The cortical neurophysiological signature of amyotrophic lateral sclerosis.

The progressive loss of motor function characteristic of amyotrophic lateral sclerosis is associated with widespread cortical pathology extending beyond primary motor regions. Increasing muscle weakness reflects a dynamic, variably compensated brain network disorder. In the quest for biomarkers to accelerate therapeutic assessment, the high temporal resolution of magnetoencephalography is uniquely able to non-invasively capture micro-magnetic fields generated by neuronal activity across the entire cortex simultaneously. This study examined task-free magnetoencephalography to characterize the cortical oscillatory signature of amyotrophic lateral sclerosis for having potential as a pharmacodynamic biomarker. Eight to ten minutes of magnetoencephalography in the task-free, eyes-open state was recorded in amyotrophic lateral sclerosis (n = 36) and healthy age-matched controls (n = 51), followed by a structural MRI scan for co-registration. Extracted magnetoencephalography metrics from the delta, theta, alpha, beta, low-gamma, high-gamma frequency bands included oscillatory power (regional activity), 1/f exponent (complexity) and amplitude envelope correlation (connectivity). Groups were compared using a permutation-based general linear model with correction for multiple comparisons and confounders. To test whether the extracted metrics could predict disease severity, a random forest regression model was trained and evaluated using nested leave-one-out cross-validation. Amyotrophic lateral sclerosis was characterized by reduced sensorimotor beta band and increased high-gamma band power. Within the premotor cortex, increased disability was associated with a reduced 1/f exponent. Increased disability was more widely associated with increased global connectivity in the delta, theta and high-gamma bands. Intra-hemispherically, increased disability scores were particularly associated with increases in temporal connectivity and inter-hemispherically with increases in frontal and occipital connectivity. The random forest model achieved a coefficient of determination (R2) of 0.24. The combined reduction in cortical sensorimotor beta and rise in gamma power is compatible with the established hypothesis of loss of inhibitory, GABAergic interneuronal circuits in pathogenesis. A lower 1/f exponent potentially reflects a more excitable cortex and a pathology unique to amyotrophic lateral sclerosis when considered with the findings published in other neurodegenerative disorders. Power and complexity changes corroborate with the results from paired-pulse transcranial magnetic stimulation. Increased magnetoencephalography connectivity in worsening disability is thought to represent compensatory responses to a failing motor system. Restoration of cortical beta and gamma band power has significant potential to be tested in an experimental medicine setting. Magnetoencephalography-based measures have potential as sensitive outcome measures of therapeutic benefit in drug trials and may have a wider diagnostic value with further study, including as predictive markers in asymptomatic carriers of disease-causing genetic variants.

Data-independent acquisition proteomics of cerebrospinal fluid implicates endoplasmic reticulum and inflammatory mechanisms in amyotrophic lateral sclerosis.

While unbiased proteomics of human cerebrospinal fluid (CSF) has been used successfully to identify biomarkers of amyotrophic lateral sclerosis (ALS), high-abundance proteins mask the presence of lower abundance proteins that may have diagnostic and prognostic value. However, developments in mass spectrometry (MS) proteomic data acquisition methods offer improved protein depth. In this study, MS with library-free data-independent acquisition (DIA) was used to compare the CSF proteome of people with ALS (n = 40), healthy (n = 15) and disease (n = 8) controls. Quantified protein groups were subsequently correlated with clinical variables. Univariate analysis identified 7 proteins, all significantly upregulated in ALS versus healthy controls, and 9 with altered abundance in ALS versus disease controls (FDR < 0.1). Elevated chitotriosidase-1 (CHIT1) was common to both comparisons and was proportional to ALS disability progression rate (Pearson r = 0.41, FDR-adjusted p = 0.035) but not overall survival. Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1; upregulated in ALS versus healthy controls) was proportional to disability progression rate (Pearson r = 0.53, FDR-adjusted p = 0.003) and survival (Kaplan Meier log-rank p = 0.013) but not independently in multivariate proportional hazards models. Weighted correlation network analysis was used to identify functionally relevant modules of proteins. One module, enriched for inflammatory functions, was associated with age at symptom onset (Pearson r = 0.58, FDR-adjusted p = 0.005) and survival (Hazard Ratio = 1.78, FDR = 0.065), and a second module, enriched for endoplasmic reticulum proteins, was negatively correlated with disability progression rate (r = -0.42, FDR-adjusted p = 0.109). DIA acquisition methodology therefore strengthened the biomarker candidacy of CHIT1 and UCHL1 in ALS, while additionally highlighted inflammatory and endoplasmic reticulum proteins as novel sources of prognostic biomarkers.

