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Ruturaj R. Masvekar, PhD Neuroimmunological Diseases Section (NDS)

Cerebrospinal Fluid Biomarkers Link Toxic Astrogliosis and Microglial Activation to Multiple Sclerosis Severity. Ruturaj R. Masvekar, PhD Neuroimmunological Diseases Section (NDS) National Institute of Allergy and Infectious Diseases (NIAID) National Institutes of Health (NIH).

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Ruturaj R. Masvekar, PhD Neuroimmunological Diseases Section (NDS)

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  1. Cerebrospinal Fluid Biomarkers Link Toxic Astrogliosis and Microglial Activation to Multiple Sclerosis Severity Ruturaj R. Masvekar, PhD Neuroimmunological Diseases Section (NDS) National Institute of Allergy and Infectious Diseases (NIAID) National Institutes of Health (NIH)

  2. The efficacy of immunomodulatory treatments in MS decreases with age and disease evolution Age (years) Front Neurol. 2017; 8:577. 

  3. Alternative intrathecal processes underlying disability progression during late stages of MS • Mitochondrial dysfunction, ER stress, hypoxia etc. • Aberrant microglial and astroglial activation • Degeneration of oligodendrocytes • Axonal damage and neuronal apoptosis Currently we lack the biomarkers that could measure these CNS cell-specific intrathecal processes in living subjects

  4. Objectives • Identify and validate CSF biomarkers reflective of CNS cell-specific intrathecal processes • Assemble related biomarkers into clusters to decrease dimensionality and to better understand biology behind them • Investigate whether such CNS cell type-enriched clusters differ in MS patients during disease evolution • Investigate whether these clusters correlate with clinical measures of disability, CNS tissue destruction and MS severity

  5. Identification of CNS cell type-enriched biomarkers Primary Human Neurons (ScienCell) Primary Human Astrocytes (ScienCell) Oligodendrocytes differentiated from hPSCs Human Microglia Cell Line (CHME5) Human Brain Endothelial Cell Line (HCMEC/D3) 24 hours cell-culture supernatants Slow Off-rate Modified DNA Aptamers assay (SOMAscan; measuring 1,302 proteins) Cell type-enriched biomarker: absolute release of this protein by a specific cell type was at least 5-fold higher than the release by any other cell types 40 neuron-, 73 astrocyte-, 81 oligodendrocyte-, 18 microglia-, and 38 endothelial cell type-enriched biomarkers

  6. Identification of CNS cell type-enriched biomarker clusters • Proteins that are secreted together by identical CNS cell types under identical physiological or pathological conditions will have strong correlations in the CSF samples collected from diverse group of individuals Patients’ CSF SOMAscan (n = 431; HD = 42, NIND = 57, CIS = 20, RR-MS = 127, SP-MS = 72 and PP-MS = 113) Variable cluster analysis CNS cell type-enriched biomarkers Cluster score (principal component score) CNS cell type-enriched biomarker clusters 7 neuron-, 20 astrocyte-, 19 oligodendrocyte-, 7 microglia-, and 11 endothelial cell type-enriched biomarker clusters

  7. Analysis of differences in cluster scores across disease diagnostic subgroups All Patients (n = 431) Training Cohort (n = 217) Validation Cohort (n = 214) Age-adjusted cluster scores compared among disease diagnostic subgroups Statistically significant differences (ANOVA; p < 0.05) Only statistically significant differences assessed in independent validation cohort

  8. Astrocyte cluster 8 and microglia cluster 2 significantly elevated in all MS subgroups Validation Cohort Astrocyte Cluster 8 Microglia Cluster 2 Age-Adjusted Cluster Score

  9. Selected clinical measures of disability, CNS tissue destruction and MS severity • Disability: • EDSS: Expanded Disability Status Scale (Discrete, 0-10) • CombiWISE: Combinatorial Weight-Adjusted Disability Scale (Continuous 0-100; Front Neurol. 2016; 7:131) • CNS tissue destruction • COMRIS-CTD: Composite MRI scale of CNS tissue destruction   (Mult Scler Relat Disord. 2015; 4(6):526-35) • MS severity • MSSS: Multiple Sclerosis Severity Score (EDSS-based) • ARMSS: Age Related Multiple Sclerosis Severity (EDSS-based) • MS-DSS: Multiple Sclerosis Disease Severity Scale (CombiWISE-based) (Developed using machine-learning, can predict future rates of disability progression; Front Neurol. 2017; 8:598) • CombiWISE, COMRIS-CTD and MS-DSS available free at: https://bielekovalab.shinyapps.io/msdss/ Courtesy of Multiple Sclerosis Association of America MS Severity: how fast disability progresses

  10. Correlation analysis between cluster scores and selected clinical measures Only MS Patients (n = 312) Training Cohort (n = 160) Validation Cohort (n = 152) Analyze correlations between age-adjusted cluster scores and selected clinical measures Statistically significant correlations (Spearman correlations; p < 0.01) Only statistically significant correlations assessed in independent validation cohort

  11. Astrocyte cluster 8 and microglial cluster 2 significantly correlated with selected clinical measures Validation Cohort Disability CNS tissue destruction MS severity r = 0.26 p = 0.0002 r = 0.36 p < 0.0001 r = 0.19 p = 0.0082 CombiWISE COMRIS-CTD MS-DSS r = 0.27 p = 0.0002 r = 0.36 p < 0.0001 r = 0.28 p = 0.0001 CombiWISE COMRIS-CTD MS-DSS Age-Adjusted Cluster Score Age-Adjusted Cluster Score Age-Adjusted Cluster Score

  12. Abnormally activated microglia induce toxic (A1) astrocytes & both are associated with MS lesions Nature. 2017; 541(7638):481-487. Immunity. 2017; 46(6):957-967.

  13. Conclusions & Future Directions STOP STOP + Abnormally Activated Microglia ? Pro-Inflammatory Cytokines Disability, CNS Tissue Destruction and MS Severity Quiescent (Resting) Astrocytes Toxic (A1) Astrocytes ? Drug Library Screening

  14. Acknowledgements Neuroimmunological Diseases Section (NDS) Chief: Bibi Bielekova Lab: Chris Barbour Paav Hannikainen Kayla Jackson Peter Kosa Ruturaj Masvekar Linh Pham Jon Phillips Elena Romm Mihael Varosanec Clinical: Tiffany Hauser Mary Sandford Alison Wichman Michelle Woodland Special thanks to NDS patients and their families https://www.niaid.nih.gov/research/bibi-bielekova-md

  15. Questions • Poster: P281; Friday, March 1, 2019; 11:15 AM – 1:00 PM • Contacts: • Ruturaj R. Masvekar: ruturaj.masvekar@nih.gov • Bibi Bielekova: bibi.bielekova@nih.gov • Article:

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