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Chun-Ting Lee and colleagues at the U.S. National Institutes of Heath investigated the mechanism of cocaine's effect on foetal brain development. Previously, it had been observed that exposure of the developing brain to cocaine can cause neurological and behavioural abnormalities in babies born to mothers who use the drug during pregnancy. Dr. Lee's findings indicate that a byproduct of cocaine metabolism inhibits the development of nerve cells by way of interfering with cyclin A, a protein involved in regulating cell division.
Scientist Live spoke with Dr. Lee and discussed his research.
What prompted your lab to study the effects of cocaine on neural progenitor cells?
Cocaine abuse during pregnancy is an important public health issue as it exposes several hundred thousand infants per year to cocaine in the United States alone. Our research was incited by some interesting findings coming out about the effects of cocaine. It was discovered that neonatal exposure to cocaine causes neocortical cytoarchitectural and neurobehavioural alterations in the developing brain; however, it remained unclear what cell types are targeted by cocaine in the developing cerebral cortex or what molecular changes cause these abnormalities. It was also shown that cocaine affects cerebral neocortical cytoarchitecture in primates only if administered during neocortical neuronogenesis, the period when proliferation of neural progenitors is most active (Lidow MS, 2001). The specific actions of cocaine in the second trimester and the decrease of neuron numbers in the cortex suggested that cocaine may affect important cellular functions of neural progenitor cells. Previously, our lab had developed a rat central nervous system progenitor cell line, AF5, which can be studied in culture but retains normal cell growth regulatory mechanisms. Using this line, we decided to tackle the question of why cocaine was causing these deficits by trying to identify the molecular mechanisms which are involved in cocaine-induced proliferation inhibition of neural progenitor cells.
Can you describe your methods and the rationale behind it?
We first treated AF5 cells with cocaine to find that their proliferation is reduced by quantification of the number of cells. To rule out an increase in cell death as the cause of reduced numbers, we examined the extracellular lactate dehydrogenase (LDH) activity and numbers of apoptotic nuclei immunoreactive for single-strand DNA, but did not observe changes in either.
Then, to determine where in the cell cycle cocaine was having its effect, we measured the cell cycle distribution by Fluorescence Activated Cell Sorting (FACS) and found that the G1-to-S phase transition was interrupted. Next, to find a molecule that cocaine could mediate to disrupt this transition, we used a microarray with various cell cycle-related genes. We found that cyclin A2 was significantly down-regulated by cocaine, and confirmed this finding in human foetal neural progenitor cells and developing rat brains using RT-PCR and western blot analysis. Reversing cyclin A down-regulation by gene transfer counteracted the proliferation inhibition caused by cocaine in AF5 cells, suggesting that cocaine-induced down-regulation of cyclin A contributes to the proliferation inhibition seen in cocaine-exposed AF5 cells.
The next step was to find out why cyclin A causes this cell cycle arrest. Using western blot analysis, we screened 11 candidate molecules known to regulate cyclin A expression to see which one was affected by cocaine, and found only the molecule ATF4 was changed. To see what causes the change in ATF4, we looked at the pathways that control it, and again using western blot analysis, we found the eIF2α-ATF4 pathway, an ER stress pathway, was the cause. Looking for the origin of these changes, we discovered a cocaine-induced accumulation of reactive oxygen species, which is a result of the N-oxidation of cocaine via cytochrome P450, and triggers ER stress signals, which activate the eIF2α-ATF4 pathway. To confirm the role of cocaine N-oxidative metabolism, we administered the p450 inhibitor cimetidine, both in vitro and in vivo, to see if it would prevent the disruption in G1-to-S phase transition caused by a decrease in cyclin A.
Discuss the significance of cyclin A2. What happens to cyclin A2 when exposed to cocaine? Why?
The decrease in cyclin A2 expression is the direct cause of the G1-to-S phase transition arrest seen in prenatal cocaine exposure as it controls genes that regulate this transition. The interruption of this phase transition is significant because it means the cells will essentially halt the cell cycle and so will stop multiplying, leading to a decrease in the total number of neural precursor cells that will eventually form the neurons in the brain. Cocaine-induced accumulation of reactive oxygen species, which involves N-oxidation of cocaine via cytochrome P450, promotes cyclin A2 down-regulation by causing endoplasmic reticulum stress, which in turn activates the eIF2α-ATF4 signalling pathway. Down-regulation of cyclin A2 may thus constitute the mechanism underlying cocaine-induced inhibition of neural progenitor cell proliferation.
How does cimetidine interact with cocaine and cyclin A2 in rats?
Cimetidine belongs to a class of drugs called histamine H2 receptor antagonists, and is often prescribed in humans for the treatment for certain gastric diseases. Cimetidine is also a p450 inhibitor. The N-oxidative pathway of cocaine metabolism is the pathway that is dependent on cytochrome p450; it is also the pathway that generates ROS and activates the cascade of events that lead to the G1-to-S phase transition arrest. Cimetidine does not interact with cocaine or cyclin A2 directly; however, it prevents cocaine-induced proliferation inhibition by blocking the N-oxidative pathway for cocaine metabolism and thus prevents the ER stress that causes a decrease in the expression of cyclin A2.
Can you place your research in the context of related research and current theories regarding cocaine and the nervous system?
More and more studies are focusing on the disrupting effects of cocaine on neural progenitor cells since they play an important role in the brain development. While it has been shown previously that cocaine changes the cellular mechanisms of human neural progenitor cells (e.g., Noonan et al., 2008) and also inhibits primate neural progenitor cell proliferation in the developing brain (Lidow et al., 2001), the mechanism of both are not clear. We are the first to describe one of the mechanisms involved in this action. Also, we point to the importance of the P450 system in the brain which was previously examined primarily in the liver where the majority of drug metabolism occurs.
Finally, what is the next step in your research?
Future research will examine the consequences of cocaine-induced inhibition of neural progenitor cell proliferation by looking at the cortical changes in developing rat foetal brains. We would also like to further examine P450-dependent oxidative metabolism of cocaine, to see if it causes changes in the cellular mechanisms of other types of cells in the brain, like astrocytes. Finally, we would like to study cytochrome p450s to explore their expression, metabolic capacities, and roles in different stages of neural differentiation. Eventually, it will also be necessary to perform long-term animal studies, using rats and other animal models, to determine whether it will be possible to employ cimetidine or other drugs to prevent the adverse effects of cocaine on brain development.