Premorbid brain structure influences risk of amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a disease of the motor network associated with brain structure and functional connectivity alterations that are implicated in disease progression. Whether such changes have a causal role in ALS, fitting with a postulated influence of premorbid cerebral architecture on the phenotypes associated with neurodegenerative disorders is not known. METHODS: This study considered causal effects and shared genetic risk of 2240 structural and functional MRI brain scan imaging-derived phenotypes (IDPs) on ALS using two sample Mendelian randomisation, with putative associations further examined with extensive sensitivity analysis. Shared genetic predisposition between IDPs and ALS was explored using genetic correlation analysis. RESULTS: Increased white matter volume in the cerebral hemispheres was causally associated with ALS. Weaker causal associations were observed for brain stem grey matter volume, parieto-occipital white matter surface and volume of the left thalamic ventral anterior nucleus. Genetic correlation was observed between ALS and intracellular volume fraction and isotropic free water volume fraction within the posterior limb of the internal capsule. CONCLUSIONS: This study provides evidence that premorbid brain structure, in particular white matter volume, contributes to the risk of ALS.

Primary care blood tests show lipid profile changes in pre-symptomatic amyotrophic lateral sclerosis.

Multiple sources of evidence suggest that changes in metabolism may precede the onset of motor symptoms in amyotrophic lateral sclerosis. This study aimed to seek evidence for alterations in the levels of blood indices collected routinely in the primary care setting prior to the onset of motor symptoms in amyotrophic lateral sclerosis. Premorbid data, measured as part of routine health screening, for total cholesterol, high-density and low-density lipoprotein cholesterol, triglyceride, glycated haemoglobin A1c and creatinine were collected retrospectively from (i) a cohort of amyotrophic lateral sclerosis patients attending a specialist clinic (n = 143) and (ii) from primary care-linked data within UK Biobank. Data were fitted using linear mixed effects models with linear b-splines to identify inflection points, controlling for age and sex. In specialist amyotrophic lateral sclerosis clinic cases, models indicated decreasing levels of total and low-density lipoprotein cholesterol prior to an inflection point in the years before symptom onset (total cholesterol 3.25 years, low-density lipoprotein cholesterol 1.25 years), after which they stabilized or rose. A similar pattern was observed in amyotrophic lateral sclerosis cases within UK Biobank, occurring several years prior to diagnosis (total cholesterol 7 years, low-density lipoprotein cholesterol 7.25 years), differing significantly from matched controls. High-density lipoprotein cholesterol followed a similar pattern but was less robust to sensitivity analyses. Levels of triglyceride remained stable throughout. Glycated haemoglobin temporal profiles were not consistent between the clinic and biobank cohorts. Creatinine level trajectories prior to amyotrophic lateral sclerosis did not differ significantly from controls but decreased significantly in the symptomatic period after an inflection point of 0.25 years after symptom onset (clinic cohort) or 0.5 years before diagnosis (UK Biobank). These data provide further evidence for a pre-symptomatic period of dynamic metabolic change in amyotrophic lateral sclerosis, consistently associated with alterations in blood cholesterols. Such changes may ultimately contribute to biomarkers applicable to population screening and for pathways guiding the targeting of preventative therapy.

Limited value of serum neurofilament light chain in diagnosing amyotrophic lateral sclerosis.

A biomarker specific for the diagnosis of amyotrophic lateral sclerosis must be sensitive across a spectrum of clinical heterogeneity. Neurofilament light chain levels in amyotrophic lateral sclerosis correlate with the rate of disability progression. Previous attempts to establish a diagnostic role for neurofilament light chain have been limited to comparison with healthy individuals or controls with alternative diagnoses unlikely to be confused with amyotrophic lateral sclerosis in real-world clinical practice. In a tertiary amyotrophic lateral sclerosis referral clinic, at first visit, serum was taken for neurofilament light chain measurement after prospectively recording the clinical diagnosis as 'amyotrophic lateral sclerosis', 'primary lateral sclerosis', 'alternative' or 'currently uncertain'. Of 133 referrals, 93 patients were diagnosed with amyotrophic lateral sclerosis (median neurofilament light chain 218.1 pg/ml, interquartile range 130.7-311.9), three primary lateral sclerosis (65.6, 51.5-106.9) and 19 alternative diagnoses (45.2, 13.5-71.9) at first visit. Of 18 initially uncertain diagnoses, eight were subsequently diagnosed with amyotrophic lateral sclerosis (98.5, 45.3-300.1). Neurofilament light chain ≥110.9 pg/ml had a positive predictive value of 0.92 for amyotrophic lateral sclerosis; <110.9 pg/ml had a negative predictive value of 0.48. In a specialized clinic, neurofilament light chain is largely confirmatory to clinical judgement in diagnosing amyotrophic lateral sclerosis and has limited ability to exclude alternative diagnoses. The current, important, value of neurofilament light chain is its potential to stratify patients with amyotrophic lateral sclerosis by disease activity and as a biomarker in therapeutic trials.

