Biomarker For Amyotrophic Lateral Sclerosis Research Articles

Inhibitory interneurons show early dysfunction in a SOD1 mouse model of amyotrophic lateral sclerosis.

Few studies in amyotrophic lateral sclerosis (ALS) measure effects of the disease on inhibitory interneurons synapsing onto motoneurons (MNs). However, inhibitory interneurons could contribute to dysfunction, particularly if altered before MN neuropathology, and establish a long-term imbalance of inhibition/excitation. We directly assessed excitability and morphology of glycinergic (GlyT2 expressing) ventral lumbar interneurons from SOD1G93AGlyT2eGFP (SOD1) and wild-type GlyT2eGFP (WT) mice on postnatal days 6-10. Patch clamp revealed dampened excitability in SOD1 interneurons, including depolarized persistent inward currents (PICs), increased voltage and current threshold for firing action potentials, along with a marginal decrease in afterhyperpolarization duration. Primary neurites of ventral SOD1 inhibitory interneurons were larger in volume and surface area than WT. GlyT2 interneurons were then divided into threesubgroups based on location: (1) interneurons within 100μm of the ventral white matter, where Renshaw cells (RCs) are located, (2) interneurons interspersed with MNs in lamina IX, and (3) interneurons in the intermediate ventral area including laminae VII and VIII. Ventral interneurons in the RC area were the most profoundly affected, exhibiting more depolarized PICs and larger primary neurites. Interneurons in lamina IX had depolarized PIC onset. In lamina VII-VIII, interneurons were least affected. In summary, inhibitory interneurons show very early region-specific perturbations poised to impact excitatory/inhibitory balance of MNs, modify motor output and provide early biomarkers of ALS. Therapeutics like riluzole that universally reduce CNS excitability could exacerbate the inhibitory dysfunction described here. KEY POINTS: Spinal inhibitory interneurons could contribute to amyotrophic lateral sclerosis (ALS) pathology, but their excitability has never been directly measured. We studied the excitability and morphology of glycinergic interneurons in early postnatal transgenic mice (SOD1G93A GlyT2eGFP). Interneurons were less excitable and had marginally smaller somas but larger primary neurites in SOD1mice. GlyT2 interneurons were analysed according to their localization within the ventral spinal cord. Interestingly, the greatest differences were observed in the most ventrally located interneurons. We conclude that inhibitory interneurons show presymptomatic changes that may contribute to excitatory/inhibitory imbalance in ALS.

Amyotrophic lateral sclerosis: a neurodegenerative disorder poised for successful therapeutic translation.

Amyotrophic lateral sclerosis (ALS) is a devastating disease caused by degeneration of motor neurons. As with all major neurodegenerative disorders, development of disease-modifying therapies has proven challenging for multiple reasons. Nevertheless, ALS is one of the few neurodegenerative diseases for which disease-modifying therapies are approved. Significant discoveries and advances have been made in ALS preclinical models, genetics, pathology, biomarkers, imaging and clinical readouts over the last 10-15 years. At the same time, novel therapeutic paradigms are being applied in areas of high unmet medical need, including neurodegenerative disorders. These developments have evolved our knowledge base, allowing identification of targeted candidate therapies for ALS with diverse mechanisms of action. In this Review, we discuss how this advanced knowledge, aligned with new approaches, can enable effective translation of therapeutic agents from preclinical studies through to clinical benefit for patients with ALS. We anticipate that this approach in ALS will also positively impact the field of drug discovery for neurodegenerative disorders more broadly.

