Objective: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that is incurable and ultimately fatal. Few therapeutic options are available to patients. In this study, we explored differences in microbiome composition associated with ALS. Methods: We compared the gut microbiome and inflammatory marker profiles of ALS patients (n=10) to those of their spouses (n=10). Gut microbiome profiles were determined by 16S rRNA gene sequencing. Results: The gut microbial communities of the ALS patients were more diverse and were deficient in Prevotella spp. compared with those of their spouses. In contrast, healthy couples (n=10 couples of the opposite sex) recruited from the same geographic region as the patient population did not exhibit these differences. Stool and plasma inflammatory markers were similar between ALS patients and their spouses. Predictive analysis of microbial enzymes revealed that ALS patients had decreased activity in several metabolic pathways, including carbon metabolism, butyrate metabolism, and systems involving histidine kinase and response regulators. Conclusions: ALS patients exhibit differences in their gut microbial communities compared with spouse controls. Our findings suggest that modifying the gut microbiome, such as via amelioration of Prevotella spp. deficiency, and/or altering butyrate metabolism may have translational value for ALS treatment.

The G4C2 hexanucleotide repeat expansion mutation in the C9orf72 gene is the most common genetic cause underlying both amyotrophic lateral sclerosis and frontotemporal dementia. Pathologically, these two neurodegenerative disorders are linked by the common presence of abnormal phosphorylated TDP-43 neuronal cytoplasmic inclusions. We compared the number and size of phosphorylated TDP-43 inclusions and their morphology in hippocampi from patients dying with sporadic versus C9orf72-related amyotrophic lateral sclerosis with pathologically defined frontotemporal lobar degeneration with phosphorylated TDP-43 inclusions, the pathological substrate of clinical frontotemporal dementia in patients with amyotrophic lateral sclerosis. In sporadic cases, there were numerous consolidated phosphorylated TDP-43 inclusions that were variable in size, whereas inclusions in C9orf72 amyotrophic lateral sclerosis/frontotemporal lobar degeneration were quantitatively smaller than those in sporadic cases. Also, C9orf72 amyotrophic lateral sclerosis/frontotemporal lobar degeneration homogenized brain contained soluble cytoplasmic TDP-43 that was largely absent in sporadic cases. To better understand these pathological differences, we modelled TDP-43 inclusion formation in fibroblasts derived from sporadic or C9orf72-related amyotrophic lateral sclerosis/frontotemporal dementia patients. We found that both sporadic and C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia patient fibroblasts showed impairment in TDP-43 degradation by the proteasome, which may explain increased TDP-43 protein levels found in both sporadic and C9orf72 amyotrophic lateral sclerosis/frontotemporal lobar degeneration frontal cortex and hippocampus. Fibroblasts derived from sporadic patients, but not C9orf72 patients, demonstrated the ability to sequester cytoplasmic TDP-43 into aggresomes via microtubule-dependent mechanisms. TDP-43 aggresomes in vitro and TDP-43 neuronal inclusions in vivo were both tightly localized with autophagy markers and, therefore, were likely to function similarly as sites for autophagic degradation. The inability for C9orf72 fibroblasts to form TDP-43 aggresomes, together with the observations that TDP-43 protein was soluble in the cytoplasm and formed smaller inclusions in the C9orf72 brain compared with sporadic disease, suggests a loss of protein quality control response to sequester and degrade TDP-43 in C9orf72-related diseases.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive muscular atrophy and respiratory failure. The G4C2 repeat expansion in the C9orf72 gene is the most prevalent genetic risk for ALS. Mutation carriers (C9ALS) display variability in phenotypes such as age-at-onset and duration, suggesting the existence of additional genetic factors. Here we introduce a three-step gene discovery strategy to identify genetic factors modifying the risk of both C9ALS and sporadic ALS (sALS) using limited samples. We first identified 135 candidate genetic modifiers of C9ALS using whole-genome sequencing (WGS) of extreme C9ALS cases diagnosed ~30 years apart. We then performed an unbiased genetic screen using a Drosophila model of the G4C2 repeat expansion with the genes identified from WGS analysis. This genetic screen identified the novel genetic interaction between G4C2 repeat-associated toxicity and 18 genetic factors, suggesting their potential association with C9ALS risk. We went on to test if 14 out of the 18 genes, those which were not known to be risk factors for ALS previously, are also associated with ALS risk in sALS cases. Gene-based-statistical analyses of targeted resequencing and WGS were performed. These analyses together reveal that rare variants in MYH15 represent a likely genetic risk factor for ALS. Furthermore, we show that MYH15 could modulate the toxicity of dipeptides produced from expanded G4C2 repeat. Our study presented here demonstrates the power of combining WGS with fly genetics to facilitate the discovery of fundamental genetic components of complex traits with a limited number of samples.

