Prenatal exposure of the developing brain to cocaine causes morphological and behavioral abnormalities. Recent studies indicate that cocaine-induced proliferation inhibition and/or apoptosis in neural progenitor cells may play a pivotal role in causing these abnormalities. To understand the molecular mechanism through which cocaine inhibits cell proliferation in neural progenitors, we sought to identify the molecules that are responsible for mediating the effect of cocaine on cell cycle regulation.
Methods and Findings
Microarray analysis followed by quantitative real-time reverse transcription PCR was used to screen cocaine-responsive and cell cycle-related genes in a neural progenitor cell line where cocaine exposure caused a robust anti-proliferative effect by interfering with the G1-to-S transition. Cyclin A2, among genes related to the G1-to-S cell cycle transition, was most strongly down-regulated by cocaine. Down-regulation of cyclin A was also found in cocaine-treated human primary neural and A2B5+ progenitor cells, as well as in rat fetal brains exposed to cocaine in utero. Reversing cyclin A down-regulation by gene transfer counteracted the proliferation inhibition caused by cocaine. Further, we found that cocaine-induced accumulation of reactive oxygen species, which involves N-oxidation of cocaine via cytochrome P450, promotes cyclin A down-regulation by causing an endoplasmic reticulum (ER) stress response, as indicated by increased phosphorylation of eIF2α and expression of ATF4. In the developing rat brain, the P450 inhibitor cimetidine counteracted cocaine-induced inhibition of neural progenitor cell proliferation as well as down-regulation of cyclin A.
Our results demonstrate that down-regulation of cyclin A underlies cocaine-induced proliferation inhibition in neural progenitors. The down-regulation of cyclin A is initiated by N-oxidative metabolism of cocaine and consequent ER stress. Inhibition of cocaine N-oxidative metabolism by P450 inhibitors may provide a preventive strategy for counteracting the adverse effects of cocaine on fetal brain development.
Investigating the mechanism of cocaine's effect on fetal brain development, Chun-Ting Lee and colleagues find that down-regulation of cyclin A by a cocaine metabolite inhibits neural proliferation.
Every year, cocaine abuse by mothers during pregnancy exposes thousands of unborn infants (fetuses) to this powerful and addictive stimulant. Maternal cocaine abuse during early pregnancy increases the risk of miscarriage; its use during late pregnancy slows the baby's growth and can trigger premature labor. Babies exposed to cocaine shortly before birth are often irritable and have disturbed sleep patterns. They can also be very sensitive to sound and touch and consequently hard to comfort. These problems usually resolve spontaneously within the first few weeks of life but some permanent birth defects are also associated with frequent cocaine abuse during pregnancy. In particular, babies exposed to cocaine before birth sometimes have small heads—an abnormality that generally indicates a small brain—and, although they usually have normal intelligence, the development of their thinking skills and language is often delayed, and they can have behavioral problems.
Why Was This Study Done?
Exposure to cocaine before birth clearly interferes with some aspects of brain development. More specifically, it reduces the number and position of neurons (the cells that transmit information in the form of electrical impulses around the body) within the brain. All neurons develop from neural progenitor cells, and previous research suggests that cocaine exposure before birth inhibits the proliferation of these cells in the developing brain. It would be useful to understand exactly how cocaine affects neural progenitor cells, because it might then be possible to prevent the drug's adverse effects on brain development. In this study, therefore, the researchers investigate the molecular mechanism that underlies cocaine's effect on neural progenitor cells.
What Did the Researchers Do and Find?
When the researchers investigated the effects of cocaine on AF5 cells (rat neural progenitor cells that grow indefinitely in the laboratory), they found that concentrations of cocaine similar to those measured in fetal brains after maternal drug exposure inhibited the proliferation of AF5 cells by blocking the “G1-to-S transition.” This is a stage that cells have to pass through between each round of cell division (the production of two daughter cells from one parent cell). Next, the researchers showed that cocaine-treated AF5 cells made much less cyclin A2, a protein that controls the G1-to-S transition, than untreated cells. Cocaine also decreased cyclin A2 levels in neural progenitor cells freshly isolated from human fetal brains and in fetal rat brains exposed to the drug while in their mother's womb. Treatment of AF5 cells with a cyclin A2 expression vector (a piece of DNA that directs the production of cyclin A2) counteracted the down-regulation of cyclin A2 and restored AF5 proliferation in the presence of cocaine. Other experiments indicate that the reduction of cyclin A2 by cocaine in AF5 cells involves the accumulation of “reactive oxygen species,” by-products of the breakdown of cocaine by a protein that is a member of a family of proteins called cytochrome P450. Finally, treatment of pregnant rats with cimetidine (which inhibits the action of cytochrome P450) counteracted both the inhibition of neural progenitor cell proliferation and the cyclin A2 down-regulation that cocaine exposure induced in the brains of their unborn pups.
What Do These Findings Mean?
These findings show that the cocaine-induced inhibition of neural progenitor cell proliferation involves, at least in part, interfering with the production (that is, causing down-regulation) of cyclin A2. They also show that this down-regulation is induced by the breakdown of cocaine by cytochrome P450, and that in both a rat cell line and in fetal rats, the cytochrome P450 inhibitor cimetidine (a drug that is already used clinically for stomach problems) can block the adverse effects of cocaine on the proliferation of neural progenitor cells. These findings need to be confirmed in animals more closely related to people than rats, and the long-term effects of cimetidine need to be investigated, in particular its effects on cocaine toxicity. Nevertheless these results raise the possibility that giving cimetidine or other drugs with similar effects to pregnant women who are addicted to cocaine might prevent some of the harm that their drug habit does to their unborn children, although it is not clear whether there is a dosage of cimetidine that might be both safe and adequate for this purpose.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050117.
A PLoS Medicine Perspective article by Steven Hyman further discusses this study
The US National Institute on Drug Abuse provides a fact sheet on cocaine (in English and Spanish)
The UK charity Release provides information and advice to the public and professionals about the law and drugs, including information about cocaine
MedlinePlus also provides a list of links to information about cocaine (in English and Spanish)
The March of Dimes Foundation, a US nonprofit organization for the improvement of child health, provides information about illicit drug use during pregnancy (in English and Spanish)
The Organization of Teratology Information Specialists also provides a fact sheet on cocaine and pregnancy (in English, Spanish, and French)