Neuroinflammation-Related Proteins NOD2 and Spp1 Are Abnormally Upregulated in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown etiology and poorly understood pathophysiology. There is no specific biomarker either for diagnosis or prognosis. The aim of our study was to investigate differentially expressed proteins in the CSF and serum from patients with ALS to determine their role in the disease process and evaluate their utility as diagnostic or prognostic biomarkers. We performed mass spectrometry in the CSF from 3 patients with ALS and 3 healthy controls (HCs). The results were compared with motor cortex dysregulated transcripts obtained from 11patients with sporadic ALS and 8 HCs. Candidate proteins were tested using ELISA in the serum of 123 patients with ALS, 30 patients with Alzheimer disease (AD), 28 patients with frontotemporal dementia (FTD), and 102 HCs. Patients with ALS, AD, and FTD were prospectively recruited from January 2003 to December 2020. A group of age-matched HCs was randomly selected from the Sant Pau Initiative on Neurodegeneration cohort of the Sant Pau Memory Unit. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and osteopontin (Spp1) were differentially expressed in the CSF and the motor cortex transcriptome of patients with ALS compared with that in HCs (p < 0.05). NOD2 and Spp1 levels were significantly higher in sera from patients with ALS than in HCs (p < 0.001). Receiver operating characteristic analysis showed an area under the curve of 0.63 for NOD2 and 0.81 for Spp1. NOD2 levels were significantly lower in patients with AD and FTD than in patients with ALS (p < 0.0001), but we found no significant differences in Spp1 levels between patients with ALS, AD (p = 0.51), and FTD (p = 0.42). We found a negative correlation between Spp1 levels and ALS functional rating scale (r = -0.24, p = 0.009). Our discovery-based approach identified NOD2 as a novel biomarker in ALS and adds evidence to the contribution of Spp1 in the disease process. Both proteins are involved in innate immunity and autophagy and are increased in the serum from patients with ALS. Our data support a relevant role of neuroinflammation in the pathophysiology of the disease and may identify targets for disease-modifying treatments in ALS. Further longitudinal studies should investigate the diagnostic and prognostic value of NOD2 and Spp1 in clinical practice.

Proteomics analysis indicates the involvement of immunity and inflammation in the onset stage of SOD1-G93A mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron degenerative disease, and the pathogenic mechanism that underlies ALS is still unclear. We analyzed the differentially expressed proteins (DEPs) in the spinal cord between SOD1-G93A transgenic mice at the onset stage and non-transgenic (NTG) littermates based on 4D label-free quantitative proteomics (4D-LFQ) with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In our study, 189 DEPs were screened, of which 166 were up-regulated and 23 down-regulated. Clusters of Orthologous Groups (COG)/ EuKaryotic Orthologous Groups (KOG) classification, subcellular localization annotation, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, clustering analysis and protein-protein interaction (PPI) network analyses were performed. Parallel reaction monitoring (PRM) analysis validated 48 proteins from immunity and inflammation-related pathways of KEGG. We described the function and distribution of DEPs, most of which were involved in the following pathways: complement and coagulation cascades, antigen processing and presentation, NF-kappa B signaling pathway, Retinoic acid-inducible gene I (RIG) -I-like receptor signaling pathway, the extracellular matrix-receptor (ECM-receptor) interaction, focal adhesion, phagosome and lysosome. PPI network analysis identified Fn1, Fga, Serpina1e and Serpina3n as potential biomarkers. Our discoveries broaden the view and expand our understanding of immunity and inflammation in ALS. SignificanceThis study gives a comprehensive description of DEPs in the spinal cord proteomics of SOD1-G93A mice at the onset period. Compared with a previous study focusing on progressive stage, we showed that immunity and inflammation play an important role at the onset stage of ALS. Several pathways validated by PRM bring new insight to the pathological mechanisms of ALS. The participation of RIG-I-like signaling pathway in ALS and potential biomarkers Fga, Fn1, Serpina1e and Serpina3n are supplements to existing knowledge.

