Prediction Model of Amyotrophic Lateral Sclerosis by Deep Learning with Patient Induced Pluripotent Stem Cells.

In amyotrophic lateral sclerosis (ALS), early diagnosis is essential for both current and potential treatments. To find a supportive approach for the diagnosis, we constructed an artificial intelligence-based prediction model of ALS using induced pluripotent stem cells (iPSCs). Images of spinal motor neurons derived from healthy control subject and ALS patient iPSCs were analyzed by a convolutional neural network, and the algorithm achieved an area under the curve of 0.97 for classifying healthy control and ALS. This prediction model by deep learning algorithm with iPSC technology could support the diagnosis and may provide proactive treatment of ALS through future prospective research. ANN NEUROL 2021;89:1226-1233.

Neuromelanin imaging analyses of the substantia nigra in patients with Machado-Joseph disease.

PURPOSE: Machado-Joseph disease (MJD/SCA3) is the most frequent spinocerebellar ataxia worldwide. Pathological studies revealed less melanin-containing dopaminergic neurons in the substantia nigra (SN) pars compacta in MJD/SCA3 patients. The purpose of this study was to quantify the damage in the SN reported for MJD/SCA3 patients in vivo using neuromelanin MR imaging. METHODS: We retrospectively reviewed the clinical information and MR imaging in sixteen MJD/SCA3 patients and fourteen healthy controls. High-resolution T1-WI of the SN were acquired using a 3-T MR system. The neuromelanin-related contrast (NRC), which was defined as the number of pixels of high-signal-intensity areas on T1-WI in the SN, was calculated semi-automatically. The NRC values were compared between MJD/SCA3 patients and normal controls. RESULTS: The NRC values were significantly lower in MJD/SCA3 patients than in healthy controls. In MJD/SCA3 patients, a significant negative correlation existed between disease durations and NRC values. No significant difference in the NRC values was revealed between the MJD/SCA3 patients with and without parkinsonism. CONCLUSION: Neuromelanin in the SN may decrease at the early stage of the disease and continue to decrease over time with the disease duration in MJD/SCA3 patients. The NRC may be a useful biological index for monitoring MJD/SCA3.

Non-motor manifestations in ALS patients with tracheostomy and invasive ventilation.

INTRODUCTION: This study aimed to investigate non-motor manifestations in amyotrophic lateral sclerosis (ALS) patients with tracheostomy and invasive ventilation (TIV) and their relevance to disease progression. METHODS: Sixty-seven ALS patients with TIV were enrolled, and followed-up prospectively. The patients were classified at the final evaluation into two subgroups according to the duration of TIV use or disease stage measured by communication impairment. We identified non-motor manifestations and investigated their frequencies and differences across the stages. RESULTS: The non-motor manifestations were macroglossia (22.4%), unstable blood pressure (38.8%), hypothermia (26.9%), dysuria (50.7%), and hyperglycemia (12.1%). These manifestations occurred significantly more frequently in patients with TIV ≥5 years than in patients with TIV <5 years, and more in patients with severe communication impairment than in those with preserved communication ability. DISCUSSION: Non-motor manifestations are observed at a high rate in ALS patients with TIV, and are possibly related to disease progression. Muscle Nerve 57: 735-741, 2018.

Identification of Middle Chain Fatty Acyl-CoA Ligase Responsible for the Biosynthesis of 2-Alkylmalonyl-CoAs for Polyketide Extender Unit.

