Neurotrophic and neuritogenic drug leads from natural products: A combined in vitro assay for evaluation of cell viability and NGF-stimulated neuritic outgrowth in neuroscreen 1 cells

Neuritogenesis or neuritic outgrowth is a fundamental cellular differentiation process for formation of new neurons. Recent reports have indicated importance of this process in neuroregeneration and repair of damaged neurons. Neurotrophic and Neuritogenic substances, which repair damaged neurons through stimulation of neuritic outgrowth, may be important for restoration and readjustment of normal neuronal functions of the damaged neurons. Neurite outgrowth assays are important phenotypic methods to screen chemical effects on the neuronal cells, especially for discovery of low molecular weight neurotrophic agents. Bioactive small molecules with neurotrophic and neuritogenic actions hold great promise as therapeutic agents for treatment of neurodegenerative diseases and neuronal injuries by virtue of their ability to stimulate neuritic outgrowth. A combined in vitro method that measures and quantifies neurite outgrowth along with the cell viability in a single assay has been described here. This assay is simple, less expensive and a useful tool for identification of new neurotrophic agents. The stimulation of neurite outgrowth was measured by analysis of digital cell images by the NIS software in terms of multiple parameters namely, the effect of test compounds on mean neurite length, neurite length/cell, nodes/cell and number of neurites/cell. An important advantage of this method, which can be performed in a 96-well plate, is the determination of cytotoxicity and neurotrophic activity of the test compounds in a single assay. The assay has been successfully applied for screening natural as well as synthetic compounds libraries. Focused screening of a library of harmala alkaloids and other natural products has identified new natural products leads, which produced significant activation of NGF-stimulated neuritic outgrowth. The lead natural products have been investigated further for interrogation of neurotrophin signaling pathways. Acknowledgement- Supported by NIH – National Institute of General Medical Sciences #P20GM104932 (COBRE-In vitro Research Core)