Cerebrospinal Fluid Of Amyotrophic Lateral Sclerosis Patients Research Articles

The presence and clinical significance of autoantibodies in amyotrophic lateral sclerosis: a narrative review.

Amyotrophic lateral sclerosis (ALS) is a debilitating and rapidly fatal neurodegenerative disease, which is characterized by the selective loss of the upper and lower motor neurons. The pathogenesis of ALS remains to be elucidated and has been connected to genetic, environmental and immune conditions. Evidence from clinical and experimental studies has suggested that the immune system played an important role in ALS pathophysiology. Autoantibodies are essential components of the immune system. Several autoantibodies directed at antigens associated with ALS pathogenesis have been identified in the serum and/or cerebrospinal fluid of ALS patients. The aim of this review is to summarize the presence and clinical significance of autoantibodies in ALS.

Filtered Cerebrospinal Fluid From Patients With Amyotrophic Lateral Sclerosis Displays an Altered Proteome and Affects Motor Phenotype in a Mouse Model.

Cerebrospinal fluid (CSF) has been implicated in amyotrophic lateral sclerosis (ALS) due to its ability to spread inflammatory proteins throughout the nervous system. We hypothesized that filtration of the CSF could remove pathogenic proteins and prevent them from altering motor phenotypes in a mouse model. We filtered the CSF from 11 ALS patients via 100 kilodaltons (kD) molecular weight cut-off filters. We used mass spectrometry-based discovery proteomics workflows to compare protein abundances before and after filtration. To test the effects of CSF filtration on motor function, we injected groups of mice with saline, filtered ALS-CSF, or unfiltered ALS-CSF (n=12 per group) and assessed motor function via pole descent and open field tests. We identified proteins implicated in ALS pathogenesis and showed that these were removed in significant amounts in our workflow. Key filtered proteins included complement proteins, chitinases, serine protease inhibitors, and neuro-inflammatory proteins such as amyloid precursor protein, chromogranin A, and glial fibrillary acidic protein. Compared to the filtered ALS-CSF mice, unfiltered ALS-CSF mice took longer to descend a pole (10 days post-injection, 11.14 seconds vs 14.25 seconds, p = 0.02) and explored less on an open field (one day post-injection, 21.81 m vs 16.83 m, p = 0.0004). We demonstrated the ability to filter proteins from the CSF of ALS patients and identified potentially pathologic proteins that were reduced in quantity. Additionally, we demonstrated the ability of unfiltered ALS-CSF to induce motor deficits in mice on the pole descent and open field tests and showed that filtration could prevent this deficit. Given the lack of effective treatments for ALS, this could be a novel solution for patients suffering from this deadly and irreversible condition.

In FUS[1−359]‐tg mice O,S-dibenzoyl thiamine reduces muscle atrophy, decreases glycogen synthase kinase 3 beta, and normalizes the metabolome

Mutations in the gene encoding the RNA/DNA-binding protein Fused in Sarcoma (FUS) have been detected in familial amyotrophic lateral sclerosis (ALS) patients. FUS has been found to be a critical component of the oxidative damage repair complex that might explain its role in neurodegeneration. Here, we examined what impact antioxidant treatment with thiamine (vitamine B1), or its more bioavailable derivative O,S-dibenzoylthiamine (DBT), would have on the hallmarks of pathology in the FUS[1−359]‐transgenic mouse model of ALS. From 8-weeks old, in the pre-symptomatic phase of disease, animals received either thiamine, DBT (200 mg/kg/day), or vehicle for 6 weeks. We examined physiological, behavioral, molecular and histological outcomes, as well as the serum metabolome using nuclear magnetic resonance (NMR). The DBT-treated mice displayed improvements in physiological outcomes, motor function and muscle atrophy compared to vehicle, and the treatment normalized levels of brain glycogen synthase kinase-3β (GSK-3β), GSK-3β mRNA and IL-1β mRNA in the spinal cord. Analysis of the metabolome revealed an increase in the levels of choline and lactate in the vehicle-treated FUS mutants alone, which is also elevated in the cerebrospinal fluid of ALS patients, and reduced glucose and lipoprotein concentrations in the FUS[1−359]‐tg mice, which were not the case in the DBT-treated mutants. The administration of thiamine had little impact on the outcome measures, but it did normalize circulating HDL levels. Thus, our study shows that DBT therapy in FUS mutants is more effective than thiamine and highlights how metabolomics may be used to evaluate therapy in this model.