Advantages of routine next-generation sequencing over standard genetic testing in the amyotrophic lateral sclerosis clinic.

BACKGROUND: Next-generation sequencing has enhanced our understanding of amyotrophic lateral sclerosis (ALS) and its genetic epidemiology. Outside the research setting, testing is often restricted to those who report a family history. The aim of this study was to explore the added benefit of offering routine genetic testing to all patients in a regional ALS centre. METHODS: C9ORF72 expansion testing and exome sequencing was offered to consecutive patients (150 with ALS and 12 with primary lateral sclerosis [PLS]) attending the Oxford Motor Neuron Disease Clinic within a defined time period. RESULTS: A total of 17 (11.3%) highly penetrant pathogenic variants in C9ORF72, SOD1, TARDBP, FUS and TBK1 were detected, of which 10 were also found through standard clinical genetic testing pathways. The systematic approach resulted in five additional diagnoses of a C9ORF72 expansion (number needed to test [NNT] = 28), and two further missense variants in TARDBP and SOD1 (NNT = 69). Additionally, 3 patients were found to carry pathogenic risk variants in NEK1, and 13 patients harboured common missense variants in CFAP410 and KIF5A, also associated with an increased risk of ALS. We report two novel non-coding loss-of-function splice variants in TBK1 and OPTN. No relevant variants were found in the PLS patients. Patients were offered double-blinded participation, but >80% requested disclosure of the results. CONCLUSIONS: This study provides evidence that expanding genetic testing to all patients with a clinical diagnosis of ALS enhances the potential for recruitment to clinical trials, but will have direct resource implications for genetic counselling.

Profiling non-coding RNA expression in cerebrospinal fluid of amyotrophic lateral sclerosis patients.

INTRODUCTION: Objective biomarkers for the fatal neurodegenerative disease amyotrophic lateral sclerosis or motor neuron disease (ALS/MND) are critical for diagnosis, drug development, clinical trials, and insight into disease pathology. Key candidates for biomarkers present in biofluids include non-coding RNA (ncRNA) transcripts including microRNA, piwi-interacting RNA and transfer RNA. To determine if the central nervous system was the source of the dysregulated ncRNA biomarkers we previously observed in serum, we sought to identify dysregulated ncRNA candidates in cerebrospinal fluid (CSF) which may provide new insight into the disease pathology. METHODS AND MATERIALS: Small RNA sequencing (RNA-seq) was undertaken on CSF samples from healthy controls (n = 18), disease mimics (n = 8), and ALS patients (n = 40) in our Oxford Study for Biomarkers of ALS cohort, with RT-qPCR used to confirm their dysregulation. RESULTS: We found a range of ncRNA that were dysregulated in the RNA-seq screen, but these failed to be validated or detected in some cases using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, our previously identified serum ncRNA biomarker showed no change in CSF or correlation to serum. CONCLUSIONS: This study suggests the CSF may not be the source of dysregulated ncRNA in the serum and highlights the difficulty in identifying ncRNA in CSF as biomarkers for ALS.KEY MESSAGESIn this current study, we investigated the expression of non-coding RNA transcripts in the cerebrospinal fluid of ALS patients compared to healthy controls.RNA-seq identified dysregulated non-coding RNA transcripts, but these were not validated with RT-qPCR.We conclude that cerebrospinal fluid is not a suitable source of diagnostic biomarkers.