Understanding the biosynthetic mechanism of the atypical polyketide extender unit is important for the development of bioactive natural products. Reveromycin (RM) derivatives produced by Streptomyces sp. SN-593 possess several aliphatic extender units. Here, we studied the molecular basis of 2-alkylmalonyl-CoA formation by analyzing the revR and revS genes, which form a transcriptional unit with the revT gene, a crotonyl-CoA carboxylase/reductase homolog. We mainly focused on the uncharacterized adenylate-forming enzyme (RevS). revS gene disruption resulted in the reduction of all RM derivatives, whereas reintroduction of the gene restored the yield of RMs. Although RevS was classified in the fatty acyl-AMP ligase clade based on phylogenetic analysis, biochemical characterization revealed that the enzyme catalyzed the middle chain fatty acyl-CoA ligase (FACL) but not the fatty acyl-AMP ligase activity, suggesting the molecular evolution for acyl-CoA biosynthesis. Moreover, we examined the in vitro conversion of fatty acid into 2-alkylmalonyl-CoA using purified RevS and RevT. The coupling reaction showed efficient conversion of hexenoic acid into butylmalonyl-CoA. RevS efficiently catalyzed C8-C10 middle chain FACL activity; therefore, we speculated that the acyl-CoA precursor was truncated via ß-oxidation and converted into (E)-2-enoyl-CoA, a RevT substrate. To determine whether the ß-oxidation process is involved between the RevS and RevT reaction, we performed the feeding experiment using [1,2,3,4-(13)C]octanoic acid. (13)C NMR analysis clearly demonstrated incorporation of the [3,4-(13)C]octanoic acid moiety into the structure of RM-A. Our results provide insight into the role of uncharacterized RevS homologs that may catalyze middle chain FACL to produce a unique polyketide extender unit.

A rapid functional decline type of amyotrophic lateral sclerosis is linked to low expression of TTN.

OBJECTIVE: To classify the patterns of functional decline in patients with sporadic amyotrophic lateral sclerosis (ALS) and explore the genetic backgrounds that modified these patterns. METHODS: We included 465 patients with sporadic ALS in the analysis and clustered the longitudinal functional scores in the registered patients, using a mixture approach of a non-linear mixed-effects model. We conducted a genome-wide analysis of 572 983 single nucleotide polymorphisms (SNPs). We then assessed the association between the clusters of longitudinal functional scores and SNPs. RESULTS: We identified the following four clusters of longitudinal functional decline in the cases: a rapid decline cluster, an intermediate decline cluster, a sigmoidal decline cluster and a moderate decline cluster. We identified seven SNPs associated with the rapid decline cluster, using a recessive model (p=3.47-8.34×10(-8)). The OR for the probabilities of the rapid decline cluster ranged from 5.5 to 5.84. Homozygosity for the minor alleles in the seven SNPs, which constituted a linkage disequilibrium (LD) block, was associated with decreased expression of TTN (encoding Titin, a large sarcomere protein) in the expression quantitative trait loci database of a large-scale Japanese genetic variation database (p=8.6×10(-10)-1.1×10(-7)). TTN expression in immortalised lymphocyte lines was decreased in patients who were homozygous for the minor alleles compared with those who were homozygous for the major alleles (n=19 in each group, p=0.002). CONCLUSIONS: We detected an LD block associated with a rapid functional decline in patients with sporadic ALS, which is linked to decreased expression of TTN.

Predictive factors for prognosis following unsedated percutaneous endoscopic gastrostomy in ALS patients.

INTRODUCTION: This study aimed to determine the prognostic factors and the values that predict survival after percutaneous endoscopic gastrostomy (PEG) tube placement in patients with amyotrophic lateral sclerosis (ALS). METHODS: We retrospectively analyzed the correlations for 97 consecutive patients with ALS between clinical parameters and survival following PEG tube placement using the log-rank test and Cox proportional-hazards models. RESULTS: The log-rank test showed that an arterial carbon dioxide pressure (PaCO2 ) of ≤ 40 mmHg (P = 0.0054), a forced vital capacity (FVC) of ≥ 38% of predicted (P = 0.0003), and bulbar-onset (P = 0.0121) were significantly associated with better post-PEG survival. Multivariate analysis showed that the FVC and PaCO2 were associated with better post-PEG survival (P = 0.0081 and P = 0.0265, respectively). CONCLUSIONS: PEG tube placement in ALS is recommended when FVC is ≥ 38% of predicted and when PaCO2 is normal. Muscle Nerve 54: 277-283, 2016.