TDP-43 as structure-based biomarker in amyotrophic lateral sclerosis.

Pathologic alterations of Transactivation response DNA‐binding protein 43 kilo Dalton (TDP‐43) are a major hallmark of amyotrophic lateral sclerosis (ALS). In this pilot study, we analyzed the secondary structure distribution of TDP‐43 in cerebrospinal fluid of ALS patients (n = 36) compared to Parkinson´s disease patients (PD; n = 30) and further controls (Ctrl; n = 24) using the immuno‐infrared sensor technology. ALS patients could be discriminated from PD and Ctrl with a sensitivity/specificity of 89 %/77 % and 89 %/83 %, respectively. Our findings demonstrate that TDP‐43 misfolding measured by the immuno‐infrared sensor technology has the potential to serve as a biomarker candidate for ALS.

Metabo-lipidomics of Fibroblasts and Mitochondrial-Endoplasmic Reticulum Extracts from ALS Patients Shows Alterations in Purine, Pyrimidine, Energetic, and Phospholipid Metabolisms.

Amyotrophic lateral sclerosis (ALS) is characterized by a wide metabolic remodeling, as shown by recent metabolomics and lipidomics studies performed in samples from patient cohorts and experimental animal models. Here, we explored the metabolome and lipidome of fibroblasts from sporadic ALS patients (n = 13) comparatively to age- and sex-matched controls (n = 11), and the subcellular fraction containing the mitochondria and endoplasmic reticulum (mito-ER), given that mitochondrial dysfunctions and ER stress are important features of ALS patho-mechanisms. We also assessed the mitochondrial oxidative respiration and the mitochondrial genomic (mtDNA) sequence, although without yielding significant differences. Compared to controls, ALS fibroblasts did not exhibit a mitochondrial respiration defect nor an increased proportion of mitochondrial DNA mutations. In addition, non-targeted metabolomics and lipidomics analyses identified 124 and 127 metabolites, and 328 and 220 lipids in whole cells and the mito-ER fractions, respectively, along with partial least-squares-discriminant analysis (PLS-DA) models being systematically highly predictive of the disease. The most discriminant metabolomic features were the alteration of purine, pyrimidine, and energetic metabolisms, suggestive of oxidative stress and of pro-inflammatory status. The most important lipidomic feature in the mito-ER fraction was the disturbance of phosphatidylcholine PC (36:4p) levels, which we had previously reported in the cerebrospinal fluid of ALS patients and in the brain from an ALS mouse model. Thus, our results reveal that fibroblasts from sporadic ALS patients share common metabolic remodeling, consistent with other metabolic studies performed in ALS, opening perspectives for further exploration in this cellular model in ALS.

Targeting astrocyte and motor neuron specific miRNAs to prevent neuro-immune dysregulation in ALS

