James D. Berry

Intrinsic Membrane Hyperexcitability of Amyotrophic Lateral Sclerosis Patient-Derived Motor Neurons

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor nervous system. We show using multielectrode array and patch-clamp recordings that hyperexcitability detected by clinical neurophysiological studies of ALS patients is recapitulated in induced pluripotent stem cell-derived motor neurons from ALS patients harboring superoxide dismutase 1 (SOD1), C9orf72, and fused-in-sarcoma mutations. Motor neurons produced from a genetically corrected but otherwise isogenic SOD1(+/+) stem cell line do not display the hyperexcitability phenotype. SOD1(A4V/+) ALS patient-derived motor neurons have reduced delayed-rectifier potassium current amplitudes relative to control-derived motor neurons, a deficit that may underlie their hyperexcitability. The Kv7 channel activator retigabine both blocks the hyperexcitability and improves motor neuron survival in vitro when tested in SOD1 mutant ALS cases. Therefore, electrophysiological characterization of human stem cell-derived neurons can reveal disease-related mechanisms and identify therapeutic candidates.

Modulating inflammatory monocytes with a unique microRNA gene signature ameliorates murine ALS

Amyotrophic lateral sclerosis (ALS) is a progressive disease associated with neuronal cell death that is thought to involve aberrant immune responses. Here we investigated the role of innate immunity in a mouse model of ALS. We found that inflammatory monocytes were activated and that their progressive recruitment to the spinal cord, but not brain, correlated with neuronal loss. We also found a decrease in resident microglia in the spinal cord with disease progression. Prior to disease onset, splenic Ly6Chi monocytes expressed a polarized macrophage phenotype (M1 signature), which included increased levels of chemokine receptor CCR2. As disease onset neared, microglia expressed increased CCL2 and other chemotaxis-associated molecules, which led to the recruitment of monocytes to the CNS by spinal cord-derived microglia. Treatment with anti-Ly6C mAb modulated the Ly6Chi monocyte cytokine profile, reduced monocyte recruitment to the spinal cord, diminished neuronal loss, and extended survival. In humans with ALS, the analogous monocytes (CD14+CD16-) exhibited an ALS-specific microRNA inflammatory signature similar to that observed in the ALS mouse model, linking the animal model and the human disease. Thus, the profile of monocytes in ALS patients may serve as a biomarker for disease stage or progression. Our results suggest that recruitment of inflammatory monocytes plays an important role in disease progression and that modulation of these cells is a potential therapeutic approach.

Targeting miR-155 restores abnormal microglia and attenuates disease in SOD1 mice.

To investigate miR‐155 in the SOD1 mouse model and human sporadic and familial amyotrophic lateral sclerosis (ALS).

Design and Initial Results of a Multi-Phase Randomized Trial of Ceftriaxone in Amyotrophic Lateral Sclerosis

Objectives Ceftriaxone increases expression of the astrocytic glutamate transporter, EAAT2, which might protect from glutamate-mediated excitotoxicity. A trial using a novel three stage nonstop design, incorporating Phases I-III, tested ceftriaxone in ALS. Stage 1 determined the cerebrospinal fluid pharmacokinetics of ceftriaxone in subjects with ALS. Stage 2 evaluated safety and tolerability for 20-weeks. Analysis of the pharmacokinetics, tolerability, and safety was used to determine the ceftriaxone dosage for Stage 3 efficacy testing. Methods In Stage 1, 66 subjects at ten clinical sites were enrolled and randomized equally into three study groups receiving intravenous placebo, ceftriaxone 2 grams daily or ceftriaxone 4 grams daily divided BID. Participants provided serum and cerebrospinal fluid for pharmacokinetic analysis on study day 7. Participants continued their assigned treatment in Stage 2. The Data and Safety Monitoring Board (DSMB) reviewed the data after the last participants completed 20 weeks on study drug. Results Stage 1 analysis revealed linear pharmacokinetics, and CSF trough levels for both dosage levels exceeding the pre-specified target trough level of 1 µM (0.55 µg/mL). Tolerability (Stages 1 and 2) results showed that ceftriaxone at dosages up to 4 grams/day was well tolerated at 20 weeks. Biliary adverse events were more common with ceftriaxone but not dose-dependent and improved with ursodeoxycholic (ursodiol) therapy. Conclusions The goals of Stages 1 and 2 of the ceftriaxone trial were successfully achieved. Based on the pre-specified decision rules, the DSMB recommended the use of ceftriaxone 4 g/d (divided BID) for Stage 3, which recently closed. Trial Registration ClinicalTrials.gov NCT00349622.

Roadmap and standard operating procedures for biobanking and discovery of neurochemical markers in ALS

Abstract Despite major advances in deciphering the neuropathological hallmarks of amyotrophic lateral sclerosis (ALS), validated neurochemical biomarkers for monitoring disease activity, earlier diagnosis, defining prognosis and unlocking key pathophysiological pathways are lacking. Although several candidate biomarkers exist, translation into clinical application is hindered by small sample numbers, especially longitudinal, for independent verification. This review considers the potential routes to the discovery of neurochemical markers in ALS, and provides a consensus statement on standard operating procedures that will facilitate multicenter collaboration, validation and ultimately clinical translation.

