Category: Preclinical Development
Purpose: Neurite outgrowth or neuritogenesis is a fundamental process in the differentiation of neurons and plays an important role in development, formation and remodeling of synapses including response to injury, repair and regeneration of damaged neurons. Understanding the mechanism of neurotrophic processes like neurite outgrowth and neuronal survival is crucial for brain development studies and treatment of various neurodegenerative disorders, which occur as a result of progressive loss of structure and/or function of neurons (1). Studies in our lab have indicated SAHA induced neurite outgrowth in Neuroscreen-1 cell via MAPK signaling pathway involving upstream kinase TrkA (2).
Cyclic AMP pathway is also implicated in the process of neurite outgrowth. This study involved understanding the role of cyclic AMP pathway in neurite outgrowth induced by SAHA to understand the mechanism for neuritogenic effect of SAHA.
Methods: NeuroScreen-1 (NS-1), a clone of PC12 (rat adrenal pheochromocytoma) cell line optimized for Nerve Growth Factor (NGF)-stimulated neuritic outgrowth, was used as a model of neuronal differentiation in this study. To understand whether SAHA mediated neurite outgrowth involves cyclic AMP pathway , we pretreated the Neuroscreen-1 cells with 200 µM adenylate cyclase inhibitor SQ 22536 followed by 1 µM SAHA with and without nerve growth factor (NGF). A spectrophotometric 96 well microplate assay combined with digital cellular image analysis, recently standardized in our lab, was employed for evaluation of cytotoxicity and neuritogenesis of SAHA.
Results: SAHA independently produced significant neurotrophic (induction of neuritic outgrowth) action like NGF in the NS-1 cells. Further, to investigate, if the mechanism for SAHA-induced differentiation of NS-1 cells is via cyclic AMP pathway, SQ22536 an adenylate cyclase inhibitors was tested. NGF mediated neuritogenesis was reduced in presence of adenylate cyclase inhibitor suggesting involvement of CAMP in NGF-mediated outgrowth. However. Surprisingly SAHA mediated neurite outgrowth in Neuroscreen-1 cells is significantly enhanced in presence of adenylate cyclase inhibitor suggesting lower level of cyclic AMP may bring about some cellular changes which is crucial for vorinostat mediated neurite out growth. Also, Vorinostat mediated neurite outgrowth may not be following the same mechanism as NGF mediated neurite outgrowth.
Conclusion: The studies reveal mechanism for neurotrophic action of SAHA and role of cyclic AMP in SAHA induced neurite outgrowth. Future research is needed to address how lower level of cyclic AMP can lead to enhanced neurite outgrowth induced by vorinostat.
1)Maruoka, H., Sasaya, H., Sugihara, K., Shimoke, K., and Ikeuchi, T. (2011). Low-molecular-weight compounds having neurotrophic activity in cultured PC12 cells and neurons. The Journal of Biochemistry 150(5), 473-475.
2) Shukla, S., Shariat-Madar, Z., Walker, L.A., and Tekwani, B.L. (2016). Mechanism for neurotropic action of vorinostat, a pan histone deacetylase inhibitor. Molecular and Cellular Neuroscience 77, 11-20.
Babu Tekwani– Birmingham, Alabama