Event Abstract Back to Event Targeting astrocyte and motor neuron specific miRNAs to prevent neuro-immune dysregulation in ALS Ana R. Vaz1, 2, Catarina Sequeira1, Daniela Vizinha1, Marta Barbosa1, Cátia Gomes1 and Dora Brites1, 2, 3* 1 Research Institute for Medicines (iMed.ULisboa), Portugal 2 Faculdade de Farmácia, Universidade de Lisboa, Department of Biochemistry and Human Biology, Portugal 3 Faculdade de Farmácia, Universidade de Lisboa, Portugal Amyotrophic lateral sclerosis (ALS) is characterized by motor neurons (MNs) loss and glial reactivity in brain and spinal cord (SC). A dysregulated expression of microRNAs (miRNAs) have been implicated in neurodegeneration and neuroinflammation in ALS. MiRNA(miR)-124 is elevated in the cerebrospinal fluid of ALS patients and in the SC of the ALS mice with the G93A mutation in human superoxide dismutase-1 (mSOD1). We demonstrated that this miRNA is upregulated in MN-NSC-34 overexpressing mSOD1 and in small extracellular vesicles isolated from cell secretome [1]. We also observed miR-155 upregulation in the SC of pe-symptomatic mSOD1 mice [2], often associated with mSOD1 microglia [3], but with unknown effect when expressed by astrocytes. In contrast, miR-146a was downregulated either in the cortical brain (CB) of symptomatic mSOD1 mice and in isolated astrocytes [4], suggesting distinct miRNA deregulation depending on the CNS region. Here, we evaluated the effects of miR-155/146a/124 modulation in astrocytic and MN function. For that, we used primary astrocytes from mSOD1 mice pups (CB or SC) and mSOD1 MN-NSC-34. Cells were transfected with anti-miR-155 (SC astrocytes), premiR-146a (CB astrocytes) or pre/anti-miR-124 (MN). We also explored whether changes in MN-miR-124 expression impact on secretome-mediated astrocytic function. Downregulation of miR-155 in mSOD1 SC astrocytes counteracted the inflammatory milieu, by decreasing TNF-α/IL-1β/HMGB1/miR-124, and increasing IL-6/IL-10/SOCS1/miR-146a expression, without changing the astrocytic aberrant phenotype (GFAPlow/Cx43high/S100Bhigh). However, miR-146a upregulation in mSOD1 CB astrocytes neutralized both the phenotypic aberrancy and the inflammatory profile. The upregulation of miR-124 in WT-MNs mimicked the pathological features of mSOD1-MNs, namely inflammatory-associated-miRNAs (increased miR-125b/decreased miR-146a/-21), synaptic dysfunction (increased synaptophysin/decreased PDS-95), neurite loss, reduced mitochondria viability and axonal transport deficits (increased dynein/decreased kinesin), while miR-124 downregulation was able to prevent those dysfunctionalities in mSOD1-MNs. The astrocytic aberrant phenotype of mSOD1 SC astrocytes was observed in WT cells upon incubation with secretome from mSOD1-MNs or pre-miR-124-WT-MN, but prevented in the presence of the secretome of anti-miR-124-mSOD1-MN. More importantly, secretome derived from anti-miR-124-mSOD1-MN rescued the aberrant phenotype of mSOD1 SC astrocytes by increasing GFAP/GLT-1 expression. Overall, our results highlight that combined modulation of specific miRNAs in different target cells may represent a promising therapeutic potential in ALS disease. Acknowledgements Funded by SCML (ALS Research Grant ELA-2015-002) and FCT (PTDC/MED-NEU/31395/2017 and LISBOA-01-0145-FEDER-031395 to DB, UID/DTP/04138/2013 to iMed.ULisboa, and fellowship ARV-SFRH/BPD/76590/2011).

Evaluation of Chitotriosidase and CC-Chemokine Ligand 18 as Biomarkers of Microglia Activation in Amyotrophic Lateral Sclerosis

Background: The development of biomarkers for use in diagnosing, monitoring disease progression and analyzing therapeutic trials response in amyotrophic lateral sclerosis (ALS) is essential. Objective: The aim of this study was to identify inflammatory factors in plasma or cerebrospinal fluid (CSF) from patients with ALS with particular attention to specific markers of microglia activation as chitotriosidase (ChT) and chemokine (C-C motif) ligand 18 (CCL18) to determine its potential as ALS biomarkers. Methods: We studied CSF and plasma samples from 32 patients and 42 healthy controls. We assayed the ChT activity by a spectrofluorometric method and protein levels of other inflammatory ­biomarkers (tumor necrosis factor [TNF]-alpha, interleukin [IL]-6 and CCL18) by enzyme-linked immunosorbent assay. CHIT1 gene polymorphism in exon 10 (c.1049_1072dup24) encoding inactive ChT enzyme was genotyped in all subjects. Results: ChT activity and TNF-alpha protein levels were significantly higher in CSF of ALS patients, but we found no correlation with the severity and progression of the disease. Nevertheless, we did not found any differences in CCL18 or IL-6 protein levels between both groups in CSF or plasma. In our sample, only 3% of subjects were homozygous carriers for the CHIT1 exon 10 duplication associated with defective enzyme. Conclusions: High ChT activity in CSF of patients with ALS may reflect microglia activation and could be a potential biomarker of the disease. We did not find any significant difference regarding CCL-18, another specific marker of microglia activation that is related with M2-like microglia phenotype. Deepening the understanding of the activation state of microglia (M1 and M2) may contribute to the knowledge about the specific role of neuroinflammation in ALS and future therapeutic strategies.