Creatine kinase and prognosis in amyotrophic lateral sclerosis: a literature review and multi-centre cohort analysis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a prognostically heterogeneous neurodegenerative disease. Blood creatine kinase (CK) level has been inconsistently reported as a prognostic biomarker and raised levels in some ALS patients have been presumed to reflect muscle wasting, which is also variable. METHODS: MEDLINE was systematically searched for papers related to CK in ALS and the relevant studies were reviewed. Using data from 222 ALS patients in a multi-centre, prospective, longitudinal cohort, survival analyses using Kaplan-Meier and Cox proportional hazards models were undertaken in relation to CK and other prognostic factors. RESULTS: Twenty-five studies investigating CK in ALS were identified, of which 10 specifically studied the link between CK and survival. Five studies observed no association, four found that higher CK levels were associated with longer survival and one, the opposite. In our cohort (n = 222), 39% of patients had a CK level above the laboratory reference range. Levels were higher in males compared to females (p < 0.001), in patients with limb versus bulbar onset of symptoms (p < 0.001) and in patients with higher lower motor neuron burden (p < 0.001). There was no significant trend in longitudinal CK values. Although a higher standardised log (CK) at first visit was associated with longer survival in univariate analysis (hazard ratio 0.75, p = 0.003), there was no significant association after adjusting for other prognostic covariates. CONCLUSION: While raised CK levels in ALS do reflect lower motor neuron denervation to a large extent, they are not independently associated with survival when measured in the symptomatic phase of the disease.

Multicentre appraisal of amyotrophic lateral sclerosis biofluid biomarkers shows primacy of blood neurofilament light chain.

The routine clinical integration of individualized objective markers of disease activity in those diagnosed with the neurodegenerative disorder amyotrophic lateral sclerosis is a key requirement for therapeutic development. A large, multicentre, clinic-based, longitudinal cohort was used to systematically appraise the leading candidate biofluid biomarkers in the stratification and potential therapeutic assessment of those with amyotrophic lateral sclerosis. Incident patients diagnosed with amyotrophic lateral sclerosis (n = 258), other neurological diseases (n = 80) and healthy control participants (n = 101), were recruited and followed at intervals of 3-6 months for up to 30 months. Cerebrospinal fluid neurofilament light chain and chitotriosidase 1 and blood neurofilament light chain, creatine kinase, ferritin, complement C3 and C4 and C-reactive protein were measured. Blood neurofilament light chain, creatine kinase, serum ferritin, C3 and cerebrospinal fluid neurofilament light chain and chitotriosidase 1 were all significantly elevated in amyotrophic lateral sclerosis patients. First-visit plasma neurofilament light chain level was additionally strongly associated with survival (hazard ratio for one standard deviation increase in log10 plasma neurofilament light chain 2.99, 95% confidence interval 1.65-5.41, P = 0.016) and rate of disability progression, independent of other prognostic factors. A small increase in level was noted within the first 12 months after reported symptom onset (slope 0.031 log10 units per month, 95% confidence interval 0.012-0.049, P = 0.006). Modelling the inclusion of plasma neurofilament light chain as a therapeutic trial outcome measure demonstrated that a significant reduction in sample size and earlier detection of disease-slowing is possible, compared with using the revised Amyotrophic Lateral Sclerosis Functional Rating Scale. This study provides strong evidence that blood neurofilament light chain levels outperform conventional measures of disease activity at the group level. The application of blood neurofilament light chain has the potential to radically reduce the duration and cost of therapeutic trials. It might also offer a first step towards the goal of more personalized objective disease activity monitoring for those living with amyotrophic lateral sclerosis.

Higher blood high density lipoprotein and apolipoprotein A1 levels are associated with reduced risk of developing amyotrophic lateral sclerosis.