Mutations of optineurin in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) has its onset in middle age and is a progressive disorder characterized by degeneration of motor neurons of the primary motor cortex, brainstem and spinal cord. Most cases of ALS are sporadic, but about 10% are familial. Genes known to cause classic familial ALS (FALS) are superoxide dismutase 1 (SOD1), ANG encoding angiogenin, TARDP encoding transactive response (TAR) DNA-binding protein TDP-43 (ref. 4) and fused in sarcoma/translated in liposarcoma (FUS, also known as TLS). However, these genetic defects occur in only about 20-30% of cases of FALS, and most genes causing FALS are unknown. Here we show that there are mutations in the gene encoding optineurin (OPTN), earlier reported to be a causative gene of primary open-angle glaucoma (POAG), in patients with ALS. We found three types of mutation of OPTN: a homozygous deletion of exon 5, a homozygous Q398X nonsense mutation and a heterozygous E478G missense mutation within its ubiquitin-binding domain. Analysis of cell transfection showed that the nonsense and missense mutations of OPTN abolished the inhibition of activation of nuclear factor kappa B (NF-kappaB), and the E478G mutation revealed a cytoplasmic distribution different from that of the wild type or a POAG mutation. A case with the E478G mutation showed OPTN-immunoreactive cytoplasmic inclusions. Furthermore, TDP-43- or SOD1-positive inclusions of sporadic and SOD1 cases of ALS were also noticeably immunolabelled by anti-OPTN antibodies. Our findings strongly suggest that OPTN is involved in the pathogenesis of ALS. They also indicate that NF-kappaB inhibitors could be used to treat ALS and that transgenic mice bearing various mutations of OPTN will be relevant in developing new drugs for this disorder.

Quantitative analysis of the features of fasciculation potentials and their relation with muscle strength in amyotrophic lateral sclerosis.

This study aimed to quantitatively analyze fasciculation potentials (FPs) and to investigate their relationship with muscle strength in amyotrophic lateral sclerosis (ALS). Fifty-one patients with sporadic ALS or progressive muscular atrophy (25 men, 26 women, mean age of 68 years) underwent needle EMG. We determined the duration, phase number, and amplitude of FPs from three muscles (upper trapezius, biceps brachii, and tibialis anterior) and examined their relations with muscle strength. In total, 878 FPs were analyzed. FP duration displayed a significant negative relation with the strength of all three muscles; the weaker muscles showed longer durations of FPs than the muscles with normal strength. The amplitude and phase number were not related with muscle strength, but there were significant correlations between the duration and amplitude of FPs in the trapezius and tibialis anterior muscles. The longer duration of FPs in muscles with weak strength suggests that the morphological changes of FPs were caused by temporal dispersion through progressively degenerating and/or immature reinnervating motor branches, and were observed uniformly in different muscles along with disease progression.

A Japanese familial ALS patient with autonomic failure and a p.Cys146Arg mutation in the gene for SOD1 (SOD1).

We describe a Japanese man with familial amyotrophic lateral sclerosis (ALS) associated with a p.Cys146Arg mutation in the copper/zinc superoxide dismutase gene (SOD1). The patient developed bulbar signs followed by rapidly progressive limb muscle weakness. The prominent clinical feature was orthostatic hypotension due to autonomic failure, which occurred after he underwent tracheostomy 1 year and 3 months after the onset. Thereafter, he required mechanical ventilation and progressed to communication stage V (totally locked-in state) 7 years after the onset. Neuropathology showed ALS with posterior column degeneration and multiple system degeneration. Severe neuronal loss in the intermediolateral nucleus was also observed. Two previously reported cases of ALS patients with autonomic failure showed severe neuronal loss in the intermediolateral nucleus in addition to degeneration of the motor neurons. Thus, autonomic failure due to neuronal loss in the intermediolateral nucleus could present in patients with ALS associated with certain mutations in SOD1.

