Proteomic investigation of ALS motor cortex identifies known and novel pathogenetic mechanisms.

The key pathological feature in ALS is death of motor neurones from the brain and spinal cord, but the molecular mechanisms underlying this degeneration remain unknown. Quantifying the motor cortex proteome in autopsy brain and comparing tissues from ALS cases and non-ALS controls is critical to understanding these mechanisms. We used Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) to characterize the proteomes of the motor cortex from ALS cases (n = 8) and control subjects (n = 8). A total of 1427 proteins were identified at a critical local false discovery rate < 5%; 187 of these exhibited significant expression differences between ALS cases and controls. Of these, 91 proteins were significantly upregulated and 96 proteins were significantly downregulated. Bioinformatics analysis revealed that these proteins are involved in molecular transport, protein trafficking, free radical scavenging, lipid metabolism, cell death and survival, nucleic acid metabolism, inflammatory response or amino acid metabolism and carbohydrate metabolism. Differentially expressed proteins were subjected to pathway analysis. This revealed abnormalities in pathways involving mitochondrial function, sirtuin signaling, oxidative phosphorylation, glycolysis, phagosome maturation, SNARE signaling, redox regulation and several others. Core analysis revealed mitochondrial dysfunction to be the top canonical pathway. The top-enriched networks involved JNK activation and inhibition of AKT signaling, suggesting that disruption of these signaling pathways could lead to demise of motor neurons in the ALS motor cortex.

Disease Reactivation After Cessation of Disease-Modifying Therapy in Patients With Relapsing-Remitting Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: To evaluate the rate of return of disease activity after cessation of multiple sclerosis (MS) disease-modifying therapy. METHODS: This was a retrospective cohort study from 2 large observational MS registries: MSBase and OFSEP. Patients with relapsing-remitting MS who had ceased a disease-modifying therapy and were followed up for the subsequent 12 months were included in the analysis. The primary study outcome was annualized relapse rate in the 12 months after disease-modifying therapy discontinuation stratified by patients who did, and did not, commence a subsequent therapy. The secondary endpoint was the predictors of first relapse and disability accumulation after treatment discontinuation. RESULTS: A total of 14,213 patients, with 18,029 eligible treatment discontinuation epochs, were identified for 7 therapies. Annualized rates of relapse (ARRs) started to increase 2 months after natalizumab cessation (month 2-4 ARR 0.47, 95% CI 0.43-0.51). Commencement of a subsequent therapy within 2-4 months reduced the magnitude of disease reactivation (mean ARR difference: 0.15, 0.08-0.22). After discontinuation of fingolimod, rates of relapse increased overall (month 1-2 ARR: 0.80, 0.70-0.89) and stabilized faster in patients who started a new therapy within 1-2 months (mean ARR difference: 0.14, -0.01 to 0.29). The magnitude of disease reactivation for other therapies was low but reduced further by commencement of another treatment 1-10 months after treatment discontinuation. Predictors of relapse were a higher relapse rate in the year before cessation, female sex, younger age, and higher EDSS score. Commencement of a subsequent therapy reduced both the risk of relapse (HR 0.76, 95% CI 0.72-0.81) and disability accumulation (0.73, 0.65-0.80). DISCUSSION: The rate of disease reactivation after treatment cessation differs among MS treatments, with the peaks of relapse activity ranging from 1 to 10 months in untreated cohorts that discontinued different therapies. These results suggest that untreated intervals should be minimized after stopping antitrafficking therapies (natalizumab and fingolimod). CLASSIFICATION OF EVIDENCE: This study provides Class III that disease reactivation occurs within months of discontinuation of MS disease-modifying therapies. The risk of disease activity is reduced by commencement of a subsequent therapy.

Association of Latitude and Exposure to Ultraviolet B Radiation With Severity of Multiple Sclerosis: An International Registry Study.

