Abuse of cocaine during pregnancy has exposed several hundred thousand infants per year to cocaine in the United States alone. A variety of disorders of central nervous system (CNS) development , e.g., intrauterine growth retardation, interference with neuronal migration and differentiation, and neurobehavioral deficits have been associated with prenatal exposure to cocaine. However, the mechanisms through which in utero cocaine exposure affects fetal brain development are unknown. This project involves investigation of the biological mechanism through which cocaine impairs development of the brain. The ultimate purpose of this project will be to develop therapies which might be used to prevent brain damage caused by the abuse of cocaine during human pregnancy.? ? Prenatal exposure to cocaine causes morphological and behavioral abnormalities in developing brains. We found that cyclin A down-regulation plays a pivotal role in cocaine-induced inhibition of neural progenitor proliferation. In developing rat fetuses, exposure to cocaine inhibited proliferation of neural progenitor cells in the ventricular proliferative zone.? ? In pregnant rats treated with cocaine, cortical size was reduced when cocaine was injected during the early or middle period of cortical neurogenesis, but there was no effect of cocaine when injected during the late period. Cell cycle progression of neural progenitor cells in the ventricular zone, but not the subventricular zone, was inhibited by cocaine.? ? To test the effect of cocaine on cell proliferation, we used the AF5 neural progenitor cell line. Cocaine caused a decrease in cell proliferation but did not cause cell death. By FACS analysis, phosphorylated H3 immunocytochemistry, and bromodeoxyuridine labeling, cocaine was found to inhibit the G1-to-S phase transition. Using cDNA microarray analysis, cyclin A2 and the downstream transcript c-myc were identified as cell cycle regulators involved in the G1/S transition which were down-regulated by cocaine. Other G1/S phase transition controllers, such as cyclin D1, D2, D3, E1, and E2 were not changed by cocaine. We therefore hypothesized that down-regulation of cyclin A2 causes cocaine-induced inhibition of cell proliferation. Cyclin A2 mRNA was also measured in primary human fetal CNS cells by quantitative real-time RT-PCR, and was found to significantly be decreased in human neural progenitor cells and oligodendrocyte progenitor cells, whereas levels were not altered in neurons and microglia. In contrast, the cyclin A2 transcript level was increased by cocaine in human astrocytes. These data suggest that cocaine specifically down-regulates cyclin A2 in progenitor cells.? ? Western blotting confirmed a reduction of cyclin A2 protein by cocaine. Cyclin A induces a conformational change in CDK2 that leads to phosphorylation. CDK2 activates pRb that in turn promotes the expression of c-Myc. By western blotting, cocaine significantly decreased the active forms of CDK2 and pRb (phosphorylated form) as well as c-Myc expression. ? ? To examine whether cyclin A is responsible for cocaine-induced inhibition of proliferation, we compensated for cocaine-induced down-regulation of cyclin A by transfecting AF5 cells with a vector encoding cyclin A (pRc/CMV-CycA). Cyclin A transfection abolished the cocaine-induced down-regulation of cyclin A as well as the inhibition of proliferation caused by cocaine. These data suggested that cocaine inhibits AF5 cell proliferation by down-regulating cyclin A.
| Anantharam, Vellareddy; Lehrmann, Elin; Kanthasamy, Arthi et al. (2007) Microarray analysis of oxidative stress regulated genes in mesencephalic dopaminergic neuronal cells: relevance to oxidative damage in Parkinson's disease. Neurochem Int 50:834-47 |
| Chen, Jia; Lee, Chun-Ting; Errico, Stacie L et al. (2007) Increases in expression of 14-3-3 eta and 14-3-3 zeta transcripts during neuroprotection induced by delta9-tetrahydrocannabinol in AF5 cells. J Neurosci Res 85:1724-33 |
| Sanchez, Joseph F; Crooks, Daniel R; Lee, Chun-Ting et al. (2006) GABAergic lineage differentiation of AF5 neural progenitor cells in vitro. Cell Tissue Res 324:1-8 |