CSF neurofilament proteins as diagnostic and prognostic biomarkers for amyotrophic lateral sclerosis.

Elevated cerebrospinal fluid (CSF), Neurofilament Light (NF-L) and phosphorylated Heavy (pNF-H) chain levels have been found in Amyotrophic Lateral Sclerosis (ALS), with studies reporting a correlation of both neurofilaments (NFs) with the disease progression. Here, we measured NF-L and pNF-H concentrations in the CSF of ALS patients from a single tertiary Center and investigated their relationship with disease-related variables. A total of 190 ALS patients (Bulbar, 29.9%; Spinal, 70.1%; M/F=1.53) and 130 controls with mixed neurological diseases were recruited. Demographic and clinical variables were recorded, and ΔFS was used to rate the disease progression. Controls were divided into two cohorts: (1) patients with non-inflammatory neurological diseases (CTL-1); (2) patients with acute/subacute inflammatory diseases and tumors, expected to lead to significant axonal and tissue damage (CTL-2). For each patient and control, CSF was taken at the time of the diagnostic work-up and stored following the published guidelines. CSF NF-L and pNF-H were assayed with commercially available ELISA-based methods. Standard curves (from independent ELISA kits) were highly reproducible for both NFs, with a coefficient of variation<20%. We found that CSF NF-L and pNF-H levels in ALS were significantly increased when compared to CTL-1 (NF-L: ALS, 4.7ng/ml vs CTL-1, 0.61ng/ml, p<0.001; pNF-H: ALS, 1.7ng/ml vs CTL-1, 0.03ng/ml, p<0.0001), but not to CTL-2. Analysis of different clinical and prognostic variables disclosed meaningful correlations with both NF-L and pNF-H levels. Our results, from a relatively large ALS cohort, confirm that CSF NF-L and pNF-H represent valuable diagnostic and prognostic biomarkers in ALS.

Small RNA Sequencing of Sporadic Amyotrophic Lateral Sclerosis Cerebrospinal Fluid Reveals Differentially Expressed miRNAs Related to Neural and Glial Activity.

Amyotrophic lateral sclerosis (ALS) is a clinical subtype of motor neurone disease (MND), a fatal neurodegenerative disease involving the loss of both the upper and lower motor neurones from the motor cortex, brainstem, and spinal cord. Identifying specific disease biomarkers would help to not only improve diagnostic delay but also to classify disease subtypes, monitor response to therapeutic drugs and track disease progression. miRNAs are small non-coding RNA responsible for regulating gene expression and ultimately protein expression and have been used as biomarkers for many cancers and neurodegenerative disorders. Investigating the detection of miRNAs in cerebrospinal fluid (CSF), the fluid that bathes the central nervous system (CNS) is a prime target for identifying potential biomarkers for ALS. This is the first study to investigate the expression of miRNAs in the CSF of ALS patients using small RNA sequencing. We detected 11 differentially expressed miRNAs in the CSF of sporadic ALS (sALS) patients related to neural and glial activity. Additionally, miRNAs involved in glucose metabolism and the regulation of oxidative stress were also identified. Detecting the presence of potential CSF derived miRNA biomarkers in sALS could open up a whole new area of knowledge to help gain a better understanding of disease pathophysiology. Additionally, with further investigation, the tracking of CSF miRNA over the disease course could be used to follow the disease progression and monitor the effect of novel therapeutics that could be personalized to an individual disease phenotype.

