Development of a central nervous system axonal myelination assay for high throughput screening

BMC Neuroscience

Methodology Article

Jay S. Tung¹  Elen S. Rosler²  Tassie L. Collins³  Jason C. Dugas⁴  Karen D. Lariosa-Willingham⁵  Dmitri Leonoudakis⁵ 

[1] Translational Medicine Center, Myelin Repair Foundation, 94085, Sunnyvale, CA, USA;Translational Medicine Center, Myelin Repair Foundation, 94085, Sunnyvale, CA, USA;Alios BioPharma, 94080, South San Francisco, CA, USA;Translational Medicine Center, Myelin Repair Foundation, 94085, Sunnyvale, CA, USA;NGM Biopharmaceuticals, Inc., 94080, South San Francisco, CA, USA;Translational Medicine Center, Myelin Repair Foundation, 94085, Sunnyvale, CA, USA;Rigel Pharmaceuticals, 94080, South San Francisco, CA, USA;Translational Medicine Center, Myelin Repair Foundation, 94085, Sunnyvale, CA, USA;Teva Pharmaceuticals, Biologics and CNS Discovery, 94063, Redwood City, CA, USA;

关键词: Myelination;    Oligodendrocyte;    High throughput;    Drug screening;    Differentiation;    Primary cell-based assay;    Image analysis;    Multiple sclerosis;    Myelin basic protein;   

DOI  :  10.1186/s12868-016-0250-2

 received in 2015-10-01, accepted in 2016-04-12,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundRegeneration 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.ResultsWe 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.ConclusionsWe 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.

【 授权许可】

CC BY   
© Lariosa-Willingham et al. 2016