Effects of ATP and NGF on Proliferation and Migration of Neural Precursor Cells.

Purinergic receptors belong to the most ancient neurotransmitter system. While their relevance in neurotransmission is well characterized, it has become clear that they have many other cellular functions. During development, they participate in regulation of proliferation and differentiation of stem cells. Here, we used rat embryonic telencephalon neurosphere cultures to detect purinergic P2 receptor subtype expression and possible synergistic actions of these receptors with NGF. Neurospheres proliferate in the presence of EGF and FGF-2; however, upon depletion of these growth factors, they migrate and differentiate into neurons and glial phenotypes. Expression patterns of P2X and P2Y receptors changed along neural differentiation. Gene expression of P2X2-7 and P2Y1,2,4,6,12,14 receptors was confirmed in undifferentiated and neural-differentiated neurospheres, with an up-regulation of P2X2 and P2X6 subtypes, together with a down-regulation of P2X4, P2X7 and P2Y subtypes upon induction to differentiation. BrdU-labeling and subsequent flow cytometry analysis was used to measure cell proliferation, which was increased by chronic exposure to NGF and increasing concentrations of ATP, in line with the expression levels of PCNA. Furthermore, a synergistic effect on proliferation was observed in conditions of co-incubation with ATP and NGF. While ATP and NGF independently promoted neural migration, no inter-relation between these factors was detected for this cellular process. As conclusion, an unknown synergism of ATP and NGF in proliferation is described. Future efforts may elucidate the underlying mechanisms of the interrelationship of ATP and NGF during neurogenesis.

Paraoxon and Pyridostigmine Interfere with Neural Stem Cell Differentiation.

Acetylcholinesterase (AChE) inhibition has been described as the main mechanism of organophosphate (OP)-evoked toxicity. OPs represent a human health threat, because chronic exposure to low doses can damage the developing brain, and acute exposure can produce long-lasting damage to adult brains, despite post-exposure medical countermeasures. Although the main mechanism of OP toxicity is AChE inhibition, several lines of evidence suggest that OPs also act by other mechanisms. We hypothesized that rat neural progenitor cells extracted on embryonic day 14.5 would be affected by constant inhibition of AChE from chronic exposure to OP or pyridostigmine (a reversible AChE blocker) during differentiation. In this work, the OP paraoxon decreased cell viability in concentrations >50 µM, as measured with the MTT assay; however, this effect was not dose-dependent. Reduced viability could not be attributed to blockade of AChE activity, since treatment with 200 µM pyridostigmine did not affect cell viability, even after 6 days. Although changes in protein expression patterns were noted in both treatments, the distribution of differentiated phenotypes, such as the percentages of neurons and glial cells, was not altered, as determined by flow cytometry. Since paraoxon and pyridostigmine each decreased neurite outgrowth (but did not prevent differentiation), we infer that developmental patterns may have been affected.

Functions of neurotrophins and growth factors in neurogenesis and brain repair.

The identification and isolation of multipotent neural stem and progenitor cells in the brain, giving rise to neurons, astrocytes, and oligodendrocytes initiated many studies in order to understand basic mechanisms of endogenous neurogenesis and repair mechanisms of the nervous system and to develop novel therapeutic strategies for cellular regeneration therapies in brain disease. A previous review (Trujillo et al., Cytometry A 2009;75:38-53) focused on the importance of extrinsic factors, especially neurotransmitters, for directing migration and neurogenesis in the developing and adult brain. Here, we extend our review discussing the effects of the principal growth and neurotrophic factors as well as their intracellular signal transduction on neurogenesis, fate determination and neuroprotective mechanisms. Many of these mechanisms have been elucidated by in vitro studies for which neural stem cells were isolated, grown as neurospheres, induced to neural differentiation under desired experimental conditions, and analyzed for embryonic, progenitor, and neural marker expression by flow and imaging cytometry techniques. The better understanding of neural stem cells proliferation and differentiation is crucial for any therapeutic intervention aiming at neural stem cell transplantation and recruitment of endogenous repair mechanisms.