BACKGROUND: Premorbid body mass index, physical activity, diabetes and cardiovascular disease have been associated with an altered risk of developing amyotrophic lateral sclerosis (ALS). There is evidence of shared genetic risk between ALS and lipid metabolism. A very large prospective longitudinal population cohort permits the study of a range of metabolic parameters and the risk of subsequent diagnosis of ALS. METHODS: The risk of subsequent ALS diagnosis in those enrolled prospectively to the UK Biobank (n=502 409) was examined in relation to baseline levels of blood high and low density lipoprotein (HDL, LDL), total cholesterol, total cholesterol:HDL ratio, apolipoproteins A1 and B (apoA1, apoB), triglycerides, glycated haemoglobin A1c (HbA1c) and creatinine, plus self-reported exercise and body mass index. RESULTS: Controlling for age and sex, higher HDL (HR 0.84, 95% CI 0.73 to 0.96, p=0.010) and apoA1 (HR 0.83, 95% CI 0.72 to 0.94, p=0.005) were associated with a reduced risk of ALS. Higher total cholesterol:HDL was associated with an increased risk of ALS (HR 1.17, 95% CI 1.05 to 1.31, p=0.006). In models incorporating multiple metabolic markers, higher LDL or apoB was associated with an increased risk of ALS, in addition to a lower risk with higher HDL or apoA. Coronary artery disease, cerebrovascular disease and increasing age were also associated with an increased risk of ALS. CONCLUSIONS: The association of HDL, apoA1 and LDL levels with risk of ALS contributes to an increasing body of evidence that the premorbid metabolic landscape may play a role in pathogenesis. Understanding the molecular basis for these changes will inform presymptomatic biomarker development and therapeutic targeting.

Advancing mechanistic understanding and biomarker development in amyotrophic lateral sclerosis.

INTRODUCTION: Proteomic analysis has contributed significantly to the study of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). It has helped to define the pathological change common to nearly all cases, namely intracellular aggregates of phosphorylated TDP-43, shifting the focus of pathogenesis in ALS toward RNA biology. Proteomics has also uniquely underpinned the delineation of disease mechanisms in model systems and has been central to recent advances in human ALS biomarker development. AREAS COVERED: The contribution of proteomics to understanding the cellular pathological changes, disease mechanisms, and biomarker development in ALS are covered. EXPERT OPINION: Proteomics has delivered unique insights into the pathogenesis of ALS and advanced the goal of objective measurements of disease activity to improve therapeutic trials. Further developments in sensitivity and quantification are expected, with application to the presymptomatic phase of human disease offering the hope of prevention strategies.

Network Analysis of the CSF Proteome Characterizes Convergent Pathways of Cellular Dysfunction in ALS.

BACKGROUND: Amyotrophic lateral sclerosis is a clinical syndrome with complex biological determinants, but which in most cases is characterized by TDP-43 pathology. The identification in CSF of a protein signature of TDP-43 network dysfunction would have the potential to inform the identification of new biomarkers and therapeutic targets. METHODS: We compared CSF proteomic data from patients with ALS (n = 41), Parkinson's disease (n = 19) and healthy control participants (n = 20). Weighted correlation network analysis was used to identify modules within the CSF protein network and combined with gene ontology enrichment analysis to functionally annotate module proteins. Analysis of module eigenproteins and differential correlation analysis of the CSF protein network was used to compare ALS and Parkinson's disease protein co-correlation with healthy controls. In order to monitor temporal changes in the CSF proteome, we performed longitudinal analysis of the CSF proteome in a subset of ALS patients. RESULTS: Weighted correlation network analysis identified 10 modules, including those enriched for terms involved in gene expression including nucleic acid binding, RNA metabolism and translation; humoral immune system function, including complement pathways; membrane proteins, axonal outgrowth and adherence; and glutamatergic synapses. Immune system module eigenproteins were increased in ALS, whilst axonal module eigenproteins were decreased in ALS. The 19 altered protein correlations in ALS were enriched for gene expression (OR 3.05, p = 0.017) and membrane protein modules (OR 17.48, p = 0.011), including intramodular hub proteins previously identified as TDP-43 interactors. Proteins decreasing over longitudinal analysis ALS were enriched in glutamatergic synapse and axonal outgrowth modules. Protein correlation network disruptions in Parkinson's disease showed no module enrichment. CONCLUSIONS: Alterations in the co-correlation network in CSF samples identified a set of pathways known to be associated with TDP-43 dysfunction in the pathogenesis of ALS, with important implications for therapeutic targeting and biomarker development.

Non-neuronal cells in amyotrophic lateral sclerosis - from pathogenesis to biomarkers.

The prevailing motor neuron-centric view of amyotrophic lateral sclerosis (ALS) pathogenesis could be an important factor in the failure to identify disease-modifying therapy for this neurodegenerative disorder. Non-neuronal cells have crucial homeostatic functions within the CNS and evidence of involvement of these cells in the pathophysiology of several neurodegenerative disorders, including ALS, is accumulating. Microglia and astrocytes, in crosstalk with peripheral immune cells, can exert both neuroprotective and adverse effects, resulting in a highly nuanced range of neuronal and non-neuronal cell interactions. This Review provides an overview of the diverse roles of non-neuronal cells in relation to the pathogenesis of ALS and the emerging potential of non-neuronal cell biomarkers to advance therapeutic development.