Identification of potentially functional modules and diagnostic genes related to amyotrophic lateral sclerosis based on the WGCNA and LASSO algorithms

Amyotrophic lateral sclerosis (ALS) is a genetically and phenotypically heterogeneous disease results in the loss of motor neurons. Mounting information points to involvement of other systems including cognitive impairment. However, neither the valid biomarker for diagnosis nor effective therapeutic intervention is available for ALS. The present study is aimed at identifying potentially genetic biomarker that improves the diagnosis and treatment of ALS patients based on the data of the Gene Expression Omnibus. We retrieved datasets and conducted a weighted gene co-expression network analysis (WGCNA) to identify ALS-related co-expression genes. Functional enrichment analysis was performed to determine the features and pathways of the main modules. We then constructed an ALS-related model using the least absolute shrinkage and selection operator (LASSO) regression analysis and verified the model by the receiver operating characteristic (ROC) curve. Besides we screened the non-preserved gene modules in FTD and ALS-mimic disorders to distinct ALS-related genes from disorders with overlapping genes and features. Altogether, 4198 common genes between datasets with the most variation were analyzed and 16 distinct modules were identified through WGCNA. Blue module had the most correlation with ALS and functionally enriched in pathways of neurodegeneration-multiple diseases’, ‘amyotrophic lateral sclerosis’, and ‘endocytosis’ KEGG terms. Further, some of other modules related to ALS were enriched in ‘autophagy’ and ‘amyotrophic lateral sclerosis’. The 30 top of hub genes were recruited to a LASSO regression model and 5 genes (BCLAF1, GNA13, ARL6IP5, ARGLU1, and YPEL5) were identified as potentially diagnostic ALS biomarkers with validating of the ROC curve and AUC value.

CSF p-tau as a potential cognition impairment biomarker in ALS.

Cerebrospinal fluid (CSF) and serum tau (t-tau, p-tau) are potential biomarkers for neurodegeneration in Alzheimer disease (AD), but their role in amyotrophic lateral sclerosis (ALS) is unclear. The aim of our study was to evaluate CSF and serum p-tau and t-tau in patients with ALS and to analyze the correlation and clinical parameters between them. CSF and serum samples were obtained from 90 patients with ALS, 48 other neurological disease (OND), and 20 with AM (ALS mimic, AM) diseases. The levels of p-tau and t-tau in the CSF and serum were assessed with an enzyme-linked immunosorbent assay, and disease progression parameters, including the duration, the ALSFRS-R score, disease progression rate (DPR), the upper motor neuron (UMN) score, the Mini-mental State Examination (MMSE) score, the Montreal Cognitive Assessment (MoCA) score, and the Edinburgh Cognitive and Behavioral ALS Screen (ECAS) results, were analyzed by registered neurologists. Statistical analyses were performed using Prism software. Compared with controls, patients with ALS displayed significantly lower levels of CSF p-tau and p-tau:t-tau ratio. The CSF p-tau level in patients with ALS and cognition impairment was higher than that in patients with ALS who did not have cognition impairment. CSF p-tau level was negatively correlated with MMSE, MoCA, and ECAS total score and the specific score of ECAS in patients with ALS and cognition impairment. The CSF p-tau level and p-tau:t-tau ratio were lower in patients with ALS than patients with OND and AM. Results suggest that CSF p-tau may be used as an index of cognition impairment in patients with ALS.

MicroRNA signatures in genetic frontotemporal dementia and amyotrophic lateral sclerosis.

MicroRNAs are promising biomarkers of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), but discrepant results between studies have so far hampered their use in clinical trials. We aim to assess all previously identified circulating microRNA signatures as potential biomarkers of genetic FTD and/or ALS, using homogeneous, independent validation cohorts of C9orf72 and GRN mutation carriers. 104 individuals carrying a C9orf72 or a GRN mutation, along with 31 controls, were recruited through the French research network on FTD/ALS. All subjects underwent blood sampling, from which circulating microRNAs were extracted. We measured differences in the expression levels of 65 microRNAs, selected from 15 published studies about FTD or ALS, between 31 controls, 17 C9orf72 presymptomatic subjects, and 29 C9orf72 patients. We also assessed differences in the expression levels of 30 microRNAs, selected from five studies about FTD, between 31 controls, 30 GRN presymptomatic subjects, and 28 GRN patients. More than half (35/65) of the selected microRNAs were differentially expressed in the C9orf72 cohort, while only a small proportion (5/30) of microRNAs were differentially expressed in the GRN cohort. In multivariate analyses, only individuals in the C9orf72 cohort could be adequately classified (ROC AUC up to 0.98 for controls versus presymptomatic subjects, 0.94 for controls versus patients, and 0.77 for presymptomatic subjects versus patients) with some of the signatures. Our results suggest that previously identified microRNAs using sporadic or mixed cohorts of FTD and ALS patients could potentially serve as biomarkers of C9orf72-associated disease, but not GRN-associated disease.

