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Format: Abstract
See comment in PubMed Commons belowBMC Neurosci. 2016 Apr 22;17:16. doi: 10.1186/s12868-016-0250-2.Lariosa-Willingham KD1,2, Rosler ES1,3, Tung JS1, Dugas JC1,4, Collins TL1,5, Leonoudakis D6,7.1Translational Medicine Center, Myelin Repair Foundation, Sunnyvale, CA, 94085, USA. 2Teva Pharmaceuticals, Biologics and CNS Discovery, Redwood City, CA, 94063, USA. 3Alios BioPharma, South San Francisco, CA, 94080, USA. 4Rigel Pharmaceuticals, South San Francisco, CA, 94080, USA. 5NGM Biopharmaceuticals, Inc., South San Francisco, CA, 94080, USA. 6Translational Medicine Center, Myelin Repair Foundation, Sunnyvale, CA, 94085, USA. dmitri.leonoudakis@tevapharm.com. 7Teva Pharmaceuticals, Biologics and CNS Discovery, Redwood City, CA, 94063, USA. dmitri.leonoudakis@tevapharm.com. Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system.
We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency.
We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.
Differentiation; Drug screening; High throughput; Image analysis; Multiple sclerosis; Myelin basic protein; Myelination; Oligodendrocyte; Primary cell-based assay
PMID: 27103572 PMCID: PMC4840960 DOI: 10.1186/s12868-016-0250-2
[PubMed - indexed for MEDLINE] Free PMC ArticleAmyloid Precursor Protein Secretases/pharmacology Animals Axons/drug effects* Axons/physiology Cell Culture Techniques Cell Differentiation/drug effects Cerebral Cortex/drug effects* Cerebral Cortex/physiology Culture Media, Conditioned/pharmacology Drug Evaluation, Preclinical/methods* Fluorescent Antibody Technique/methods High-Throughput Screening Assays/methods* Multiple Sclerosis/drug therapy* Multiple Sclerosis/physiopathology Myelin Sheath/drug effects* Myelin Sheath/physiology Nerve Regeneration/drug effects* Oligodendroglia/drug effects Oligodendroglia/physiology Rats Culture Media, Conditioned Amyloid Precursor Protein Secretases BioMed Central Europe PubMed Central PubMed Central Multiple Sclerosis - MedlinePlus Health Information
