Cerebrospinal Fluid Biomarkers Link Toxic Astrogliosis and Microglial Activation to Multiple Sclerosis Severity Ruturaj Masvekar1, Tianxia Wu2, Peter Kosa1, Christopher Barbour1, Valentina Fossati3 and Bibiana Bielekova1

1National Institute of Allergy and Infectious Diseases, 2National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD. 3The New York Stem Cell Foundation Research Institute, New York, NY.

Introduction  Identification of CNS cell type-enriched biomarkers  Correlation analysis between cluster scores and clinical measures  The efficacy of immunomodulatory treatments in MS decreases of disability, CNS tissue destruction and MS severity 1 Astrocyte Cluster 8 Microglia Cluster 2 with age and disease evolution (MMP7, SERPINA3, GZMA and CLIC1) (DSG2 & TNFRSF25) Training Cohort Validation Cohort Training Cohort Validation Cohort Spearman r 0.29 0.26 0.23 0.24 EDSS p value 2.2E-05 3.1E-04 7.8E-04 4.4E-04 Disability Spearman r 0.30 0.26 0.24 0.27 CombiWISE p value 1.2E-05 1.7E-04 4.0E-04 1.7E-04 CNS Tissue Spearman r 0.38 0.36 0.36 0.36 COMRIS-CTD Destruction p value 9.0E-09 1.1E-07 8.5E-08 1.1E-07 Spearman r 0.19 0.11 0.12 0.12 MSSS p value 0.01 0.16 0.10 0.16 Spearman r 0.27 0.25 0.21 0.24 ARMSS MS Seveirty p value 6.9E-05 5.8E-04 2.2E-03 5.8E-04 Spearman r 0.21 0.19 0.19 0.28 MS-DSS p value 2.8E-03 0.01 0.01 1.3E-04

Semi-partial Spearman correlation coefficients between cluster scores (after accounting for association with age) and clinical measures of disability (EDSS: Expanded Disability Status 40 neuron-, 73 astrocyte-, 81 oligodendrocyte-, 18 microglia-, and 38 Scale and CombiWISE2: Combinatorial Weight-Adjusted Disability Scale), CNS tissue endothelial cell type-enriched biomarkers destruction (COMRIS-CTD3: Composite MRI scale of CNS Tissue Destruction) and MS Age (years) severity (MSSS: Multiple Sclerosis Severity Score, ARMSS: Age Related Multiple Sclerosis 4  Identification of CNS cell type-enriched biomarker clusters Severity and MS-DSS : Multiple Sclerosis Disease Severity Scale). Correlation p values in the  Alternative intrathecal processes underlying disability validation cohort were adjusted to account for multiple comparisons using Benjamini Hochberg • Proteins that are secreted together by identical CNS cell types under identical False Discovery Rate method. Only clusters which had reproducible statistically significant progression during late stages of MS physiological or pathological conditions will have strong correlations in the CSF correlations (p < 0.01) with at least one clinical measure are represented. • Mitochondrial dysfunction, ER stress, hypoxia etc. samples collected from diverse group of individuals • Aberrant microglial and astroglial activation Astrocyte cluster 8 and microglial cluster 2 significantly correlated with • Degeneration of oligodendrocytes clinical measures of disability, CNS tissue destruction and MS severity • Axonal damage and neuronal apoptosis Currently we lack the biomarkers that could measure these CNS cell-specific  Abnormally activated microglia induce toxic (A1) astrocytes & intrathecal processes in living subjects both are associated with MS lesions5,6

Objectives  Identify and validate CSF biomarkers reflective of CNS cell- specific intrathecal processes 7 neuron-, 20 astrocyte-, 19 oligodendrocyte-, 7 microglia-, and 11 endothelial cell type-enriched biomarker clusters  Assemble related biomarkers into clusters to decrease dimensionality and to better understand biology behind them  Analysis of differences in cluster scores across disease diagnostic subgroups  Investigate whether such CNS cell-enriched clusters differ in MS Astrocyte Cluster 8 Microglia Cluster 2 MMP7 Matrix Metalloproteinase 7 DSG2 Desmoglein 2 patients during disease evolution SERPINA3 Serpin Family A Member 3 TNFRSF25 Tumor Necrosis Factor Receptor GZMA Granzyme A Superfamily Member 25 CLIC1 Chloride Intracellular Channel Protein 1  Investigate whether these clusters correlate with clinical measures of disability, CNS tissue destruction and MS severity Conclusions and Future Directions

Methods and Results  Research subjects

HD CIS NIND RR-MS SP-MS PP-MS - Adjusted Cluster Score

Training Cohort (N = 217) 19 10 28 66 38 56 Age Validation Cohort (N = 214) 23 10 29 61 34 57 Total (N = 431) 42 20 57 127 72 113 After accounting for associations with age cluster scores were compared among disease diagnostic subgroups in training cohort. Statistically significant differences (ANOVA; p < 0.05) All subjects’ (n = 431) were divided into two cohorts: Training Cohort (n = 217) and Validation were then assessed in the independent validation cohort. Only clusters which had reproducible Cohort (n = 214). Gender-ratio, age-, disease diagnostics subgroups-, clinical measures were statistically significant differences are represented (Validation Cohort). comparable in the two cohorts. HD: healthy donors, CIS: clinically isolated syndrome, NIND: non-inflammatory neurological disorder, RR-MS: relapsing-remitting MS, SP-MS: secondary- Astrocyte cluster 8 and microglia cluster 2 significantly elevated in all MS progressive MS and PP-MS: primary-progressive MS. subgroups compared to HD and NIND subgroups References Acknowledgements 1) Weideman AM et al. Meta-analysis of the Age-Dependent Efficacy of Multiple Sclerosis Treatments. Front Neurol. 2017; 8:577. The study was supported by the intramural research program of the National Institute of 2) Kosa P et al. Development of a Sensitive Outcome for Economical Drug Screening for Progressive Multiple Sclerosis Treatment. Front Neurol. 2016; 7:131. Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). 3) Kosa P et al. Novel composite MRI scale correlates highly with disability in multiple sclerosis patients. Mult Scler Relat Disord. 2015; 4(6):526-535. 4) Weideman AM et al. New Multiple Sclerosis Disease Severity Scale Predicts Future Accumulation of Disability. Front Neurol. 2017; 8:598. 5) Liddelow SA et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature. 2017; 541(7638):481-487. 6) Liddelow SA & Barres BA. Reactive Astrocytes: Production, Function, and Therapeutic Potential. Immunity. 2017; 46(6):957-967.