O.07 TDP43 accumulates in intramuscular nerve bundles of ALS patients

Amyotrophic lateral sclerosis (ALS) shows progressive muscle weakness and atrophy caused by neuronal death associate with TAR DNA-binding protein 43 (TDP43). Meanwhile, Awaji and Gold Coast criterion include an electrophysiological aspect to support clinical findings but not a pathological aspect because myopathological findings of ALS are not considered as specific. However, the advantage of the Awaji criteria is less pronounced in patients with spinal-onset ALS. The Gold Coast criteria enables us to diagnose spinal onset ALS patients and needs to allow for a specific diagnosis including a pathological aspect. Here, we aimed to identify and characterize the histopathology of peripheral axons in muscles of ALS patients. The post-mortem study included 10 sporadic ALS (SALS) patients with TDP43 pathology and 12 patients with non-ALS disease. The muscle biopsy cohort considered 450 patients and enrolled 114 as without family history of ALS or other neuromuscular diseases and not diagnosed with muscle diseases at biopsy. They ensure clinical follow-up in minimum one year. Exclusions were as follows: 51 for not screening clinical records after biopsy, 282 diagnosed with muscular diseases, and 3 harboring known causative genes of ALS. Muscle tissues were evaluated by histochemistry and immunohistochemical analysis. Ten SALS patients exhibited axonal phosphorylated TDP43 (pTDP43)-positive accumulations in intramuscular nerve bundles; 12 non-ALS patients did not. Among the muscle biopsy cohort, 71 patients exhibited intramuscular nerve bundles; 43 did not. Of the former, 33 presenting pTDP43-positive nerve bundles were later diagnosed with ALS. The remaining 38 showed no pTDP43-positive bundles and did not develop ALS. Among those without evident nerve bundles, 3 were later diagnosed with ALS. Among ALS patients in the biopsy cohort, 9 having pTDP43-positive bundles showed only lower motor neuron symptoms at biopsy. Axonal pTDP43 accumulations might be characteristic for ALS patients. As such findings precede clinical fulfillment of the Gold Coast criteria, TDP43 in nerve bundles might be a novel diagnostic biomarker for ALS.

Blood biomarkers in ALS: challenges, applications and novel frontiers.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease among adults. With diagnosis reached relatively late into the disease process, extensive motor cell loss narrows the window for therapeutic opportunities. Clinical heterogeneity in ALS and the lack of disease-specific biomarkers have so far led to large-sized clinical trials with long follow-up needed to define clinical outcomes. In advanced ALS patients, there is presently limited scope to use imaging or invasive cerebrospinal fluid (CSF) collection as a source of disease biomarkers. The development of more patient-friendly and accessible blood biomarker assays is hampered by analytical hurdles like the matrix effect of blood components. However, blood also provides the opportunity to identify disease-specific adaptive changes of the stoichiometry and conformation of target proteins and the endogenous immunological response to low-abundance brain peptides, such as neurofilaments (Nf). Among those biomarkers under investigation in ALS, the change in concentration before or after diagnosis of Nf has been shown to aid prognostication and to allow the a priori stratification of ALS patients into smaller sized and clinically more homogeneous cohorts, supporting more affordable clinical trials. Here, we discuss the technical hurdles affecting reproducible and sensitive biomarker measurement in blood. We also summarize the state of the art of non-CSF biomarkers in the study of prognosis, disease progression, and treatment response. We will then address the potential as disease-specific biomarkers of the newly discovered cryptic peptides which are formed down-stream of TDP-43 loss of function, the hallmark of ALS pathobiology.