Lipidomics Reveals Cerebrospinal-Fluid Signatures of ALS

Amyotrophic lateral sclerosis (ALS), the commonest adult-onset motor neuron disorder, is characterized by a survival span of only 2–5 years after onset. Relevant biomarkers or specific metabolic signatures would provide powerful tools for the management of ALS. The main objective of this study was to investigate the cerebrospinal fluid (CSF) lipidomic signature of ALS patients by mass spectrometry to evaluate the diagnostic and predictive values of the profile. We showed that ALS patients (n = 40) displayed a highly significant specific CSF lipidomic signature compared to controls (n = 45). Phosphatidylcholine PC(36:4), higher in ALS patients (p = 0.0003) was the most discriminant molecule, and ceramides and glucosylceramides were also highly relevant. Analysis of targeted lipids in the brain cortex of ALS model mice confirmed the role of some discriminant lipids such as PC. We also obtained good models for predicting the variation of the ALSFRS-r score from the lipidome baseline, with an accuracy of 71% in an independent set of patients. Significant predictions of clinical evolution were found to be correlated to sphingomyelins and triglycerides with long-chain fatty acids. Our study, which shows extensive lipid remodelling in the CSF of ALS patients, provides a new metabolic signature of the disease and its evolution with good predictive performance.

Cerebrospinal fluid from patients with amyotrophic lateral sclerosis inhibits sonic hedgehog function.

Sonic hedgehog (Shh) is a morphogen essential to the developing nervous system that continues to play an important role in adult life by contributing to cell proliferation and differentiation, maintaining blood-brain barrier integrity, and being cytoprotective against oxidative and excitotoxic stress, all features of importance in amyotrophic lateral sclerosis (ALS). ALS is a fatal disease characterized by selective loss of motor neurons due to poorly understood mechanisms. Evidence indicates that Shh might play an important role in ALS, and that Shh signaling might be also adversely affected in ALS. Since little is known about the functional status of Shh pathway in patients with ALS, we therefore sought to determine whether Shh protein levels or biological activity in cerebrospinal fluid (CSF) was less in ALS patients than controls, and whether these measures could be correlated with ALS disease severity and disease progression, and with other CSF analytes of biological interest in ALS. Comparing Shh levels in the CSF of normal controls (n = 13), neurological controls (n = 12), and ALS patients (n = 9) measured by ELISA, we found that CSF Shh levels were not different between controls and ALS patients. However, when assessing Shh biological activity in CSF using in vitro cell-based assays, which measure Shh activity as inducible Gli-driven luminescence, we found that in the presence of exogenous recombinant Shh or the Shh agonist, purmorphamine, the inducible activity of CSF was significantly augmented in the control groups as expected, but not in the ALS group, suggesting the presence of an inhibitor of Shh signaling in ALS CSF samples. Since purmorphamine acts on Smoothened, downstream of Shh and its receptor Patched, the inhibitory action is downstream of Smoothened. Our results also demonstrated that while the inhibitory effect of ALS CSF on Shh signaling did not correlate significantly with ALS disease characteristics, the levels of IL-1β and TNF-α did. In addition to being significantly elevated in ALS CSF, these cytokines negatively correlated with the disease duration, whereas GDF11 was a favorable predictor of ALS clinical score. We also found that TNF-α significantly inhibited Shh biological activity in vitro, potentially suggesting a novel role of TNF-α in ALS pathogenesis. Collectively, this is the first report demonstrating that Shh signaling in CSF of ALS patients is compromised.

Reduced levels of alpha-1-antitrypsin in cerebrospinal fluid of amyotrophic lateral sclerosis patients: a novel approach for a potential treatment.

BackgroundAmyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative motor neuron disease that involves activation of the immune system and inflammatory response in the nervous system. Reduced level of the immuno-modulatory and anti-inflammatory protein alpha-1-antitrypsin (AAT) is associated with inflammation-related pathologies. The objective of the present is to determine AAT levels and IL-23 in the cerebrospinal fluid (CSF) of ALS patients and control group.FindingsCSF samples from newly diagnosed ALS patients and age-matched controls were analyzed for AAT and IL-23 by ELISA and magnetic luminex screening, respectively. A statistically significant reduction of 45 % in mean AAT levels was observed in the CSF of ALS patients (21.4 μg/ml) as compared to the control group (mean 38.8 μg/ml, p = 0.013). A statistically significant increase of 30.8 % in CSF mean levels of the pro-inflammatory cytokine IL-23 was observed in ALS patients (1647 pg/ml) in comparison to the controls (1259 pg/ml, p = 0.012). A negative correlation coefficient (r = −0.543) was obtained by linear regression analysis of the two measured parameters (p = 0.036).ConclusionsReduced AAT and elevated IL-23 CSF levels support the notion of neuroinflammatory process occurring in ALS patients. Increasing AAT levels in the patients’ nervous system should be further investigated as a new therapeutic approach and a novel potential tool for ALS treatment.

