High-Content Phenotypic Screening and Triaging Strategy to Identify Small Molecules Driving Oligodendrocyte Progenitor Cell Differentiation

Abstract

Multiple Sclerosis is a demyelinating disease of the CNS and the primary cause of neurological disability in young adults. Loss of myelinating oligodendrocytes leads to neuronal dysfunction and death and is an important contributing factor to this disease. Endogenous oligodendrocyte precursor cells (OPCs), which on differentiation are responsible for replacing myelin, are present in the adult CNS. As such, therapeutic agents that can stimulate OPCs to differentiate and remyelinate demyelinated axons under pathologic conditions may improve neuronal function and clinical outcome. We describe the details of an automated, cell-based, morphometric-based, high-content screen that is used to identify small molecules eliciting the differentiation of OPCs after 3 days. Primary screening was performed using rat CG-4 cells maintained in culture conditions that normally support a progenitor cell-like state. From a library of 73,000 diverse small molecules within the Sanofi collection, 342 compounds were identified that increased OPC morphological complexity as an indicator of oligodendrocyte maturation. Subsequent to the primary high-content screen, a suite of cellular assays was established that identified 22 nontoxic compounds that selectively stimulated primary rat OPCs but not C2C12 muscle cell differentiation. This rigorous triaging yielded several chemical series for further expansion and bio-or cheminformatics studies, and their compelling biological activity merits further investigation.

Potential Benefit of the Charge-Stabilized Nanostructure Saline RNS60 for Myelin Maintenance and Repair

“…However, 5 days after plating with two applications of RNS60, we detected higher numbers O4 and MBP expressing cells under RNS60 treatment compared to control medium (N1) condition. We believe our results are in line with many other studies that have attempted pharmacological promotion of OPCs differentiation 32 33 . Finally, RNS60 did not alter the rate of OPC proliferation.…”

Drug discovery for remyelination and treatment of MS

“…Phenotypic screens are enjoying something of a renaissance in terms of projects aimed at identifying interventions to treat diseases of the CNS (Prior et al, ; Pruss, ). Indeed, eight studies to date have taken the broadly similar approach of screening compound libraries for their ability to promote the differentiation of oligodendrocytes in vitro (Deshmukh et al, ; Joubert et al, ; Lariosa‐Willingham et al, ; Mei et al, ; Najm et al, ; Peppard et al, ; Porcu et al, ) (Table ). The results of these studies have already generated new compounds and targets of great promise (see Tables , ).…”

“…Each of the eight in vitro OPC differentiation screens employed some form of automated analysis. Six of the eight screens were carried out using simple oligodendrocyteenriched 2D cultures grown in multiwell plate format, with automated image acquisition(Deshmukh et al, 2013; Joubert et al, 2010;Lariosa- Willingham et al, 2016;Najm et al, 2015;Peppard et al, 2015;Porcu et al, 2015).Subsequent image analyses included relatively simple quantification of DAPI-positive nuclei surrounded by MBP staining, as used by Deshmukh et al, to serve as a proxy for oligodendrocyte differentiation. In contrast, both Najm et al, and Lariosa-Willingham employed more complex analyses of cell morphology to estimate myelin produced per MBP-expressing oligodendrocyte, in addition to assessing differentiation.…”

An automated high-resolution in vivo screen in zebrafish to identify chemical regulators of myelination

“…The fact that many demyelinated lesions contain OPCs that do not differentiate has prompted efforts to identify strategies to promote oligodendrocyte differentiation. Indeed, recent unbiased phenotypic screens have led to the identification of several hit compounds that promote oligodendrocyte differentiation in vitro ( Deshmukh et al, 2013 ; Joubert et al, 2010 ; Mei et al, 2014 ; Mei et al, 2016 ; Najm et al, 2015 ; Peppard et al, 2015 ; Porcu et al, 2015 ). A number of these hits have been validated in vivo ( Cole et al, 2017 ), and one, clemastine, has even shown efficacy in improving optic nerve function in patients with MS ( Green et al, 2017 ).…”