Objective: It is essential to develop predictive algorithms for Amyotrophic Lateral Sclerosis (ALS) disease progression to allow for efficient clinical trials and patient care. The best existing predictive models rely on several months of baseline data and have only been validated in clinical trial research datasets. We asked whether a model developed using clinical research patient data could be applied to the broader ALS population typically seen at a tertiary care ALS clinic. Methods: Based on the PRO-ACT ALS database, we developed random forest (RF), pre-slope, and generalized linear (GLM) models to test whether accurate, unbiased models could be created using only baseline data. Secondly, we tested whether a model could be validated with a clinical patient dataset to demonstrate broader applicability. Results: We found that a random forest model using only baseline data could accurately predict disease progression for a clinical trial research dataset as well as a population of patients being treated at a tertiary care clinic. The RF Model outperformed a pre-slope model and was similar to a GLM model in terms of root mean square deviation at early time points. At later time points, the RF Model was far superior to either model. Finally, we found that only the RF Model was unbiased and was less subject to overfitting than either of the other two models when applied to a clinic population. Interpretation: We conclude that the RF Model delivers superior predictions of ALS disease progression.

Introduction/aims: Improved functional outcome measures in amyotrophic lateral sclerosis (ALS) would aid ALS trial design and help hasten drug discovery. We evaluate the longitudinal performance of the Rasch-Built Overall Amyotrophic Lateral Sclerosis Disability Scale (ROADS) compared to the Amyotrophic Lateral Sclerosis Functional Rating Scale Revised for Self-Entry (ALSFRS-RSE) as patient reported outcomes of functional status in people with ALS. Methods: Participants completed the ROADS and the ALSFRS-RSE questionnaires at baseline, 3-, 6-, and 12- mo using Research Electronic Data Capture as part of a prospective, longitudinal, remote, online survey study of fatigue in ALS from 9/2020 to 12/2021. The scales were compared cross-sectionally (at baseline) and longitudinally. Correlation coefficients, coefficients of variation, and descriptive statistics were assessed. Results: A total of 182 adults with ALS consented to the study. This volunteer sample was comprised of predominantly White, non-Hispanic, non-smoking participants. Consented participant survey completion was approximately 90% at baseline and greater than 40% at 12 mo. The ALSFRS-RSE and the ROADS had high, significant agreement at 3 and 6 mo by Cohen's kappa ≥71% (p < 0.001); the number of functional increases or plateaus on the two scales were not significantly different; and the coefficient of variation of functional decline was similar at the 6-month mark, though higher for the ROADS at 3 mo and lower at 12 mo. Discussion: Although the ROADS performed similarly to the ALSFRS-RSE in an observational cohort, it has psychometric advantages, such as Rasch-modeling and unidimensionality. It merits further investigation as a patient reported outcome of overall disability and efficacy outcome measure in ALS trials.

Background Coformulated sodium phenylbutyrate/taurursodiol (PB/TURSO) was shown to prolong survival and slow functional decline in amyotrophic lateral sclerosis (ALS). Objective Determine whether PB/TURSO prolonged tracheostomy/ventilation-free survival and/or reduced first hospitalisation in participants with ALS in the CENTAUR trial. Methods Adults with El Escorial Definite ALS ≤18 months from symptom onset were randomised to PB/TURSO or placebo for 6 months. Those completing randomised treatment could enrol in an open-label extension (OLE) phase and receive PB/TURSO for ≤30 months. Times to the following individual or combined key events were compared in the originally randomised treatment groups over a period spanning trial start through July 2020 (longest postrandomisation follow-up, 35 months): death, tracheostomy, permanent assisted ventilation (PAV) and first hospitalisation. Results Risk of any key event was 47% lower in those originally randomised to PB/TURSO (n=87) versus placebo (n=48, 71% of whom received delayed-start PB/TURSO in the OLE phase) (HR=0.53; 95% CI 0.35 to 0.81; p=0.003). Risks of death or tracheostomy/PAV (HR=0.51; 95% CI 0.32 to 0.84; p=0.007) and first hospitalisation (HR=0.56; 95% CI 0.34 to 0.95; p=0.03) were also decreased in those originally randomised to PB/TURSO. Conclusions Early PB/TURSO prolonged tracheostomy/PAV-free survival and delayed first hospitalisation in ALS. Trial registration number NCT03127514; NCT03488524.

