Molecular Characterization of NS-1 with Robust Growth and Enhanced Phenotypic Properties

Basic research for neurodegenerative disease largely relies on cell line studies, which also act as the primary platform for drug screening in translational medicine. Neuroscreen-1 (NS-1), is a sub-clone of PC12, a noradrenergic cell line that shows dopaminergic properties are widely used in neurobiology, neurotoxicology, and drug discovery studies [1,2]. 1,2

Differentiation of PC12 by NGF is well documented. NGF treatment on PC12 cell leads to cessation of cell division, induction of neurite outgrowth, and production of electrically excitable cells in culture, mimicking the characteristics of sympathetic-like neurons[6] 6. In addition to the neuroprotective and neuro-restorative properties of NGF, dysregulation in NGF signaling has been positively correlated with Alzheimer’s disease (AD)[7] 7, epilepsy[9] 9, and cancer[12] 12. AD is characterized by death of forebrain cholinergic neurons, resulting from imbalance in expression of NGF, Pro NGF, TrkA, p75NTR ( Mufson 2008). NGF regulates proliferation and differentiation of neuronal cells via activation of tyrosine protein kinase (TrkA) receptor, and downstream signaling molecules that include Ras/MAP kinase cascade, IP3-dependent Ca2+ release, and PI3K/Akt pathways[13] 13. Furthermore, NGF increases expression of ChAT and VAChT, cholinergic markers are required for cholinergic neurotransmission[14,15] 14,15. ChAT enables the synthesis of acetylcholine (ACh) from acetyl-CoA and choline, whereas VAChT acts as a membrane transporter loading ACh into secretory vesicle and makes it available for secretion[16] 16. Decrease in ChAT and VAChT is suggested to play a role in the progression of AD[17,18] 17,18.

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Muscarinic acetyl choline receptors (Chrms) GPCR found in the cholinergic system signal through binding of acetylcholine and are involved in learning and memory[19,20] 20, and thus been implicated in neuronal diseases [22], [23] [24] 22,23. Similarly, adenosine receptors (AR) mediates the actions of adenosine in CNS by release of neurotransmitters and synaptic plasticity[25] 25. Among four members, A1 receptor is prevalent in the synaptic regions whereas A2A receptor is localized in the striatum and olfactory bulb[26] 26. The A2A receptor has been shown to modulate NGF-induced neurite outgrowth (NOG) in PC12 and neuritogenesis in primary hippocampal neuron in association with translin-associated protein X (TRAX)[27] 27.

NS-1 cell line has been used as a neuronal cell model to study the chemical dependent initiation, progression, inhibition, and toxicity in neurite outgrowth assays[2,5,28,29] 29,30. Despite increasing interest in the use of NS-1 as a substitute model for PC12, investigations of alterations in growth and differentiation characteristic has been limited at the molecular level. Understanding traits of NS-1 gene expression in differentiated and na??ve states is critical in defining the representative neuronal subtype for utility as a relevant substitute experimental model for PC12. . In the present study, we compared the gene and protein expression profiles by analysis of neuronal molecular markers by qPCR and immunoblot analyses. We conclude that changes in gene and protein expression could account for enhanced phenotypic properties of NS-1, and that the expression of neuronal markers support the use of NS-1 as an alternative and substitute cell model to PC12.

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