Theme 06 - Tissue Biomarkers

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with a variable clinical presenta- tion and rate of disease progression. There is no coherent strategy to develop disease biomarkers for early diagnosis in atypical cases or for phenotypic stratification. Several proteomic studies on affected tissues and fluids from ALS individu-als have recently emerged. Beside target-driven approaches to identify biomarkers, as shown for neurofilaments, an unbiased methodology to mine the wealth of these prote- omic data studies could dissect the best candidate biomarkers for validation in ALS biofluids (1). Objectives: To build a comprehensive dataset incorporating proteomic studies from tissues and biofluids collected from patients with ALS that could serve as testbed for a bioinfor- matic analysis and for the identification of a molecular signature of the disease. To analyse bioinformatically available datasets and identify an ALS proteomic signature that can guide further investigations into informative biomarkers in biological fluids. Methods: ALS Mass spectrometry and SomaScan data from a range of human studies fulfilling pre-defined selection criteria have been collected from publicly available repositories. Data integration relies on overcoming limitations intrinsic to these heterogeneous datasets, including accessibility, study size, data representation and tissue type. Datasets are individually ana- lysed with comprehensive bioinformatics including principal component analysis and data clustering. Once proteins with a disease-specific pattern of expression are identified within different study populations in silico, validation of their utility as biomarkers will be undertaken on our longitudinal ALS samples. Results: The first part of the project has focused on data col-lection, evaluation and cleaning. From 81 ALS studies, 13 human tissue/fluid and 12 cell lines studies have been selected for analyses. 30 of the 81 studies do not provide supplementary information and 9 have shared only signifi- cant discoveries. At this stage of the project, volcano plots and heat maps have been used to aid proteomics biomarkers visualisation. Compared to controls proteins levels, MMP-9, OLR, Calgranulin B, and S100A6 genes have been differen-tially regulated across some of the ALS studies. Discussion: There are limited publicly available data which are difficult to access. Most of the information found in the literature is about finite results but not raw data. As expected, there is not an established shared template to col-lect Mass spectrometry data, complicating data comparison and analysis. However, we have built a dataset that encom-passes tissue/biofluids and cell lines which shows promising results. Our work is currently in progress but it is expected to deliver potentially novel protein candidates that may have a role as ALS biomarkers and could be tested in our large lon- gitudinal biofluids collection. Results: Approximately 50% of the patients presented with CK levels above the 99th percentile cut-off. 68% of the patients with ALS had CM-MB levels above the 99th percentile cut-off. CK levels are significantly elevated only in female patients. CK- MB levels are significantly elevated in ALS patients, particularly among male patients. We performed subgroup analyses and clinical correlations. Only patients with spinal-onset ALS showed significantly elevated CK serum levels. Patients with spinal and bulbar-onset ALS showed significantly elevated CK- MB serum levels. Patients with PLS and benign fasciculation syndrome always showed normal CK-MB serum levels. In contrast to cTnT, CK-MB levels in ALS patients stabilize over time. The CM-MB level in serum was positively correlated with CK and cTnT serum levels and slightly correlated with the bulbar sub-score in the ALSFRS-R and disease duration. Conclusions: We propose that CK-MB elevation in ALS is of non-cardiac origin and may serve as a marker of lower moto- neuron or skeletal muscle involvement. CK-MB levels may thus be helpful in defining restricted phenotypes of ALS such as PLS and may also have value as a prognostic marker. Further research is necessary to determine the biological origin of the CK-MB elevation and to confirm its validity as a diagnostic marker. proxies neuroinflam- prognostic amyotrophic sclerosis the cellular contribute to upregulation to be fully identified. We suggesting ALS in Background: We previously presented that calretinin (CR), a cytoplasmic calcium-binding protein, is closely associated with massive microglial infiltration in anterolateral funiculi outside the corticospinal tracts (ALFoC) in ALS spinal cord, and that CR-stimulated microglia to Background: Amyotrophic lateral sclerosis (ALS) is a progressive, devastating neurodegenerative disorder. Neuroinflammation is involved in the pathogenesis and progression of ALS. Cytokines are deeply involved in the inflam- matory process. We aimed to