CSF extracellular vesicle proteomics demonstrates altered protein homeostasis in amyotrophic lateral sclerosis.

BACKGROUND: Extracellular vesicles (EVs) released by neurons and glia reach the cerebrospinal fluid (CSF). Studying the proteome of CSF-derived EVs offers a novel perspective on the key intracellular processes associated with the pathogenesis of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and a potential source from which to develop biomarkers. METHODS: CSF EVs were extracted using ultrafiltration liquid chromatography from ALS patients and controls. EV size distribution and concentration was measured using nanoparticle tracking analysis and liquid chromatography-tandem mass spectrometry proteomic analysis performed. RESULTS: CSF EV concentration and size distribution did not differ between ALS and control groups, nor between a sub-group of ALS patients with or without an associated hexanucleotide repeat expansion (HRE) in C9orf72. Univariate proteomic analysis identified downregulation of the pentameric proteasome-like protein Bleomycin hydrolase in ALS patients, whilst Gene Ontology enrichment analysis demonstrated downregulation of proteasome core complex proteins (8/8 proteins, normalized enrichment ratio -1.77, FDR-adjusted p = 0.057) in the ALS group. The sub-group of ALS patients associated with the C9orf72 HRE showed upregulation in Ubiquitin-like modifying-activating protein 1 (UBA1) compared to non-C9orf72 cases. CONCLUSIONS: Proteomic analysis of CSF EVs in ALS detects intracellular alterations in protein homeostatic mechanisms, previously only identified in pathological tissues. This supports the wider use of CSF EVs as a source of novel biomarkers reflecting key and potentially druggable pathological intracellular pathway alterations in ALS.

An ALS-linked mutation in TDP-43 disrupts normal protein interactions in the motor neuron response to oxidative stress.

TDP-43 pathology is a key feature of amyotrophic lateral sclerosis (ALS), but the mechanisms linking TDP-43 to altered cellular function and neurodegeneration remain unclear. We have recently described a mouse model in which human wild-type or mutant TDP-43 are expressed at low levels and where altered stress granule formation is a robust phenotype of TDP-43M337V/- expressing cells. In the present study we use this model to investigate the functional connectivity of human TDP-43 in primary motor neurons under resting conditions and in response to oxidative stress. The interactome of human TDP-43WT or TDP-43M337V was compared by mass spectrometry, and gene ontology enrichment analysis identified pathways dysregulated by the M337V mutation. We found that under normal conditions the interactome of human TDP-43WT was enriched for proteins involved in transcription, translation and poly(A)-RNA binding. In response to oxidative stress, TDP-43WT recruits proteins of the endoplasmic reticulum and endosomal-extracellular transport pathways, interactions which are reduced in the presence of the M337V mutation. Specifically, TDP-43M337V impaired protein-protein interactions involved in stress granule formation including reduced binding to the translation initiation factors Poly(A)-binding protein and Eif4a1 and the endoplasmic reticulum chaperone Grp78. The M337V mutation also affected interactions involved in endosomal-extracellular transport and this this was associated with reduced extracellular vesicle secretion in primary motor neurons from TDP-43M337V/- mice and in human iPSCs-derived motor neurons. Taken together, our analysis highlights a TDP-43 interaction network in motor neurons and demonstrates that an ALS associated mutation may alter the interactome to drive aberrant pathways involved in the pathogenesis of ALS.

Identification of a potential non-coding RNA biomarker signature for amyotrophic lateral sclerosis.

