placebo group. However, in the open-label extension period, the ropinirole group showed significant suppression of ALSFRS-R decline and an additional 27.9 weeks of disease-progression-free survival. iPSC-derived motor neurons from participants showed dopamine D2 receptor expression and a potential involvement of the SREBP2-cholesterol pathway in therapeutic effects. Lipid peroxide represents a clinical surrogate marker to assess disease progression and drug efficacy. Limitations include small sample sizes and high attrition rates in the open-label extension period, requiring further validation.
