Towards a blood-based biomarker for remyelination: Glial cell death and associated byproducts in demyelinating disease

Abstract

Over the past few decades, an explosion of interest in the use of accessible biofluids to identify and track molecular disease has revolutionized the fields of oncology, prenatal medicine, and others. Technological advances in signal detection and epigenetic profiling have allowed for novel applications of liquid biopsies to diseases without characteristic mutational profiles, including many degenerative, ischaemic, infectious, autoimmune, and inflammatory disorders. These events have paved the way for a wide array of neurological conditions to benefit from enhanced diagnostic, prognostic, and treatment abilities through the use of liquid biomarkers: a “liquid biopsy” approach. Two demyelinating conditions that stand to benefit from this approach are multiple sclerosis (MS) and progressive multifocal leukoencephalopathy (PML). MS is a chronic neuroinflammatory and neurodegenerative disease affecting over two million people worldwide and is a major cause of neurological disability, particularly in young adults. PML is a rare, severe demyelinating condition resulting from the reactivation of the JC polyomavirus in immunocompromised patients. In both MS and PML, there is a clinical need for relevant fluid biomarkers to predict/monitor disease progression and to assess treatment response, including for novel therapies targeting myelin repair. While several potential remyelinating agents have proceeded to clinical trials, methods for accurately and non-invasively assessing changes in remyelination status remain limited. Recent studies have found that cell-free DNA (cfDNA) released from dying neurons and glia can be detected in the bloodstream using cell-specific methylation patterns. A better understanding of remyelination-associated glial cell turnover dynamics may aid in the discovery of relevant liquid biomarkers to meet this clinical need. In the work described herein, I sought to apply this epigenetic liquid biopsy approach to characterize potential circulating biomarkers that may be indicative of demyelination and remyelination, as well as neuroinflammation and neurodegeneration. I did this using a combination of histology-based characterization studies in an animal model of demyelination/remyelination and analysis of clinical samples from patients with demyelinating disease. Using the animal model of demyelination/remyelination, I found that the repairing lesion environment is marked by a high degree of glial cell death, particularly that of oligodendrocyte progenitor cells (OPCs) and microglia. These results prompted my effort to identify differentially methylated regions in OPCs and microglia by isolating cells from human brain tissue and whole-genome bisulfite-sequencing, resulting in the identification of novel methylation-sensitive ddPCR targets. Next, I completed a thorough, unbiased, sequencing-based tissue/cell-of-origin analysis of plasma from MS and PML patients to characterize the contributions of a range of tissue-/cell-types to the circulating cfDNA milieu. After building a 200+ sample repository containing plasma from healthy volunteers (HVs), MS patients, and PML patients, I extracted cfDNA from each sample, completed quality assessment using fragment size distribution and concentration measures, and completed bisulfite conversion followed by whole-genome bisulfite-sequencing. After completion of extraction of CpG methylation statuses and methylation signature deconvolution, I obtained circulating cfDNA cell-of-origin compositional profiles for each sequenced sample. Using this approach, I found that PML patients have increased bulk nuclear and mitochondrial cfDNA levels compared to HVs and MS patients, consistent with increased disease severity, as well as increased plasma cfDNA source heterogeneity. Furthermore, by comparing circulating cfDNA cell-of-origin profiles between demyelinating diseases and course descriptors, I revealed compositional differences indicative of differential cell turnover dynamics. Collectively, this work provides insight into the cellular dynamics occurring during myelin repair, identifies candidate blood-based biomarkers of remyelination, and provides comprehensive epigenomic sequencing data that may be used to identify additional clinically relevant biomarkers and to better understand the underlying biology and cell-types involved in human demyelinating disease.