Objective biomarkers for the clinically heterogeneous adult-onset neurodegenerative disorder amyotrophic lateral sclerosis are crucial to facilitate assessing emerging therapeutics and improve the diagnostic pathway in what is a clinically heterogeneous syndrome. With non-coding RNA transcripts including microRNA, piwi-RNA and transfer RNA present in human biofluids, we sought to identify whether non-coding RNA in serum could be biomarkers for amyotrophic lateral sclerosis. Serum samples from our Oxford Study for Biomarkers in motor neurone disease/amyotrophic lateral sclerosis discovery cohort of amyotrophic lateral sclerosis patients (n = 48), disease mimics (n = 16) and age- and sex-matched healthy controls (n = 24) were profiled for non-coding RNA expression using RNA-sequencing, which showed a wide range of non-coding RNA to be dysregulated. We confirmed significant alterations with reverse transcription-quantitative PCR in the expression of hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-92a-3p, hsa-piR-33151, TRV-AAC4-1.1 and TRA-AGC6-1.1. Furthermore, hsa-miR-206, a previously identified amyotrophic lateral sclerosis biomarker, showed a binary-like pattern of expression in our samples. Using the expression of these non-coding RNA, we were able to discriminate amyotrophic lateral sclerosis samples from healthy controls in our discovery cohort using a random forest analysis with 93.7% accuracy with promise in predicting progression rate of patients. Importantly, cross-validation of this novel signature using a new geographically distinct cohort of samples from the United Kingdom and Germany with both amyotrophic lateral sclerosis and control samples (n = 156) yielded an accuracy of 73.9%. The high prediction accuracy of this non-coding RNA-based biomarker signature, even across heterogeneous cohorts, demonstrates the strength of our approach as a novel platform to identify and stratify amyotrophic lateral sclerosis patients.

CSF chitinases before and after symptom onset in amyotrophic lateral sclerosis.

OBJECTIVE: To evaluate the CSF levels of chitinase proteins during the presymptomatic and early symptomatic phases of amyotrophic lateral sclerosis (ALS). METHODS: CSF samples were obtained from 16 controls, 55 individuals at-risk for ALS (including 18 carrying a mutation in C9ORF72, 33 in SOD1), 12 ALS patients, and 7 phenoconverters (individuals diagnosed with ALS during follow-up). At-risk individuals and phenoconverters were enrolled through the Pre-fALS study, which includes individuals carrying an ALS-associated gene mutation without disease manifestations at initial assessment. Longitudinal CSF collections, where possible, took place every 3-12 months for ALS patients and every 1-2 years for others. CSF levels of chitotriosidase 1 (CHIT1), chitinase-3-like protein 1 (CHI3L1, YKL-40) and chitinase-3-like protein 2 (CHI3L2, YKL-39) were measured by ELISA, along with CHIT1 activity. Longitudinal changes in at-risk individuals and phenoconverters were fitted to linear mixed effects models. RESULTS: Slowly rising levels of CHIT1 were observed over time in the at-risk individuals (slope 0.059 log10 [CHIT1] per year, P < 0.001). Among phenoconverters, CHIT1 levels and activity rose more sharply (0.403 log10 [CHIT1] per year, P = 0.005; 0.260 log10 [CHIT1 activity] per year, P = 0.007). Individual levels of both CHI3L1 and CHI3L2 remained relatively stable over time in all participant groups. INTERPRETATION: The CHIT1 neuroinflammatory response is a feature of the late presymptomatic to early symptomatic phases of ALS. This study does not suggest a long prodrome of upregulated glial activity in ALS pathogenesis, but strengthens the place of CHIT1 as part of a panel of biomarkers to objectively assess the impact of immune-modulatory therapeutic interventions in ALS.

CSF chitinase proteins in amyotrophic lateral sclerosis.

OBJECTIVE: To evaluate the classifier performance, clinical and biochemical correlations of cerebrospinal fluid (CSF) levels of the chitinase proteins Chitotriosidase-1 (CHIT1), Chitinase-3-like protein 1 (CHI3L1) and Chitinase-3-like protein 2 (CHI3L2) in amyotrophic lateral sclerosis (ALS). METHODS: CSF levels of CHIT1, CHI3L1, CHI3L2, phosphorylated neurofilament heavy chain (pNFH) and C-reactive protein were measured by ELISA in a longitudinal cohort of patients with ALS (n=82), primary lateral sclerosis (PLS, n=10), ALS-mimic conditions (n=12), healthy controls (n=25) and asymptomatic carriers of ALS-causing genetic mutations (AGC; n=5). RESULTS: CSF CHIT1, CHI3L1 and CHI3L2 were elevated in patients with ALS compared with healthy controls (p<0.001) and ALS-mimics (CHIT1, p<0.001; CHI3L1, p=0.017; CHI3L2, p<0.001). CHIT1 and CHI3L2 were elevated in ALS compared with PLS (CHIT1, p=0.021; CHI3L1, p=0.417; CHI3L2, p<0.001). Chitinase levels were similar in AGCs and healthy controls. Chitinase proteins distinguished ALS from healthy controls (area under the curve (AUC): CHIT1 0.92; CHI3L1 0.80; CHI3L2 0.90), mimics (AUC: CHIT1 0.84; CHI3L1 0.73; CHI3L2 0.88) and, to a lesser extent, PLS (AUC: CHIT 0.73; CHI3L1 0.51; CHI3L2 0.82) but did not outperform pNFH. CHIT1 and CHI3L2 correlated with disease progression rate (Pearson's r=0.49, p<0.001; r=0.42, p<0.001, respectively). CHI3L1 correlated with degree of cognitive dysfunction (r=-0.25, p=0.038). All chitinases correlated with pNFH. CHIT1 levels were associated with survival in multivariate models. Chitinase levels were longitudinally stable. CONCLUSIONS: CSF chitinase proteins may have limited value as independent diagnostic and stratification biomarkers in ALS, but offer a window into non-autonomous mechanisms of motor neuronal loss in ALS, specifically in assessing response to therapies targeting neuroinflammatory pathways.

