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


RESULTS
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.


CONCLUSIONS
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.
Translational Medicine Center, Myelin Repair Foundation, Sunnyvale, CA, 94085, USA.|Teva Pharmaceuticals, Biologics and CNS Discovery, Redwood City, CA, 94063, USA.
Karen D
KD
Lariosa-Willingham
Translational Medicine Center, Myelin Repair Foundation, Sunnyvale, CA, 94085, USA.|Alios BioPharma, South San Francisco, CA, 94080, USA.
Elen S
ES
Rosler
Translational Medicine Center, Myelin Repair Foundation, Sunnyvale, CA, 94085, USA.
Jay S
JS
Tung
Translational Medicine Center, Myelin Repair Foundation, Sunnyvale, CA, 94085, USA.|Rigel Pharmaceuticals, South San Francisco, CA, 94080, USA.
Jason C
JC
Dugas
Translational Medicine Center, Myelin Repair Foundation, Sunnyvale, CA, 94085, USA.|NGM Biopharmaceuticals, Inc., South San Francisco, CA, 94080, USA.
Tassie L
TL
Collins
Translational Medicine Center, Myelin Repair Foundation, Sunnyvale, CA, 94085, USA. dmitri.leonoudakis@tevapharm.com.|Teva Pharmaceuticals, Biologics and CNS Discovery, Redwood City, CA, 94063, USA. dmitri.leonoudakis@tevapharm.com.
Dmitri
D
Leonoudakis
D053829:Amyloid Precursor Protein Secretases / Q000494:pharmacology; D000818:Animals; D001369:Axons / Q000187:drug effects* / Q000502:physiology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002540:Cerebral Cortex / Q000187:drug effects* / Q000502:physiology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D009103:Multiple Sclerosis / Q000188:drug therapy* / Q000503:physiopathology; D009186:Myelin Sheath / Q000187:drug effects* / Q000502:physiology; D009416:Nerve Regeneration / Q000187:drug effects*; D009836:Oligodendroglia / Q000187:drug effects / Q000502:physiology; D051381:Rats
D017077:Culture Media, Conditioned; D053829:Amyloid Precursor Protein Secretases
Differentiation; Drug screening; High throughput; Image analysis; Multiple sclerosis; Myelin basic protein; Myelination; Oligodendrocyte; Primary cell-based assay