MiRNA extracted from extracellular vesicles is a robust biomarker of amyotrophic lateral sclerosis

Background and objectivesWe examined miRNA biomarkers for ALS extracted from extracellular vesicles in blood samples using a large and diverse patient and control population. Different blood collection and storage protocols by different investigators could impact repeatability of miRNA analysis. We tested the hypotheses that miRNA extracted from extracellular vesicles using immunoaffinity purification techniques are robust and repeatable across investigators, laboratories and in a broad ALS population. MethodsDe-identified patient blood plasma samples obtained from the U.S. National ALS Biorepository were compared with plasma from non-ALS controls. Extracellular vesicles were extracted and isolated using L1CAM immunoaffinity purification. Total RNA was extracted, and miRNA quantified using qPCR following careful quality control measures. Gene fold expressions of eight miRNAs were compared using a Mann-Whitney two-tailed test. ResultsOne hundred blinded, blood plasma samples were analyzed. Thirty-five men and 15 women with ALS were compared with controls consisting of 30 men and 20 women. None of the ALS patient cohort reported family members with ALS suggesting sporadic ALS. Five of the eight biomarkers previously published were found to significantly discriminate ALS patient samples from control samples. DiscussionThe methods used in this study provide a repeatable measure of miRNA biomarkers that statistically differentiate ALS patient samples from control samples. The broad inclusion criteria for both the ALS patient cohort and controls along with the collection of blood samples by different investigators suggest that these methods are robust and represent good candidates for further research and development aimed at clinical application.

The Role of Sphingomyelin and Ceramide in Motor Neuron Diseases.

Amyotrophic Lateral Sclerosis (ALS), Spinal Bulbar Muscular Atrophy (SBMA), and Spinal Muscular Atrophy (SMA) are motor neuron diseases (MNDs) characterised by progressive motor neuron degeneration, weakness and muscular atrophy. Lipid dysregulation is well recognised in each of these conditions and occurs prior to neurodegeneration. Several lipid markers have been shown to predict prognosis in ALS. Sphingolipids are complex lipids enriched in the central nervous system and are integral to key cellular functions including membrane stability and signalling pathways, as well as being mediators of neuroinflammation and neurodegeneration. This review highlights the metabolism of sphingomyelin (SM), the most abundant sphingolipid, and of its metabolite ceramide, and its role in the pathophysiology of neurodegeneration, focusing on MNDs. We also review published lipidomic studies in MNDs. In the 13 studies of patients with ALS, 12 demonstrated upregulation of multiple SM species and 6 demonstrated upregulation of ceramides. SM species also correlated with markers of clinical progression in five of six studies. These data highlight the potential use of SM and ceramide as biomarkers in ALS. Finally, we review potential therapeutic strategies for targeting sphingolipid metabolism in neurodegeneration.

Quantitative muscle ultrasound assessment using automatic thresholding methods in amyotrophic lateral sclerosis

ObjectiveTo evaluate the usefulness of automatic thresholding methods for quantitative assessments of muscle echogenicity in amyotrophic lateral sclerosis (ALS) patients. MethodsThirty-one ALS patients and 31 matched healthy controls underwent ultrasound examination of the biceps brachii, rectus femoris and tibialis anterior muscles. Muscle echogenicity was evaluated using grayscale analysis and the 16 automatic thresholding methods of ImageJ program. The diagnostic value and correlations between ultrasound parameters and muscle strength were investigated. ResultsMean grayscale values (GSV) and mean hyperechoic fractions of 8 out of the 16 automatic thresholding methods were significantly different between patients and controls in all 3 muscles (p < 0.05 for all). Four thresholding methods (Default, Li, Moments, Otsu) showed a significant correlation between hyperechoic fractions and muscle strength, and diagnostic accuracy comparable or superior to GSVs. Otsu method was the only technique that detected ultrasound changes in normal strength muscles of ALS patients. ConclusionsOur findings support the utility of automatic thresholding methods in muscle echogenicity studies as a supplementary ultrasound image analysis in ALS. SignificanceIn an era of advances in developing neurophysiological diagnostic tools and biomarkers in ALS, muscle ultrasonography and echogenicity analysis using automatic thresholding methods could be effectively implemented in clinical research.