BACKGROUND AND OBJECTIVES: The severity of multiple sclerosis (MS) varies widely among individuals. Understanding the determinants of this heterogeneity will help clinicians optimize the management of MS. The aim of this study was to investigate the association between latitude of residence, UV B radiation (UVB) exposure, and the severity of MS. METHODS: This observational study used the MSBase registry data. The included patients met the 2005 or 2010 McDonald diagnostic criteria for MS and had a minimum dataset recorded in the registry (date of birth, sex, clinic location, date of MS symptom onset, disease phenotype at baseline and censoring, and ≥1 Expanded Disability Status Scale score recorded). The latitude of each study center and cumulative annualized UVB dose at study center (calculated from National Aeronautics and Space Administration's Total Ozone Mapping Spectrometer) at ages 6 and 18 years and the year of disability assessment were calculated. Disease severity was quantified with Multiple Sclerosis Severity Score (MSSS). Quadratic regression was used to model the associations between latitude, UVB, and MSSS. RESULTS: The 46,128 patients who contributed 453,208 visits and a cumulative follow-up of 351,196 patient-years (70% women, mean age 39.2 ± 12 years, resident between latitudes 19°35' and 56°16') were included in this study. Latitude showed a nonlinear association with MS severity. In latitudes <40°, more severe disease was associated with higher latitudes (ß = 0.08, 95% CI 0.04-0.12). For example, this translates into a mean difference of 1.3 points of MSSS between patients living in Madrid and Copenhagen. No such association was observed in latitudes <40° (ß = -0.02, 95% CI -0.06 to 0.03). The overall disability accrual was faster in those with a lower level of estimated UVB exposure before the age of 6 years (ß = - 0.5, 95% CI -0.6 to 0.4) and 18 years (ß = - 0.6, 95% CI -0.7 to 0.4), as well as with lower lifetime UVB exposure at the time of disability assessment (ß = -1.0, 95% CI -1.1 to 0.9). DISCUSSION: In temperate zones, MS severity is associated with latitude. This association is mainly, but not exclusively, driven by UVB exposure contributing to both MS susceptibility and severity.

Natalizumab, Fingolimod and Dimethyl Fumarate Use and Pregnancy-Related Relapse and Disability in Women With Multiple Sclerosis.

OBJECTIVE: To investigate pregnancy-related disease activity in a contemporary multiple sclerosis (MS) cohort. METHODS: Using data from the MSBase Registry, we included pregnancies conceived after 31 Dec 2010 from women with relapsing-remitting MS or clinically isolated syndrome. Predictors of intrapartum relapse, and postpartum relapse and disability progression were determined by clustered logistic regression or Cox regression analyses. RESULTS: We included 1998 pregnancies from 1619 women with MS. Preconception annualized relapse rate (ARR) was 0.29 (95% CI 0.27-0.32), fell to 0.19 (0.14-0.24) in third trimester, and increased to 0.59 (0.51-0.67) in early postpartum. Among women who used fingolimod or natalizumab, ARR before pregnancy was 0.37 (0.28-0.49) and 0.29 (0.22-0.37), respectively, and increased during pregnancy. Intrapartum ARR decreased with preconception dimethyl fumarate use. ARR spiked after delivery across all DMT groups. Natalizumab continuation into pregnancy reduced the odds of relapse during pregnancy (OR 0.76 per month [0.60-0.95], p=0.017). DMT re-initiation with natalizumab protected against postpartum relapse (HR 0.11 [0.04-0.32], p<0.0001). Breastfeeding women were less likely to relapse (HR 0.61 [0.41-0.91], p=0.016). 5.6% of pregnancies were followed by confirmed disability progression, predicted by higher relapse activity in pregnancy and postpartum. CONCLUSION: Intrapartum and postpartum relapse probabilities increased among women with MS after natalizumab or fingolimod cessation. In women considered to be at high relapse risk, use of natalizumab before pregnancy and continued up to 34 weeks gestation, with early re-initiation after delivery is an effective option to minimize relapse risks. Strategies of DMT use have to be balanced against potential fetal/neonatal complications.

Elevated plasma levels of D-serine in some patients with amyotrophic lateral sclerosis.

