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. 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, an effect which we found to be related to cyclin A down-regulation. 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, as described in our Annual report on immortalized cell lines. 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 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. In the developing rat brain, cocaine causes an inhibition of neural progenitor proliferation in the highly active ventricular zone only, while also causing down-regulation of cyclin A expression. We have also demonstrated that this effect of cocaine occurs by a pathway involving oxidative stress of the endoplasmic reticulum (ER), and consequent phosphorylation of E1F-2alpha and upregulation of ATF4. Oxidative metabolism of cocaine by neural progenitor cells results in ER stress, leading to cyclin A down-regulation and inhibition of cell proliferation. Inhibition of cocaine oxidative metabolism by the P450 inhibitors cimetidine or SKF-525A results prevents the generation of reactive oxygen species, ATF4 upregulation, cyclin A inhibition, and cell proliferation in vitro. In animals, cimetidine blocked the cocaine-induced inhibition of cell proliferation in the ventricular zone, while also inhibiting cocaine-induced upregulation of ATF4 and down-regulation of cyclin A. Therefore, the P450 inhibitor cimetidine normalizes the effects of cocaine on neural progenitor cell proliferation both in vitro and in an animal model. Cocaine decreased cyclin A expression in neural progenitor cells and A2B5+ progenitor cells, but in astrocytes cocaine increased cyclin A expression. Cell type-specific responses to cocaine were further further explored. Gene expression profiles were examined in cells obtained from the human fetal cerebral cortex at 20 weeks gestation. Cells were treated with 100 gM cocaine in vitro for 24 hr, followed by gene expression analysis using a human neural/stem cell/drug abuse-focused cDNA array, with verification by quantitative real-time RT-PCR. Cocaine was found to influence transcription of distinct categories of genes in a cell type-specific manner. In neural and A2B5+ progenitor cells cocaine down-regulated cytoskeleton-related genes including ezrin, 2 actin, 3d tubulin and 8 tubulin. In contrast, cocaine modulated immune and cell death-related genes in microglia and astrocytes. In microglia, cocaine up-regulated the immunoregulatory and pro-apoptotic genes IL-1 and BAX. In astrocytes, cocaine down-regulated the immune response gene glucocorticoid receptor and up-regulated the anti-apoptotic genes 14-3-3 and HVEM. Therefore, cell types comprising the developing neocortex showed differential responses to cocaine. Experiments in developing rats showed that cocaine alters the distribution of developing GABAergic and glutamatergic neurons. Cocaine decreased the Tuj1+ layer of the neocortex when administered during early periods of neocortical neurogenesis. Glutamatergic neurons in the VZ were increased by cocaine treatment, but concomitantly decreased in the outer laminae. Tangential migration of GABA+ cells was also disrupted by cocaine. Therefore, cocaine causes cytoskeletal abnormalities leading to disturbances in neural differentiation and migration, and inhibition of proliferation, in progenitor cells. In glial cells, cocaine causes changes in transcription suggestive of altered immune and apoptotic responses. We are currently using a model for human neocortical development, based on human embryonic stem cells and induced pluripotent stem cells, to examine the mechanisms responsible for the effects of cocaine on brain development in a human system. Two specific cytochrome P450 enzymes, cyp3a43 and cyp3a5, are being tested using shRNA, to examine cortical layer formation, neuronal generation, and cell migration. Pathways similar to those identified in vitro, involving ROS generation, ROS generation, and ER stress, are being examined.
|Lee, Chun-Ting; Chen, Jia; Kindberg, Abigail A et al. (2016) CYP3A5 Mediates Effects of Cocaine on Human Neocorticogenesis: Studies using an In Vitro 3D Self-Organized hPSC Model with a Single Cortex-Like Unit. Neuropsychopharmacology :|
|Lee, Chun-Ting; Bendriem, Raphael M; Freed, William J (2015) A new technique for modeling neuronal connectivity using human pluripotent stem cells. Restor Neurol Neurosci :|
|Lee, Chun-Ting; Bendriem, Raphael M; Kindberg, Abigail A et al. (2015) Functional consequences of 17q21.31/WNT3-WNT9B amplification in hPSCs with respect to neural differentiation. Cell Rep 10:616-32|
|Kindberg, Abigail A; Bendriem, Raphael M; Spivak, Charles E et al. (2014) An in vitro model of human neocortical development using pluripotent stem cells: cocaine-induced cytoarchitectural alterations. Dis Model Mech 7:1397-405|
|Lee, Chun-Ting; Chen, Jia; Worden, Lila T et al. (2011) Cocaine causes deficits in radial migration and alters the distribution of glutamate and GABA neurons in the developing rat cerebral cortex. Synapse 65:21-34|
|Lee, Chun-Ting; Chen, Jia; Hayashi, Teruo et al. (2008) A mechanism for the inhibition of neural progenitor cell proliferation by cocaine. PLoS Med 5:e117|