The PRO-ACT database Design, initial analyses, and predictive features

Objective: To pool data from completed amyotrophic lateral sclerosis (ALS) clinical trials and create an open-access resource that enables greater understanding of the phenotype and biology of ALS. Methods: Clinical trials data were pooled from 16 completed phase II/III ALS clinical trials and one observational study. Over 8 million de-identified longitudinally collected data points from over 8,600 individuals with ALS were standardized across trials and merged to create the Pooled Resource Open-Access ALS Clinical Trials (PRO-ACT) database. This database includes demographics, family histories, and longitudinal clinical and laboratory data. Mixed effects models were used to describe the rate of disease progression measured by the Revised ALS Functional Rating Scale (ALSFRS-R) and vital capacity (VC). Cox regression models were used to describe survival data. Implementing Bonferroni correction, the critical p value for 15 different tests was p = 0.003. Results: The ALSFRS-R rate of decline was 1.02 (±2.3) points per month and the VC rate of decline was 2.24% of predicted (±6.9) per month. Higher levels of uric acid at trial entry were predictive of a slower drop in ALSFRS-R (p = 0.01) and VC (p < 0.0001), and longer survival (p = 0.02). Higher levels of creatinine at baseline were predictive of a slower drop in ALSFRS-R (p = 0.01) and VC (p < 0.0001), and longer survival (p = 0.01). Finally, higher body mass index (BMI) at baseline was associated with longer survival (p < 0.0001). Conclusion: The PRO-ACT database is the largest publicly available repository of merged ALS clinical trials data. We report that baseline levels of creatinine and uric acid, as well as baseline BMI, are strong predictors of disease progression and survival.

Bulbar and speech motor assessment in ALS: Challenges and future directions

Abstract Bulbar motor deterioration due to amyotrophic lateral sclerosis (ALS) leads to the eventual impairment of speech and swallowing functions. Despite these devastating consequences, no standardized diagnostic procedure for assessing bulbar dysfunction in ALS exists and adequate objective markers of bulbar deterioration have not been identified. In this paper, we consider objective measures of speech motor function, which show promise for forming the basis of a comprehensive, quantitative bulbar motor assessment in ALS. These measures are based on the assessment of four speech subsystems: respiratory, phonatory, articulatory, and resonatory. The goal of this research is to design a non-invasive, comprehensive bulbar motor assessment instrument intended for early detection, monitoring of disease progression, and clinical trial application. Preliminary data from an ongoing study of bulbar motor decline are presented, which demonstrate the potential clinical efficacy of the speech subsystem approach.

Biochemical alterations associated with ALS

Abstract Our objective was to identify metabolic pathways affected by ALS using non-targeted metabolomics in plasma, comparing samples from healthy volunteers to those from ALS patients. This discovery could become the basis for the identification of therapeutic targets and diagnostic biomarkers of ALS. Two distinct cross-sectional studies were conducted. Plasma was collected from 62 (Study 1) and 99 (Study 2) participants meeting El Escorial criteria for possible, probable, or definite ALS; 69 (Study 1) and 48 (Study 2) healthy controls samples were collected. Global metabolic profiling was used to detect and evaluate biochemical signatures of ALS. Twenty-three metabolites were significantly altered in plasma from ALS patients in both studies. These metabolites include biochemicals in pathways associated with neuronal change, hypermetabolism, oxidative damage, and mitochondrial dysfunction, all of which are proposed disease mechanisms in ALS. The data also suggest possible hepatic dysfunction associated with ALS. In conclusion, the data presented here provide insight into the pathophysiology of ALS while suggesting promising areas of focus for future studies. The metabolomics approach can generate novel hypotheses regarding ALS disease mechanisms with the potential to identify therapeutic targets and novel diagnostic biomarkers.

The Combined Assessment of Function and Survival (CAFS): A new endpoint for ALS clinical trials

Abstract Our objective was to describe a new endpoint for amyotrophic lateral sclerosis (ALS), the Combined Assessment of Function and Survival (CAFS). CAFS ranks patients’ clinical outcomes based on survival time and change in the ALS Functional Rating Scale–Revised (ALSFRS-R) score. Each patients outcome is compared to every other patients outcome, assigned a score, and the summed scores are ranked. The mean rank score for each treatment group can then be calculated. A higher mean CAFS score indicates a better group outcome. Historically, ALS clinical trials have assessed survival and function as independent endpoints. Combined endpoints have been used in other diseases to decrease the confounding effect of mortality on analysis of functional outcomes. We explored the application of a similar approach in ALS, the CAFS endpoint, which was used as a pre-specified secondary analysis in a phase II study of dexpramipexole. Those results and some hypothetical examples based on modeling exercises are presented here. CAFS is the primary endpoint of a dexpramipexole phase III study in ALS. In conclusion, the CAFS is a robust statistical tool for ALS clinical trials and appropriately accounts for and weights mortality in the analysis of function. Trial registration: ClinicalTrials.gov identifier: NCT01281189.

FUS is sequestered in nuclear aggregates in ALS patient fibroblasts

Mutations in the nuclear RNA-binding protein FUS can cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Study of ALS patient fibroblasts reveals FUS protein aggregated in the nucleus and its regulation of RNA polymerase II disrupted. Thus mutant FUS need not be aggregated in the cytoplasm to have deleterious consequences.