A Multiplex Protein Panel Applied to Cerebrospinal Fluid Reveals Three New Biomarker Candidates in ALS but None in Neuropathic Pain Patients.

The objective of this study was to develop and apply a novel multiplex panel of solid-phase proximity ligation assays (SP-PLA) requiring only 20 μL of samples, as a tool for discovering protein biomarkers for neurological disease and treatment thereof in cerebrospinal fluid (CSF). We applied the SP-PLA to samples from two sets of patients with poorly understood nervous system pathologies amyotrophic lateral sclerosis (ALS) and neuropathic pain, where patients were treated with spinal cord stimulation (SCS). Forty-seven inflammatory and neurotrophic proteins were measured in samples from 20 ALS patients and 15 neuropathic pain patients, and compared to normal concentrations in CSF from control individuals. Nineteen of the 47 proteins were detectable in more than 95% of the 72 controls. None of the 21 proteins detectable in CSF from neuropathic pain patients were significantly altered by SCS. The levels of the three proteins, follistatin, interleukin-1 alpha, and kallikrein-5 were all significantly reduced in the ALS group compared to age-matched controls. These results demonstrate the utility of purpose designed multiplex SP-PLA panels in CSF biomarker research for understanding neuropathological and neurotherapeutic mechanisms. The protein changes found in the CSF of ALS patients may be of diagnostic interest.

Immunoablation and Stem Cell Transplantation in Amyotrophic Lateral Sclerosis: The Ultimate Test for the Autoimmune Pathogenesis Hypothesis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by a progressive death of motor neurons for which there is no cure or effective treatment. The cause of ALS and the specific mechanisms of neuronal death remain unknown. However, considerable evidence supports the existence of autoimmune mechanisms contributing to pathogenesis in ALS, including biochemical, morphological, pharmacological, and physiological studies performed in animal models, cell culture, or with ex vivo preparations (1–6). Typical hallmarks of autoimmunity, such as circulating immune complexes, higher frequency of a particular histocompatibility type, or association with other autoimmune diseases, have also been reported (7, 8). An important marker of autoimmunity is the degree of T-lymphocytic infiltration in the anterior horn of the spinal cord from ALS patients (9, 10). Using monoclonal antibodies against T-cells, B-cells, and macrophages, almost 80% of the specimens show a cellular mononuclear infiltration. The cellular composition of the spinal cord inflammation consists of subsets of suppressor or cytotoxic T-cells and macrophages in the anterior and lateral corticospinal tracts and anterior horns (10). T-helper cells are also observed in proximity to corticospinal tract degeneration (11). Hence, inflammation in ALS spinal cord and brain appears to be primarily due to T-cells and macrophages (12), and aberrant macrophage activity is believed by many investigators to contribute to the pathology underlying ALS. This may explain the recent promising results of an ALS phase 2 clinical trial of NP001, a regulator of inflammatory macrophage activity (13). Although the predefined endpoints in this study did not reach statistical significance, administration of NP001 was associated with cessation in disease progression in 27% of patients, approximately 2.5 times greater than the percentage in patients on placebo. Two major plasma markers of inflammation, interleukin-18 (IL-18) and lipopolysaccharide (LPS), differentiated NP001 responders from non-responders, suggesting that the subgroup of patients with greater baseline biomarkers of neuroinflammation experienced the most benefit (13). Additional evidence pointing toward pathologic involvement of autoimmune processes has been the finding that immunoglobulins from ALS patients have been shown to cause apoptosis of motor neurons in primary spinal cord cultures (14) and that passive transfer of immunoglobulins to mice caused abnormalities at motor end-plates and degeneration of motor neurons (4, 15). These findings suggest that antibodies can contribute to disease pathogenesis. Increased levels of interleukins IL-17 and IL-23 have also been found in serum and cerebrospinal fluid of ALS patients (16). This increment is thought to be a sign of T-helper 17 (Th17) activation, a subset of T-cells suggested to be crucial in destructive autoimmunity. Astrocytes have also been shown to participate in the pathogenesis of ALS by producing a microenvironment toxic to motor neurons through increased neuroinflammation, oxidative damage, and glutamate excitotoxicity (17, 18). Overactivated astroglia produce high levels of protein S100B and other proinflammatory factors, which exacerbate neuroinflammation. The extracellular effects of S100B vary, depending on the concentration attained; at nanomolar concentrations, S100B is trophic to neurons, but at micromolar concentrations, S100B causes neuronal apoptosis (19, 20). Many of the effects of S100B on neurons are transduced by the receptor for advanced glycation end-products (RAGE), which participates in the pathophysiology of brain inflammatory disorders by regulating several inflammation-related events, including activation and migration of microglia and neutrophils to inflammatory sites (19–21). Extravasation of S100B into the systemic circulation can also trigger a pathologic autoimmune reaction with circulating antibodies that may re-enter the CNS to initiate an autoimmune response (22). Hence, S100B can be viewed as an astrocytic endokine that can act as an immunoregulator to participate in inflammation and autoimmunity. Additional support for the autoimmune pathogenesis hypothesis is the finding that ALS has recently been included in the spectrum of neurologic manifestations associated with voltage-gated potassium channel (VGKC) autoimmunity (23–25).