Thoughtful clinical trial design is critical for efficient therapeutic development, particularly in the field of amyotrophic lateral sclerosis (ALS), where trials often aim to detect modest treatment effects among a population with heterogeneous disease progression. Appropriate outcome measure selection is necessary for trials to provide decisive and informative results. Investigators must consider the outcome measure’s reliability, responsiveness to detect change when change has actually occurred, clinical relevance, and psychometric performance. ALS clinical trials can also be performed more efficiently by utilizing statistical enrichment techniques. Innovations in ALS prediction models allow for selection of participants with less heterogeneity in disease progression rates without requiring a lead-in period, or participants can be stratified according to predicted progression. Statistical enrichment can reduce the needed sample size and improve study power, but investigators must find a balance between optimizing statistical efficiency and retaining generalizability of study findings to the broader ALS population. Additional progress is still needed for biomarker development and validation to confirm target engagement in ALS treatment trials. Selection of an appropriate biofluid biomarker depends on the treatment mechanism of interest, and biomarker studies should be incorporated into early phase trials. Inclusion of patients with ALS as advisors and advocates can strengthen clinical trial design and study retention, but more engagement efforts are needed to improve diversity and equity in ALS research studies. Another challenge for ALS therapeutic development is identifying ways to respect patient autonomy and improve access to experimental treatment, something that is strongly desired by many patients with ALS and ALS advocacy organizations. Expanded access programs that run concurrently to well-designed and adequately powered randomized controlled trials may provide an opportunity to broaden access to promising therapeutics without compromising scientific integrity or rushing regulatory approval of therapies without adequate proof of efficacy.

Approximately 16,000 persons in the United States are affected by amyotrophic lateral sclerosis (ALS). 1 Persons with ALS (PALS) have distinctive pathophysiological risks and health determinants that may increase their vulnerability for infection with severe‐acute‐respiratory‐syndrome‐associated‐coronavirus (SARS‐CoV‐2) and severe Coronavirus‐2019 (COVID‐19) outcome.2, 3 Data on the prevalence and impact of COVID‐19 in this population is scarce. 4

There is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. However, discovery of a G 4 C 2 repeat expansion in the C9ORF72 gene as the most common genetic cause of ALS has opened up new avenues for therapeutic intervention for this form of ALS. G 4 C 2 repeat expansion RNAs and proteins of repeating dipeptides synthesized from these transcripts are believed to play a key role in C9ORF72-associated ALS (c9ALS). Therapeutics that target G 4 C 2 RNA, such as antisense oligonucleotides (ASOs) and small molecules, are thus being actively investigated. A limitation in moving such treatments from bench to bedside is a lack of pharmacodynamic markers for use in clinical trials. We explored whether poly(GP) proteins translated from G 4 C 2 RNA could serve such a purpose. Poly(GP) proteins were detected in cerebrospinal fluid (CSF) and in peripheral blood mononuclear cells from c9ALS patients and, notably, from asymptomatic C9ORF72 mutation carriers. Moreover, CSF poly(GP) proteins remained relatively constant over time, boding well for their use in gauging biochemical responses to potential treatments. Treating c9ALS patient cells or a mouse model of c9ALS with ASOs that target G 4 C 2 RNA resulted in decreased intracellular and extracellular poly(GP) proteins. This decrease paralleled reductions in G 4 C 2 RNA and downstream G 4 C 2 RNA-mediated events. These findings indicate that tracking poly(GP) proteins in CSF could provide a means to assess target engagement of G 4 C 2 RNA-based therapies in symptomatic C9ORF72 repeat expansion carriers and presymptomatic individuals who are expected to benefit from early therapeutic intervention. 2017

Objectives: The goals of this study were to characterize clinical and electrophysiologic findings of subjects with upper motor neuron disease and to explore feasibility of clinical trials in this population. Methods: Twenty northeast ALS consortium (NEALS) sites performed chart reviews to identify active clinical pure upper motor neuron disease patients. Patients with hereditary spastic paraplegia (HSP) or meeting revised El Escorial electrodiagnostic criteria for ALS were excluded. Patients were classified into two groups according to the presence or absence of minor electromyography (EMG) abnormalities. Results: 233 subjects with upper motor neuron disease were identified; 217 had available EMG data. Normal EMGs were seen in 140 subjects, and 77 had minor denervation. Mean disease duration was 84 (±80) months for the entire cohort with no difference seen between the two groups. No difference was seen in clinical symptoms, disability, or outcome measures between the two groups after correcting for multiple comparisons. Conclusions: Minor EMG abnormalities were not associated with phenotypic differences in a clinical upper motor neuron disease population. These findings suggest that subtle EMG abnormalities can not necessarily be used as a prognostic tool in patients with clinical upper motor neuron disease. This study also demonstrates the availability of a large number of patients with upper motor neuron diseases within the NEALS network and suggests feasibility for conducting clinical trials in this population.