Association of ubiquitin carboxy-terminal hydrolase-L1 in cerebrospinal fluid with clinical severity in a cohort of patients with Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is an acute immune-mediated polyneuropathy. Although its pathogenic mechanism has been revealed and various therapeutic trials have been performed, a proportion of patients experience the severe sequelae associated with GBS. In this paper, we investigated whether the amount of the neuron-specific protein, ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1), in the cerebrospinal fluid of patients with GBS was correlated with the clinical course of the disease. UCH-L1 protein levels were greater in patients with GBS than in controls. The patients with GBS whose UCH-L1 protein levels were higher than those of the controls presented with more severe symptoms at peak. UCH-L1 protein levels tended to become elevated as the total protein levels were increased; however, elevated UCH-L1 without an increase in total protein might be correlated with severe disease course (bedridden or ventilator supported). These results suggest that UCH-L1 could be a biomarker associated with the severity of the disease at the acute phase of GBS.

A Japanese patient with familial ALS and a p.K510M mutation in the gene for FUS (FUS) resulting in the totally locked-in state.

We describe a Japanese patient with familial amyotrophic lateral sclerosis (ALS) and a p.K510M mutation in the fused in sarcoma gene (FUS). The patient's condition was characterized clinically by an early onset and rapid progression. The patient eventually required mechanical ventilation and progressed to the totally locked-in state. Neuropathologically, multiple system degeneration with many FUS-immunoreactive structures was observed. The involvement of the globus pallidus, subthalamic nucleus, substantia nigra, cerebellar efferent system, and both upper and lower motor neurons in the present patient was comparable to that described for ALS patients with different mutations in FUS, all of whom progressed to the totally locked-in state. However, the patient also exhibited degeneration of the cerebellar afferent system and posterior column. Furthermore, the appearance of non-compact FUS-immunoreactive neuronal cytoplasmic inclusions and many FUS-immunoreactive glial cytoplasmic inclusions were unique to the present patient. These features suggest that the morphological characteristics of the FUS-immunoreactive structures and distribution of the lesions vary with the diversity of mutations in FUS.

Neck weakness is a potent prognostic factor in sporadic amyotrophic lateral sclerosis patients.

OBJECTIVE: To clarify the emergence of muscle weakness in regions of the body that affect survival, and deterioration in activities of daily living (ADL) in amyotrophic lateral sclerosis (ALS) patients. METHODS: We conducted a multicentre-based prospective cohort study of patients with ALS. We enrolled 401 sporadic patients with ALS. Death or the introduction of invasive ventilation was defined as the primary endpoint, and the time to five clinical markers of ADL deterioration associated with bulbar paralysis or limb weakness were defined as ADL milestones. Muscle weakness was assessed in the neck flexor muscles; the bilateral abductors of the shoulders; the bilateral wrist extensor muscles; the bilateral flexor muscles of the hips; and the bilateral ankle dorsiflexion muscles. We performed Cox proportional hazards regression analyses for the primary endpoint and the five ADL milestones, adjusting for known covariate prognostic factors for ALS. RESULTS: The Medical Research Council (MRC) score for the neck flexors was the most significant prognostic factor for the primary endpoint (HR 0.74, p<0.001), loss of speech (HR 0.66, p<0.001), and loss of swallowing function (HR 0.73, p<0.001), and was one of the significant prognostic factors for loss of upper limb function, difficulty turning in bed, and loss of walking ability (p=0.001, 0.002, and 0.008, respectively). The MRC score for the neck flexors was also a significant prognostic factor for covariates of the previously reported prognostic factors. CONCLUSIONS: Neck weakness is an independent prognostic factor for survival and deterioration in ADL in Patients with ALS.

Novel G37V mutation of SOD1 gene in autopsied patient with familial amyotrophic lateral sclerosis.

We report a novel missense mutation (G37V) in exon 2 of the superoxide dismutase-1 gene in a 63-years-old Japanese male with purely lower motor neuron disease. His disease duration was 14 months, and he died of respiratory failure. The disease in this patient with the G37V mutation showed a rapid progression, although patients with G37R mutation are known to have a long survival.

Reduction rate of body mass index predicts prognosis for survival in amyotrophic lateral sclerosis: a multicenter study in Japan.