D-serine is an endogenous co-agonist with glutamate for activation of the N-methyl-D-aspartate receptor (NMDAR). D-serine exacerbates neuronal death and is elevated in the spinal cord from patients with sporadic/familial ALS. The present study was undertaken to examine whether plasma levels of D-serine of patients with ALS are different from those of healthy controls. Levels of D-serine in plasma (30 patients and 30 controls) were measured by high-performance liquid chromatography mass spectrometry. Plasma levels of D-serine in ALS patients (mean 39.27 ± 28.61 ng/ml) were significantly higher (p = 0.0293) than those of healthy control subjects (mean 21.07 ± 14.03 ng/ml) as well as previously reported values for healthy controls; ∼43% of patients had plasma D-serine levels that were 2 to 4-folds higher than those of controls. There was no association of plasma D-serine levels with disability, the duration of disease or with the age of subjects. In conclusion, we show that D-serine levels are elevated in the plasma of some ALS patients. Since D-serine serves as a co-agonist/activator of NMDAR, increases in D-serine could have a direct influence on glutamatergic neurotransmission and potentially contribute to excitotoxicity in some ALS patients.

Real-world effectiveness of cladribine for Australian patients with multiple sclerosis: An MSBase registry substudy.

BACKGROUND/OBJECTIVE: Observational clinical data from cladribine-treated patients with relapsing forms of multiple sclerosis (MS) were recorded in the Australian MS registry powered by the MSBase registry platform (5-year follow-up) and analysed to complement information from the pivotal cladribine clinical trials in MS. METHODS: A cohort of 90 cladribine-treated patients with follow-up data reported by treating physicians and recorded in the Australian MSBase registry (database lock February 2016) were examined. Clinical data included Expanded Disability Status Scale (EDSS) scores, relapses and other disease-modifying drugs (DMDs) administered before and after cladribine treatment. RESULTS: Mean age on starting cladribine was 47 years; mean age at MS onset was 34 years, and median baseline EDSS score was 5.25. Disability trajectories in patients with sufficient follow-up suggested an overall increasing trend prior to cladribine treatment which was reduced during the 2-year post-treatment. Approximately 80% of patients were EDSS progression-free, 65% remained relapse-free after 2 years and median time to next DMD was 1.7 years. CONCLUSION: These observational data suggest a disease-modifying effect in this cohort of relapsing MS patients characterised by older and more disabled patients. Since these data represent a single-arm cohort, clinical trials and larger comparative post-marketing studies are needed to validate and extend these findings.

Disability outcomes of early cerebellar and brainstem symptoms in multiple sclerosis.

BACKGROUND: Cerebellar and brainstem symptoms are common in early stages of multiple sclerosis (MS) yet their prognostic values remain unclear. OBJECTIVE: The aim of this study was to investigate long-term disability outcomes in patients with early cerebellar and brainstem symptoms. METHODS: This study used data from MSBase registry. Patients with early cerebellar/brainstem presentations were identified as those with cerebellar/brainstem relapse(s) or functional system score ⩾ 2 in the initial 2 years. Early pyramidal presentation was chosen as a comparator. Andersen-Gill models were used to compare cumulative hazards of (1) disability progression events and (2) relapses between patients with and without early cerebellar/brainstem symptoms. Mixed effect models were used to estimate the associations between early cerebellar/brainstem presentations and expanded disability status scale (EDSS) scores. RESULTS: The study cohort consisted of 10,513 eligible patients, including 2723 and 3915 patients with early cerebellar and brainstem symptoms, respectively. Early cerebellar presentation was associated with greater hazard of progression events (HR = 1.37, p < 0.001) and EDSS (ß = 0.16, p < 0.001). Patients with early brainstem symptoms had lower hazard of progression events (HR = 0.89, p = 0.01) and EDSS (ß = -0.06, p < 0.001). Neither presentation was associated with changes in relapse risk. CONCLUSION: Early cerebellar presentation is associated with unfavourable outcomes, while early brainstem presentation is associated with favourable prognosis. These presentations may be used as MS prognostic markers and guide therapeutic approach.