Iron quantitative analysis of motor combined with bulbar region in M1 cortex may improve diagnosis performance in ALS.

To explore whether the combined analysis of motor and bulbar region of M1 on susceptibility-weighted imaging (SWI) can be a valid biomarker for amyotrophic lateral sclerosis (ALS). Thirty-two non-demented ALS patients and 35 age- and gender-matched healthy controls (HC) were retrospectively recruited. SWI and 3D-T1-MPRAGE images were obtained from all individuals using a 3.0-T MRI scan. The bilateral posterior band of M1 was manually delineated by three neuroradiologists on phase images and subdivided into the motor and bulbar regions. We compared the phase values in two groups and performed a stratification analysis (ALSFRS-R score, duration, disease progression rate, and onset). Receiver operating characteristic (ROC) curves were also constructed. ALS group showed significantly increased phase values in M1 and the two subregions than the HC group, on the all and elderly level (p < 0.001, respectively). On all-age level comparison, negative correlations were found between phase values of M1 and clinical score and duration (p < 0.05, respectively). Similar associations were found in the motor region (p < 0.05, respectively). On both the total (p < 0.01) and elderly (p < 0.05) levels, there were positive relationships between disease progression rate and M1 phase values. In comparing ROC curves, the entire M1 showed the best diagnostic performance. Combining motor and bulbar analyses as an integral M1 region on SWI can improve ALS diagnosis performance, especially in the elderly. The phase value could be a valuable biomarker for ALS evaluation. • Integrated analysis of the motor and bulbar as an entire M1 region on SWI can improve the diagnosis performance in ALS. • Quantitative analysis of iron deposition by SWI measurement helps the clinical evaluation, especially for the elderly patients. • Phase value, when combined with the disease progression rate, could be a valuable biomarker for ALS.

Longitudinal monitoring of amyotrophic lateral sclerosis by diffusion tensor imaging: Power calculations for group studies.

IntroductionDiffusion tensor imaging (DTI) can be used to map disease progression in amyotrophic lateral sclerosis (ALS) and therefore is a promising candidate for a biomarker in ALS. To this end, longitudinal study protocols need to be optimized and validated regarding group sizes and time intervals between visits. The objective of this study was to assess the influences of sample size, the schedule of follow-up measurements, and measurement uncertainties on the statistical power to optimize longitudinal DTI study protocols in ALS.Patients and methodsTo estimate the measurement uncertainty of a tract-of–interest-based DTI approach, longitudinal test-retest measurements were applied first to a normal data set. Then, DTI data sets of 80 patients with ALS and 50 healthy participants were analyzed in the simulation of longitudinal trajectories, that is, longitudinal fractional anisotropy (FA) values for follow-up sessions were simulated for synthetic patient and control groups with different rates of FA decrease in the corticospinal tract. Monte Carlo simulations of synthetic longitudinal study groups were used to estimate the statistical power and thus the potentially needed sample sizes for a various number of scans at one visit, different time intervals between baseline and follow-up measurements, and measurement uncertainties.ResultsFrom the simulation for different longitudinal FA decrease rates, it was found that two scans per session increased the statistical power in the investigated settings unless sample sizes were sufficiently large and time intervals were appropriately long. The positive effect of a second scan per session on the statistical power was particularly pronounced for FA values with high measurement uncertainty, for which the third scan per session increased the statistical power even further.ConclusionWith more than one scan per session, the statistical power of longitudinal DTI studies can be increased in patients with ALS. Consequently, sufficient statistical power can be achieved even with limited sample sizes. An improved longitudinal DTI study protocol contributes to the detection of small changes in diffusion metrics and thereby supports DTI as an applicable and reliable non-invasive biomarker in ALS.