evaluate the levels of inflammation-related cytokines in patients with ALS. Methods: A total of 103 participants (62 ALS patients and 41 matched controls) were recruited from two independent neuromuscular referral centers in Huashan Hospital Fudan University and Fujian Medical University Union Hospital. The Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) was used for clinical assessment. In the first stage, the levels of serum cytokines were measured using an 18-plex Luminex kit. In the validation stage, the levels of target cytokines were measured by commercial ELISA kits. The levels of serum neurofilament light chain (NFL) were measured by the ultra-sensitive Single-molecule array (SIMOA) HD-X platform. Results: In the first stage, only the serum IL-18 levels were significantly elevated in ALS patients (13.31 [10.10 – 16.36] vs. 9.43 [6.63 – 13.32], p ¼ 0.016). ROC analysis revealed an AUC estimated at 0.70 (CI 0.55 – 0.84). Meanwhile, group by medium age (55 years old), the IL-21 levels were lower in elder ALS patients. Correlation analysis revealed that IL-5, IL-13, and IL-18 were positively associated with the disease progression rate. In the validation stage, serum IL-18 was sig- nificantly elevated in the serum of ALS patients as well (167.67 [148.25 – 175.59] vs. 116.44 [102.43 – 122.19], p ¼ 7.3 (cid:2) 10 -5 ). None of the cytokines correlated with serum NFL levels in both two stages. Conclusion: We have described a group of serum cytokines profiles in ALS patients. These cytokines have the potential to be a biomarker in the immune process of ALS. Background: Extracellular vesicles (EVs) are known to be secreted by mammalian fluids, unicellular organisms, and cell culture. Based on their biogenesis, size, and surface indica-tors, EVs are spherical vesicles that are primarily divided into microvesicles, exosomes, and apoptotic bodies. These molecules are membrane-bound entities with diameters between 50 and 1000nm that are secreted into the extracellular envir-onment by a variety of cell types (1). Objectives: In this study, we found the differentiated prote- ome in EVs of familial SOD1mutant ALS patients from healthy SOD1mutant and other ALS patients with unknown genetics, as well as healthy controls. The EVs carrying the non-coding RNAs, nucleic acids, proteins, and lipids are crucial since these EVs are cargo molecules passing the blood-brain bar- rier in both directions (2). Methods: By ultracentrifugation after serial centrifugation, from all groups were isolated from the blood plasma of mutant ALS patients, SOD1 mutant healthy controls, sporadic ALS patients neurological, and healthy controls. By utilizing anti-CD63 in Western Blotting and nanoparticle tracking analysis, EV purification After verification, RIPA was used to extract from each volunteer ’ s EV samples. Utilizing label-free quantifica-tion, used determine the profile. Maxquant Software MS data, the annotation to assess the similarities and differences of distinct (DEPs). Gene whole analyses using for gene. Some of the down-regulated proteins are SERPINC1, SERPINA6, ACTBL2, PTPRC. Discussion: To determine whether molecular characteristics contribute to the prevention of illness, we analyzed the proteome of extracellular vesicles from fALS patients with the mutant SOD1 gene and the healthy kins of these patients. As a consequence, we predicted and enriched the pathways using Cytoscape extracellular exosomes, carbon metabolism pathways, long-term depression, gap junction, serotonergic, and cholinergic synapse, as well as pathways for the innate immune system. Background: Epidemiological evidence suggest that changes in metabolism including alterations in HDL and LDL cholesterol may precede the onset of motor symptoms in amyotrophic lateral sclerosis (1,2). This study aimed to seek evidence for alterations in the levels of blood indices Objectives: To model the trajectory of primary care blood biomarkers prior to symptom onset or diagnosis in ALS patients. Methods: Data for total cholesterol, high density and low-density lipoprotein cholesterol (HDL and LDL), triglyceride, glycated haemoglobin (HbA1c) and creatinine measured as part of routine health screening were collected retrospect- ively from (1) a cohort of ALS patients attending a specialist clinic ( n ¼ 143) and (2) from primary care-linked data within UK Biobank for ALS patients and age- and sex-matched con- trols. Data were fitted using linear mixed effects models with linear b-splines to identify inflection points, controlling for age, sex. Results: In ALS clinic cases, models indicated decreasing levels of total, LDL and HDL cholesterol prior to an inflection point in the years before symptom onset (total cholesterol 3.25 years, LDL cholesterol 1.25 years, HDL cholesterol 0.5 years), after which they stabilized or rose. A similar pattern was observed in ALS cases within UK Biobank, occurring years prior to diagnosis (total cholesterol 7 years, LDL choles- ter