Elevated Levels of Homocysteinesulfinic Acid in the Plasma of Patients with Amyotrophic Lateral Sclerosis: A Potential Source of Excitotoxicity?

OBJECTIVES: Excitotoxicity is thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). One possible source of excitotoxicity is the presence of sulphur amino acids (SAAs). In the brain of subjects with ALS, there are increased levels of taurine. In the metabolism of methionine to taurine, excitatory sulphur amino acids (SAAs) are formed. These could potentially contribute to excitotoxicity in ALS. The present study has examined whether plasma levels of SAAs in 38 ALS patients differ from those of 30 healthy controls. METHODS: Plasma levels of SAAs were measured by liquid chromatography mass spectrometry. RESULTS: There were no significant changes in plasma cysteic acid, cysteine sulfinic acid, and homocysteic acid in ALS patients compared to healthy subjects. Significant elevations in plasma homocysteinesulfinic acid (HCSA) levels (p < 0.0001) were observed in the ALS patients (75.91 ± 15.38 nM) compared to healthy controls (54.06 ± 8.503 nM); 50% of the ALS patients had HCSA levels that were 1.5-2-folds higher than those of controls. Plasma levels of HCSA differed significantly (p = 0.0440) between patients with bulbar onset and spinal onset (68.57 ± 14.20 vs. 79.30 ± 14.95 nM, respectively). CONCLUSION: HCSA is elevated in the blood of subjects with ALS. Since HCSA can be transported from the blood to the CNS by active transport, has neurotransmitter properties, and can activate synaptic receptors including NMDAR and metabotropic glutamate receptor, it is possible that increases in HCSA could influence glutamatergic neurotransmission and potentially contribute to excitotoxicity in some ALS patients.

Plasma from some patients with amyotrophic lateral sclerosis exhibits elevated formaldehyde levels.

INTRODUCTION: The aim of this study was to determine whether patients with amyotrophic lateral sclerosis (ALS) exhibit higher plasma levels of formaldehyde (FA) than controls, and to look for alterations in levels of FA precursors. METHODS: We studied 40 heathy controls and 50 ALS patients from the Motor Neuron Disease clinic at the Royal Brisbane & Women's Hospital. Plasma FA levels were quantified using a FA detection assay. Trimethylamine (TMA) and trimethylamine oxide (TMAO) in plasma were quantified by multiple reaction monitoring (MRM) mass spectrometry. Plasma levels in patients and controls were compared using Mann-Whitney U test and Spearman's correlation test was used to assess the correlation between levels of FA, TMA, TMAO and other variables. RESULTS: The levels of plasma FA were significantly greater in ALS subjects than controls. TMA and TMAO levels were not significantly different between healthy controls and patients, but were greater in ALS subjects with elevated FA levels than those with normal levels. Of note, levels of TMA and TMAO demonstrated a significant positive correlation with plasma FA levels (spearman's correlation coefficients of TMA with FA [r = 0.451, p = 0.010] and TMAO [r = 0.401, p = 0.023]). There was no association of FA levels with disability measured with the ALS functional rating scale, with the duration of disease or with the age of the subjects. CONCLUSION: Elevated FA is found in some patients with ALS. FA is neurotoxic and could contribute to disease pathogenesis.

Mass spectrometry analysis of plasma from amyotrophic lateral sclerosis and control subjects.

Mass spectrometry was used to study blood samples from patients with amyotrophic lateral sclerosis (ALS) and healthy controls. Addenbrooke's cognitive examination-III (ACE-III) was used to test for cognitive impairment (CI). Nano liquid chromatography and time of flight mass spectrometry (MS) were performed on samples from 42 ALS patients and 18 healthy controls. SWATH™ proteomic analysis was utilized to look for differences between groups. Western blot analysis was used to study levels of 4 proteins, selected as being of possible interest in ALS, in the MS discovery cohort and a second validation group of 10 ALS patients and 10 healthy controls. INGENUITY PATHWAY ANALYSIS (IPA) was applied to the final proteomic data. Between ALS patients and controls, there were significant differences in the expression of 30 proteins. Between controls and ALS patients without CI, there were significant differences in 15 proteins. Between controls and ALS patients with CI, there were significant differences in 32 proteins. Changes in levels of gelsolin, clusterin, and CD5L were validated by using western blot analysis in the discovery cohort. Changes in the expression of gelsolin, clusterin, and ficolin 3 were replicated in a validation group. In ALS, the LXR/RXR and coagulation pathways were downregulated whereas the complement pathway was upregulated. The proteomic data were used to produce two new networks, centered on IL1 and on NFkB, which showed altered levels in ALS. This study highlights the usefulness of MS of blood samples as a tool to study ALS.

