Phenotypic assays to identify agents that induce reactive gliosis: a counter-screen to prioritize compounds for preclinical animal studies.

Abstract

Astrocyte phenotypes change in a process called reactive gliosis after traumatic central nervous system (CNS) injury. Astrogliosis is characterized by expansion of the glial fibrillary acidic protein (GFAP) cytoskeleton, adoption of stellate morphologies, and differential expression of some extracellular matrix molecules. The astrocytic response immediately after injury is beneficial, but in the chronic injury phase, reactive astrocytes produce inhibitory factors (i.e., chondroitin sulfate proteoglycans [CSPGs]) that limit the regrowth of injured axons. There are no drugs that promote axon regeneration or functional recovery after CNS trauma in humans. To develop novel therapeutics for the injured CNS, we screened various libraries in a phenotypic assay to identify compounds that promote neurite outgrowth. However, the effects these compounds have on astrocytes are unknown. Specifically, we were interested in whether compounds could alter astrocytes in a manner that mimics the glial reaction to injury. To test this hypothesis, we developed cell-based phenotypic bioassays to measure changes in (1) GFAP morphology/localization and (2) CSPG expression/immunoreactivity from primary astrocyte cultures. These assays were optimized for six-point dose-response experiments in 96-well plates. The GFAP morphology assay is suitable for counter-screening with a Z-factor of 0.44±0.03 (mean±standard error of the mean; N=3 biological replicates). The CSPG assay is reproducible and informative, but does not satisfy common metrics for a "screenable" assay. As proof of principle, we tested a small set of hit compounds from our neurite outgrowth bioassay and identified one that can enhance axon growth without exacerbating the deleterious characteristics of reactive gliosis.