Total tau is increased, but phosphorylated tau not decreased, in cerebrospinal fluid in amyotrophic lateral sclerosis

In amyotrophic lateral sclerosis (ALS), objective biomarkers are needed for early diagnosis and progression monitoring. Reduced phosphorylated tau (p-tau) in cerebrospinal fluid (CSF) has recently been proposed to provide such a biomarker in ALS. Here, we aimed to scrutinize this notion, evaluating both p-tau and total tau (t-tau) in CSF of ALS patients and control subjects. CSF p-tau and t-tau levels were measured in 60 consecutive ALS patients and 120 control subjects without neurodegenerative disease, using an established specific enzyme-linked immunosorbent assay method. Contrary to recent reports, CSF p-tau was not significantly reduced in ALS patients compared with control subjects (p = 0.287). However, CSF t-tau was significantly increased (p < 0.001). Correspondingly, the ratio of p-tau to t-tau was significantly reduced in ALS (p < 0.001). The area under the curve demonstrated poor sensitivity and specificity for p-tau, but moderate sensitivity and specificity for t-tau and p-tau/t-tau ratio. Thus, CSF p-tau by itself does not appear a suitable diagnostic biomarker for ALS, whereas CSF t-tau is a (probably unspecific) marker of the neuronal degeneration in ALS.

Cystatin C protects neuronal cells against mutant copper-zinc superoxide dismutase-mediated toxicity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. Cystatin C (CysC), an endogenous cysteine protease inhibitor, is a major protein component of Bunina bodies observed in the spinal motor neurons of sporadic ALS and is decreased in the cerebrospinal fluid of ALS patients. Despite prominent deposition of CysC in ALS, the roles of CysC in the central nervous system remain unknown. Here, we identified the neuroprotective activity of CysC against ALS-linked mutant Cu/Zn-superoxide dismutase (SOD1)-mediated toxicity. We found that exogenously added CysC protected neuronal cells including primary cultured motor neurons. Moreover, the neuroprotective property of CysC was dependent on the coordinated activation of two distinct pathways: autophagy induction through AMPK-mTOR pathway and inhibition of cathepsin B. Furthermore, exogenously added CysC was transduced into the cells and aggregated in the cytosol under oxidative stress conditions, implying a relationship between the neuroprotective activity of CysC and Bunina body formation. These data suggest CysC is an endogenous neuroprotective agent and targeting CysC in motor neurons may provide a novel therapeutic strategy for ALS.