Biomarkers for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an incurable, devastating neurodegenerative disease. Still, the diagnosis is mainly based on clinical symptoms, and the treatment options are strongly limited. However, the pipeline of potential treatments currently tested in clinical trials is promising. This review will discuss developments in ALS biomarker research and applications within the last 2 years and suggest future directions and needs. The diagnostic and prognostic utility of neurofilaments, a general marker for axoneuronal degeneration, has been confirmed by further studies in patients with ALS, and neurofilaments are finding their way into routine diagnostic and clinical trials. Additionally, there have been advancements in developing and implementing disease-specific biomarkers, especially in patients with a genetic variant, such as SOD1 or C9orf72 . Here, biomarkers have already been used as target markers and outcome parameters for novel treatment approaches. In addition, several novel biomarkers have shown encouraging results but should be discussed in the context of their early stage of assay and clinical establishment. The first biomarkers have found their way into clinical routine in ALS. In light of an increasing pipeline of potential treatments, further progress in discovering and implementing novel and existing biomarkers is crucial.

Increased surface P2X4 receptors by mutant SOD1 proteins contribute to ALS pathogenesis in SOD1-G93A mice

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron (MN) disease characterized by protein misfolding and aggregation leading to cellular degeneration. So far neither biomarker, nor effective treatment has been found. ATP signaling and P2X4 receptors (P2X4) are upregulated in various neurodegenerative diseases. Here we show that several ALS-related misfolded proteins including mutants of SOD1 or TDP-43 lead to a significant increase in surface P2X4 receptor density and function in vitro. In addition, we demonstrate in the spinal the cord of SOD1-G93A (SOD1) mice that misfolded SOD1-G93A proteins directly interact with endocytic adaptor protein-2 (AP2); thus, acting as negative competitors for the interaction between AP2 and P2X4, impairing constitutive P2X4 endocytosis. The higher P2X4 surface density was particularly observed in peripheral macrophages of SOD1 mice before the onset and during the progression of ALS symptoms positioning P2X4 as a potential early biomarker for ALS. P2X4 expression was also upregulated in spinal microglia of SOD1 mice during ALS and affect microglial inflammatory responses. Importantly, we report using double transgenic SOD1 mice expressing internalization-defective P2X4mCherryIN knock-in gene or invalidated for the P2X4 gene that P2X4 is instrumental for motor symptoms, ALS progression and survival. This study highlights the role of P2X4 in the pathophysiology of ALS and thus its potential for the development of biomarkers and treatments. We also decipher the molecular mechanism by which misfolded proteins related to ALS impact P2X4 trafficking at early pathological stage in cells expressing-P2X4.

Questioning the Association of the STMN2 Dinucleotide Repeat With Amyotrophic Lateral Sclerosis.

ObjectivesRecently, the number of dinucleotide CA repeats in an intron of the STMN2 gene was reported to be associated with an increased risk for amyotrophic lateral sclerosis (ALS). Therefore, we sought to replicate this observation in an independent group of ALS patients and a much larger control group.MethodsHere, we used whole-genome sequencing and tested the STMN2 CA repeat in a case-control cohort of the European genetic background and in genomes from various populations in the gnomAD cohort to attempt to replicate this proposed association.ResultsWe find that repeats well above the previously reported pathogenic threshold of 19 are commonly observed in unaffected individuals across different populations. Furthermore, we did not observe an association between longer STMN2 CA repeats and ALS phenotype.DiscussionIn summary, our results do not support a role of STMN2 CA repeats toward ALS risk. As TDP-43 aggregation is central to ALS pathogenesis, lowered expression of STMN2 could be used as a biomarker for ALS. Therefore, a variant associated both with the risk for ALS and the level of STMN2 expression would be clinically useful. However, for a variant to be actionable, it must be strongly replicated in independent cohorts and exceed the rigorous statistical thresholds applied.

Role of plasma phosphorylated neurofilament heavy chain (pNfH) in amyotrophic lateral sclerosis.