Malnutrition in the early stage has been reported as an independent predictor of survival in amyotrophic lateral sclerosis (ALS). We analyzed retrospectively the effect of variation of body mass index (BMI) on survival in ALS patients. In total, 77 consecutive ALS patients were enrolled from nine hospitals in Japan. Reduction rate of BMI was calculated from BMI before the disease onset and at the time of the first visit to each hospital. We analyzed the correlation between BMI reduction rate and total disease duration. Results showed that the median BMI reduction rate was 2.5 per year (interquartile range 1.3-3.8). The BMI reduction rate was significantly correlated with survival length (p <0.0001). There was also a significant difference in survival between ALS patients with a BMI reduction rate ≥ and < 2.5 (Kaplan-Meier survival analysis and the log-rank test, p < 0.0001; hazard ratio by the Cox model, 2.9816). In conclusion, faster reduction of BMI at the initial stage before the first visit to hospital predicts shorter survival length also in Japanese ALS patients.

Body Weight Gain Is Associated with the Disease Stage in Advanced Amyotrophic Lateral Sclerosis with Invasive Ventilation.

We investigated the incidence of weight gain and its related factors in patients with amyotrophic lateral sclerosis (ALS) who underwent tracheostomy and invasive ventilation (TIV). Seventy-eight patients with ALS and TIV were enrolled and followed up prospectively. We clarified the clinical profiles of patients with increased weight following TIV and examined chronological variations in their body mass index (BMI), energy intake, and serum albumin levels. Post follow-up, we determined their disease stage according to their communication impairment (stage I to V) and investigated factors associated with BMI increase following TIV. Patients with a post-TIV BMI increase ≥1.86 kg/m2 demonstrated a higher incidence of ophthalmoplegia (76.2%), total quadriplegia (61.9%), severe communication impairment (stage V; 33.3%), and hypoalbuminemia than those with a BMI increase <1.86 kg/m2. Patients with stage V communication impairment exhibited a larger and faster BMI decrease before TIV (mean -4.2 kg/m2 and -2.5 kg/m2/year, respectively); a larger BMI increase (mean +4.6 kg/m2) following TIV, despite lower energy intake; and lower albumin levels post follow-up than those with lower-stage communication impairment. Multilevel linear regression analysis demonstrated an independent association between communication impairment stages (stage V) and a post-TIV BMI increase (p = 0.030). Weight gain and hypoalbuminemia during TIV in patients with ALS were associated with the disease stage and may be attributable to the neurodegenerative processes that are peculiar to ALS.

Myoclonic Epilepsy with Ragged-red Fibers with Intranuclear Inclusions.

We herein report a case of myoclonic epilepsy with ragged-red fibers (MERRF) harboring a novel variant in mitochondrial cysteine transfer RNA (MT-TC). A 68-year-old woman presented with progressive myoclonic epilepsy with optic atrophy and peripheral neuropathy. A skin biopsy revealed p62-positive intranuclear inclusions. No mutations were found in the causative genes for diseases known to be related to intranuclear inclusions; however, a novel variant in MT-TC was found. The association between intranuclear inclusions and this newly identified MERRF-associated variant is unclear; however, the rare complication of intranuclear inclusions in a patient with typical MERRF symptoms should be noted for future studies.

Randomized phase 2 study of perampanel for sporadic amyotrophic lateral sclerosis.