Astrocyte adenosine deaminase loss increases motor neuron toxicity in amyotrophic lateral sclerosis.

As clinical evidence supports a negative impact of dysfunctional energy metabolism on the disease progression in amyotrophic lateral sclerosis, it is vital to understand how the energy metabolic pathways are altered and whether they can be restored to slow disease progression. Possible approaches include increasing or rerouting catabolism of alternative fuel sources to supplement the glycolytic and mitochondrial pathways such as glycogen, ketone bodies and nucleosides. To analyse the basis of the catabolic defect in amyotrophic lateral sclerosis we used a novel phenotypic metabolic array. We profiled fibroblasts and induced neuronal progenitor-derived human induced astrocytes from C9orf72 amyotrophic lateral sclerosis patients compared to normal controls, measuring the rates of production of reduced nicotinamide adenine dinucleotides from 91 potential energy substrates. This approach shows for the first time that C9orf72 human induced astrocytes and fibroblasts have an adenosine to inosine deamination defect caused by reduction of adenosine deaminase, which is also observed in induced astrocytes from sporadic patients. Patient-derived induced astrocyte lines were more susceptible to adenosine-induced toxicity, which could be mimicked by inhibiting adenosine deaminase in control lines. Furthermore, adenosine deaminase inhibition in control induced astrocytes led to increased motor neuron toxicity in co-cultures, similar to the levels observed with patient derived induced astrocytes. Bypassing metabolically the adenosine deaminase defect by inosine supplementation was beneficial bioenergetically in vitro, increasing glycolytic energy output and leading to an increase in motor neuron survival in co-cultures with induced astrocytes. Inosine supplementation, in combination with modulation of the level of adenosine deaminase may represent a beneficial therapeutic approach to evaluate in patients with amyotrophic lateral sclerosis.

UFLC-Derived CSF Extracellular Vesicle Origin and Proteome.

Cerebrospinal fluid (CSF) extracellular vesicles (EVs) show promise as a source of neurological disease biomarkers, although their precise origin is poorly understood. Current extraction techniques produce disappointing yield and purity. This study describes the application of ultrafiltration LC (UFLC) to CSF-EVs, compared with ultracentrifugation (UC), and explores CSF-EV origin. EVs are extracted from human CSF by UC and UFLC and characterized using nanoparticle tracking analysis, electron microscopy, and immunoblotting. EV and CSF proteomes are analyzed by LC-MS/MS. UFLC-isolated particles have size, morphology, and marker expression characteristic of EVs. UFLC provides greater EV yield (UFLC 7.90 × 108  ± SD 1.31 × 108 EVs mL-1 CSF, UC 1.06 × 108  ± 0.57 × 108 p < 0.001). UFLC enhances purity, proteomic depth (UFLC 622 ± 49, UC 298 ± 50, p = 0.001), and consistency of quantification (CV 17% vs 23%). EVs contain more intracellular proteins (Odds ratio [OR] 2.63 p < 0.001) and fewer plasma proteins than CSF (OR 0.60, p < 0.001). CSF and EV-enriched proteomes show overrepresentation of brain-specific proteins (EV OR 3.18, p < 0.001; CSF OR 3.37, p < 0.001). Overrepresentation of cerebral white matter (OR 1.99, p = 0.015) and choroid plexus proteins (OR 1.87, p<0.001) is observed in EVs. UFLC improves yield and purity of CSF-EVs. The EV-enriched proteome better reflects the intracellular and white matter proteome than whole CSF.