The phosphorylated neurofilament heavy chain (pNfH) is a promising biomarker in amyotrophic lateral sclerosis (ALS). We examined plasma pNfH concentrations in order to corroborate its role as a diagnostic and prognostic biomarker in ALS. Incident ALS cases enrolled in a population‐based registry were retrospectively selected and matched by sex and age with a cohort of healthy volunteers. Plasma pNfH levels were measured by an ELISA kit and correlated with clinical parameters. Discrimination ability of pNfH was tested using receiving operating characteristic (ROC) curves. Kaplan–Meier (KM) analysis and Cox proportional hazard models were used for survival analysis. Plasma pNfH was significantly higher in patients compared to controls. An optimal cut‐off of 39.74 pg/ml discriminated cases from controls with an elevated sensitivity and specificity. Bulbar‐onset cases had higher plasma pNfH compared to spinal onset (p = 0.0033). Furthermore, plasma pNfH positively correlated with disease progression rate (r = 0.19, p = 0.031). Baseline plasma pNfH did not influence survival in our cohort. Our findings confirmed the potential utility of plasma pNfH as a diagnostic biomarker in ALS. However, further studies with longitudinal data are needed to corroborate its prognostic value.

The cross-sectional area of the median nerve: An independent prognostic biomarker in amyotrophic lateral sclerosis

IntroductionUltrasound changes in the cross-sectional area of the median nerve (CSAmn) could be of interest as biomarkers in patients with amyotrophic lateral sclerosis (ALS). MethodsEighty-four ALS patients (51 men [60.7%]; mean 62.0 [SD 11.46] years old) and forty-six controls (27 men [58.7%]; mean 59.9 [SD 8.08] years old) of two different cohorts were recruited between September 2013 and February 2018. The CSAmn was measured bilaterally in each cohort, by two different examiners with two different ultrasound machines (one in each cohort). Its association with clinical variables (disease duration, muscle strength, disability, progression rate and tracheostomy-free survival) was assessed. ResultsThe CSAmn was smaller in patients than in controls, and the study cohort did not influence its values. A mild correlation between the strength of the wrist flexor and the CSAmn was found. In the multivariable analysis, the probability of this association being true was 90%. In the cox regression, both a faster progression rate and a larger CSAmn independently predicted poor survival (HR=4.29, [Cr.I95%: 2.71–6.80], p<0.001; and HR=1.14, [Cr.I95%: 1.03–1.25], p=0.01), after adjusting by age, body mass index, bulbar onset, and diagnostic delay. ConclusionsThe CSAmn is an easy to assess biomarker that seems reliable and reproducible. Our data also suggest that it could act as a progression and prognostic biomarker in ALS patients. Longitudinal studies with repeated measures are warranted to confirm its usefulness in the clinical practice.

Plasma taurine is an axonal excitability-translatable biomarker for amyotrophic lateral sclerosis

Although various body fluid biomarkers for amyotrophic lateral sclerosis (ALS) have been reported, no biomarkers specifically reflecting abnormalities in axonal excitability indices have currently been established. Capillary electrophoresis time-of-flight mass spectrometry and liquid chromatography time-of-flight mass spectrometry were used to perform a comprehensive metabolome analysis of plasma from seven ALS patients and 20 controls, and correlation analysis with disease phenotypes was then performed in 22 other ALS patients. Additionally, electrophysiological studies of motor nerve axonal excitability were performed in all ALS patients. In the ALS and control groups, levels of various metabolites directly associated with skeletal muscle metabolism, such as those involved in fatty acid β-oxidation and the creatine pathway, were detected. Receiver operating characteristic curve analysis of the top four metabolites (ribose-5-phosphate, N6-acetyllysine, dyphylline, 3-methoxytyrosine) showed high diagnostic accuracy (area under the curve = 0.971) in the ALS group compared with the control group. Furthermore, hierarchical cluster analysis revealed that taurine levels were correlated with the strength-duration time constant, an axonal excitability indicator established to predict survival. No significant effects of diabetes mellitus and treatment (Riluzole and Edaravone) on this relationship were detected in the study. Therefore, plasma taurine is a potential novel axonal excitability-translatable biomarker for ALS.