OBJECTIVE: To evaluate the efficacy and safety of perampanel in patients with sporadic amyotrophic lateral sclerosis (SALS). METHODS: This randomized, double-blind, placebo-controlled, multicenter, phase 2 clinical study was conducted at 12 sites. Patients with probable or definite ALS as defined by revised El Escorial criteria were enrolled. Sixty-six patients were randomly assigned (1:1:1) to receive placebo, 4 mg perampanel, or 8 mg perampanel daily for 48 weeks. Adverse events (AEs) were recorded throughout the trial period. The primary efficacy outcome was the change in Amyotrophic Lateral Sclerosis Rating Scale-Revised (ALSFRS-R) score after 48 weeks of treatment. RESULTS: One patient withdrew before starting the treatment. Of 65 patients included, 18 of 22 patients randomized to placebo (82%), 14 of 22 patients randomized to 4 mg perampanel (64%), and 7 of 21 patients randomized to 8 mg perampanel (33%) completed the trial. There was a significant difference in the change of ALSFRS-R scores [- 8.4 (95% CI - 13.9 to - 2.9); p = 0.015] between the placebo and the perampanel 8 mg group, primarily due to worsening of the bulbar subscore in the perampanel 8 mg group. Serious AEs were more frequent in the perampanel 8 mg group than in the placebo group (p = 0.0483). CONCLUSIONS: Perampanel was associated with a significant decline in ALSFRS-R score and was linked to worsening of the bulbar subscore in the 8 mg group.

Mutation screening of the DNAJC7 gene in Japanese patients with sporadic amyotrophic lateral sclerosis.

DNAJC7 has recently been identified as an amyotrophic lateral sclerosis (ALS) gene via large-scale exome analysis, and its involvement in ALS is still unclear in various populations. This study aimed to determine the frequencies and characteristics of the DNAJC7 variants in a Japanese ALS cohort. A total of 807 unrelated Japanese patients with sporadic ALS were screened via exome analysis. In total, we detected six rare missense variants and one splice-site variant of the DNAJC7 gene, which are not reported in the Japanese public database. Furthermore, the missense variants are located around the TPR domain, which is important for the function of DNAJC7. The total frequency of the DNAJC7 variants in Japanese ALS patients was estimated at 0.87%. Collectively, these results suggest that variants of DNAJC7 are rare cause of Japanese patients with sporadic ALS.

A morphometric study of the vagus nerve in amyotropic lateral sclerosis with circulatory collapse.

Amyotrophic lateral sclerosis (ALS) shows peculiar abnormalities of the autonomic nervous system, including sympathetic hyperactivity, which might result in sudden death. In general, the sympathetic hyperactivity could be caused by disruption of vagal inhibition. Our objective was to evaluate the vagus nerve morphometrically in autopsy cases of ALS with sympathetic hyperactivity and circulatory collapse (CC). We investigated 10 autopsied ALS patients, six of whom had exhibited autonomic storms or CC. We also examined 10 patients without ALS as controls, and one patient with Guillain-Barré syndrome (GBS) who died from CC, for comparison. After obtaining the visceral branch of the left vagus nerve at necropsy, we analyzed the density of the myelinated and unmyelinated fibers, and the fiber diameter distribution for each fiber. Results showed that the densities of both myelinated and unmyelinated fibers in ALS patients with or without CC were not significantly different from those in control patients. In contrast, the GBS patient showed marked reduction in the whole myelinated and large unmyelinated fiber density. In conclusion, the autonomic storms or CC due to sympathetic hyperactivity in ALS could not be ascribed to the deafferentation of the baroreflex, and more central neural pathophysiology should be investigated.

Hyperosmolar hyperglycemic state in advanced amyotrophic lateral sclerosis.

Our objective was to describe cases of hyperosmolar hyperglycemic state (HHS) in advanced amyotrophic lateral sclerosis (ALS) patients and discuss its pathophysiology. Five ventilator-dependent patients with ALS, with no previous history of diabetes, showed development of marked hyperglycemia (plasma glucose levels of 755-1544 mg/dl) after preceding infectious episodes. All patients had severe generalized muscle wasting and tetraplegia. The initial manifestations of HHS were fever, drowsiness, or polyuria. Hydration and intravenous insulin therapy were markedly effective, resulting in favorable recovery without the necessity of chronic medication for hyperglycemia in all cases. Seventy-five grams oral glucose tolerance tests performed via feeding tubes in two patients after the successful treatment of HHS revealed increased insulin resistance and diminished early-phase insulin secretion with preserved total insulin secretion. In conclusion, a marked loss of skeletal muscle, the largest glucose consumer of the human body, with background abnormality of early-phase insulin secretion, might be a causative factor of HHS in advanced ALS.