Is cerebrospinal fluid obtained for diagnostic purpose a good material for biomarker studies in amyotrophic lateral sclerosis?

The cerebrospinal fluid (CSF) used for identification of molecular biomarkers in amyotrophic lateral sclerosis (ALS) is mainly obtained from lumbar puncture (LP) performed to exclude other causes of motor neuron damage.Aim: The aim of the study was to analyze whether CSF of ALS patients obtained for diagnostic purposes is suitable for biomarker studies in the entire ALS population.Material and methods: We analyzed the medical data, LP frequency and CSF parameters in 568 ALS patients.Results: LP was performed in 34% of cases. Patients who underwent LP were significantly younger and more frequently presented limb onset ALS, there were no differences in the clinical phenotypes.Conclusion: CSF obtained for diagnostic purposes can be used for biomarkers studies in ALS.

Correlation of cerebral spinal fluid pH and HCO3- with disease progression in ALS

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disease, which is characterized by progressive degeneration of spinal and bulbar innervating motor neurons. However, the underlying mechanisms of motor neuron death remain poorly understood. Several candidate disease biomarkers have been detected in cerebrospinal fluid of ALS patients. The present study analyzed various cerebral spinal fluid gas parameters in ALS patients and compared these values to controls, as well as patients with cervical spondylosis, Parkinson syndrome, and spinocerebellar degeneration. Cerebral spinal fluid pH positively correlated with the ALS functional rating scale in total and limb-type ALS patients. In addition, cerebral spinal fluid pH positively correlated with shorter disease duration (less than 22weeks). These results suggested that cerebral spinal fluid pH provides a biomarker for ALS and could reflect mechanisms of disease progression in ALS patients.

Cystatin C: A Candidate Biomarker for Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurologic disease characterized by progressive motor neuron degeneration. Clinical disease management is hindered by both a lengthy diagnostic process and the absence of effective treatments. Reliable panels of diagnostic, surrogate, and prognostic biomarkers are needed to accelerate disease diagnosis and expedite drug development. The cysteine protease inhibitor cystatin C has recently gained interest as a candidate diagnostic biomarker for ALS, but further studies are required to fully characterize its biomarker utility. We used quantitative enzyme-linked immunosorbent assay (ELISA) to assess initial and longitudinal cerebrospinal fluid (CSF) and plasma cystatin C levels in 104 ALS patients and controls. Cystatin C levels in ALS patients were significantly elevated in plasma and reduced in CSF compared to healthy controls, but did not differ significantly from neurologic disease controls. In addition, the direction of longitudinal change in CSF cystatin C levels correlated to the rate of ALS disease progression, and initial CSF cystatin C levels were predictive of patient survival, suggesting that cystatin C may function as a surrogate marker of disease progression and survival. These data verify prior results for reduced cystatin C levels in the CSF of ALS patients, identify increased cystatin C levels in the plasma of ALS patients, and reveal correlations between CSF cystatin C levels to both ALS disease progression and patient survival.

Discovery and verification of amyotrophic lateral sclerosis biomarkers by proteomics

Recent studies using mass spectrometry have discovered candidate biomarkers for amyotrophic lateral sclerosis (ALS). However, those studies utilized small numbers of ALS and control subjects. Additional studies using larger subject cohorts are required to verify these candidate biomarkers. Cerebrospinal fluid (CSF) samples from 100 patients with ALS, 100 disease control, and 41 healthy control subjects were examined by mass spectrometry. Sixty-one mass spectral peaks exhibited altered levels between ALS and controls. Mass peaks for cystatin C and transthyretin were reduced in ALS, whereas mass peaks for posttranslational modified transthyretin and C-reactive protein (CRP) were increased. CRP levels were 5.84 +/- 1.01 ng/ml for controls and 11.24 +/- 1.52 ng/ml for ALS subjects, as determined by enzyme-linked immunoassay. This study verified prior mass spectrometry results for cystatin C and transthyretin in ALS. CRP levels were increased in the CSF of ALS patients, and cystatin C level correlated with survival in patients with limb-onset disease. Our biomarker panel predicted ALS with an overall accuracy of 82%.