A Combined In Vitro Assay for Evaluation of Neurotrophic Activity and Cytotoxicity.
Neurotrophic assays are phenotypic methods to identify molecules that stimulate differentiation of neuronal cells. Bioactive small molecules with neurotrophic actions hold great promise as therapeutic agents for the treatment of neurodegenerative diseases and neuronal injuries by virtue of their ability to stimulate neuritic outgrowth. A combined in vitro method, which measures neurotrophic activity and cytotoxicity in a single assay, has been described. This assay, performed in 96-well microplates with PC12 and Neuroscreen-1 (NS-1; a subclone of PC12) cells, is a simple tool for identification of new neurotrophic agents. Stimulation of neurite outgrowth was measured with NIS software by analysis of digital cell images as multiple parameters, namely, mean neurite length, neurite length/cell, nodes/cell, and number of neurites/cell. The assay has been standardized and validated with dose-response analysis for nerve growth factor (NGF) and mechanism-based inhibitors of NGF-induced neurite outgrowth, namely, SU6656 (an Src family kinase inhibitor) and PD98059 (a MEK inhibitor). The assay has been successfully applied for screening natural and synthetic compound libraries for cytotoxicity and neurotrophic activity. Screening of a set of harmala alkaloids identified harmine as a potential neurotrophic molecule that significantly stimulated NGF-induced neurite outgrowth in the NS-1 cells. Important advantages of this method are its simplicity and determination of cytotoxicity and neurotrophic activity in a single assay. This assay may be suitable for primary and cultured neuronal cells.
1 National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS, USA.
Narayan D
ND
Chaurasiya
1 National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS, USA.|2 Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, University MS, USA.
Surabhi
S
Shukla
1 National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS, USA.|2 Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi, University MS, USA.
Babu L
BL
Tekwani
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D000077290:Harmala Alkaloids / Q000737:chemistry / Q000494:pharmacology; D066298:In Vitro Techniques; D009414:Nerve Growth Factors / Q000737:chemistry / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects; D055495:Neurogenesis / Q000187:drug effects; D000071446:Neuronal Outgrowth / Q000187:drug effects; D016716:PC12 Cells; D051381:Rats; D015203:Reproducibility of Results
D000077290:Harmala Alkaloids; D009414:Nerve Growth Factors
PC12 cells and neuroscreen-1 (NS-1) cells; cytotoxicity; harmala alkaloids neurotrophic; nerve growth factor (NGF); neurite outgrowth; neuritogenesis