Screening for cognitive and behavioural impairment in amyotrophic lateral sclerosis: Frequency of abnormality and effect on survival.

OBJECTIVE: To screen for cognitive and behavioural impairment in people with amyotrophic lateral sclerosis (ALS) and controls with neuromuscular disease and to correlate these with clinical features. METHODS: 108 people with ALS and 60 controls with other neuromuscular diseases were recruited and assessed with the Addenbrooke's cognitive examination-III (ACE-III), the frontal assessment battery (FAB), and the executive function component of the Edinburgh cognitive and behavioural ALS screen (ECAS). The Amyotrophic lateral sclerosis-Frontotemporal dementia questionnaire (ALS-FTD-Q) and the Motor Neuron Disease Behavioural instrument (MiND-B) were administered to the caregivers of people with ALS. The prevalence of abnormalities was determined and correlated with clinical features and survival. In 37 people with ALS, serial studies were performed. RESULTS: The frequencies of cognitive impairment based on the ACE-III and FAB were 30.0% and 14.0%, in ALS and 11.7% and 3.3% in controls, respectively. Age and years of education influence the results of the ACE-III and ECAS executive function. In ALS, the frequencies of behavioural impairment based on ALS-FTD-Q and MiND-B were 32.1% and 39.4%, respectively. There is significant correlation of ALS-FTD-Q and MiND-B with the ALSFRS-R score. ALS participants with cognitive impairment measured with ACE-III had significantly shorter survival time than those without. ALS participants with behavioural impairment measured with ALS-FTD-Q had worse prognosis than those without. No significant difference was found between the first two serial cognitive tests based on ACE-III and FAB by using generalized estimating equation. CONCLUSION: There is a greater frequency of cognitive impairment in people with ALS than in patients with other neuromuscular diseases. The cognitive and behavioural tests are potential biomarkers of the prognosis of ALS. The results of cognitive tests are stable over 6months and possibly longer.

Neurofilaments as Biomarkers for Amyotrophic Lateral Sclerosis: A Systematic Review and Meta-Analysis.

BACKGROUND: To allow early diagnosis and monitoring of disease progression, there is a need for biomarkers in amyotrophic lateral sclerosis (ALS). Neurofilaments (NF) are emerging protein biomarkers in other neurological diseases, and are of possible use in ALS. OBJECTIVE: The aim of this study is to evaluate the utility of NF levels as blood or cerebrospinal fluid (CSF) biomarker in patients with ALS. METHODS: A systematic search of Pubmed, Embase and Scopus was performed. Methodological quality assessment was applied to refine the final search results. Meta-analysis of the data was performed. RESULTS: Level of NF heavy chain and light chains were significantly elevated in the CSF of ALS patients compared to healthy controls/controls without parenchymal central nervous system (CNS) involvement and ALS mimic disease patients. NF light chain level in CSF was higher in ALS patients than in neurological patients with CNS involvement (SMD = 1.352, P = 0.01). NF light chain concentration in blood was higher in ALS patients than healthy controls/controls without CNS involvement (SMD = 1.448, P<0.0001). NF heavy chain levels in CSF were negatively correlated disease duration and ALSFRS-R ((r = -0.447, P<0.0001; r = -0.486, P<0.0001). NF light chain levels in CSF were negatively correlated with disease duration (r = -0.273, P = 0.011). CONCLUSION: NF heavy and light chain levels have potential use as a marker of neural degeneration in ALS, but are not specific for the disease, and are more likely to be used as measures of disease progression.