Why Can't the Brain Shake Cocaine?

While celebrities and U.S. entanglement in the Colombian drug war keep cocaine in the headlines, a larger tragedy hides in the unseen lives of both addicts and former addicts. In 1999, 1.5 million Americans took cocaine at least once a month, according to the federal government's National Household Survey on Drug Abuse. The problem's vast size is aggravated by two stubborn realities: many addicts just can't quit, and those who do might relapse when stressed or tempted. Both groups suffer because cocaine, like an unforgettable experience, has permanently altered their brains. Biologists began investigating the neuronal changes underlying cocaine addiction many years ago. Some studies focused on what was happening on cell membranes; others tackled the less accessible molecular and genetic developments inside neurons. About five years ago, this intracellular approach largely petered out in the face of dauntingly complex biochemical pathways and proteins with unfathomable functions. A few labs persisted, however, and their explorations are starting to hit pay dirt. In March, for example, a team of 12 neuroscientists published a Nature paper detailing, for the first time, how chronic cocaine exposure affects several proteins along a critical signal transduction pathway in neurons.1 The ultimate goal of all this research is to tease out how short-term changes in intracellular proteins lead to the lifelong bodily changes that maintain addiction and cause relapse. One key protein identified in the Nature paper, cyclin-dependent kinase 5 (Cdk5), is "a very good candidate for involvement in long-term remodeling of the nervous system," notes Steven E. Hyman, director of the National Institute of Mental Health, whose lab has focused on drug addiction. Better understanding of this remodeling process could lead to new treatments, not only for cocaine addiction but also for neurological and psychiatric diseases that prey on the same brain circuits afflicted by cocaine. And because these circuits regulate several fundamental animal behaviors, findings about cocaine might help determine how the brain reacts to such stimuli as food, sex, and social interaction, says Eric J. Nestler, chairman of psychiatry at the University of Texas Southwestern Medical Center in Dallas and a principal author of the Cdk5 paper. "It's hard to imagine," he observes, "but these are weaker reinforcers than cocaine." A Raincoat Against Dopamine Though it exerts diverse effects throughout the body, cocaine scrambles the brain largely by attaching to proteins known as transporters, which sit on the membranes of presynaptic neurons. Transporters soak up excess neurotransmitter molecules that these neurons have released into synapses. When cocaine blocks this process, synapses become flooded with these molecules, particularly dopamine. The deluge is especially bad in the striatum's nucleus accumbens, where dopamine released by presynaptic neurons constantly stimulates postsynaptic neurons. Several days' worth of cocaine exposure also turns on the expression of various so-called immediate-early genes in these neurons. One such gene is fosB, which encodes a transcription factor that turns on many more genes. In a series of papers beginning in 1997,2 Nestler's lab has shown that D FosB, a FosB variant arising from alternative gene splicing, accumulates with repeated cocaine exposure. Unlike other proteins upregulated by cocaine, it can persist for a month or two. At the 1998 Society for Neuroscience meeting in Los Angeles, Nestler, then at Yale University School of Medicine, and Jingshan Chen, an assistant psychiatry professor at Yale, presented a poster describing a DNA array study of D FosB. The study found that when transgenic mice inducibly expressed this transcription factor, their striata produced considerably more Cdk5 mRNA and protein than the striata of nonexpressing mice. This result made sense, remembers Nestler, because the cdk5 gene promoter has an AP-1 site, apparently unnoticed until then, to which D FosB could bind. James A. Bibb, a research associate in the Rockefeller University lab of Paul Greengard, stopped by Chen's poster. Bibb was already exploring Cdk5's role in a signal transduction pathway that Greengard, a co-recipient of the 2000 Nobel Prize in physiology or medicine, had long studied. On seeing the poster, "I knew what I wanted to do," Bibb recalls. "Here's a case where we'd just found that Cdk5 regulated dopamine signaling, and this other group found that D FosB, which is upregulated by cocaine, could lead to changes in Cdk5. Chronic cocaine exposure was probably therefore going to change dopamine signaling by targeting Cdk5." The ensuing collaboration between the Greengard and Nestler labs benefited from the long-term bond between their two leaders. Greengard was Nestler's thesis adviser 20 years ago, and, says Nestler, "Paul is like a father to me." The two labs, joined by several others, made several important discoveries. In the first direct experimental link between cocaine and Cdk5, cocaine-injected rats had sharply higher Cdk5 mRNA and protein levels in their striata than did control animals. Cocaine also affected dopamine-stimulated signal transduction in ways that a Cdk5 upregulator would be expected to do.1 An experiment with roscovitine, a Cdk5 inhibitor, provided a possible clue to the effect of the surplus Cdk5. Rats on cocaine typically exhibit increased locomotion. When Jane R. Taylor, an associate psychiatry professor at Yale, infused roscovitine into the nucleus accumbens of cocaine-injected rats, the animals moved around even more than cocaine-injected rats infused with saline. If a Cdk5 inhibitor thus potentiated a cocaine effect, then Cdk5 might serve to dampen that effect. "If you're going to flood something with dopamine, if it's just going to pour rain, someone is going to get a raincoat," analogizes Bibb. "There's going to be a cellular response to try to keep things as normal as possible." But he acknowledges that Cdk5 is likely to act in far more complex ways than as a simple homeostatic regulator.3 DNA Arrays and Other Proteins Kent E. Vrana, an associate professor of physiology and pharmacology at Wake Forest University School of Medicine, his graduate student, Willard M. ("Bill") Freeman, and eight colleagues recently took a more global look at cocaine's intracellular effects. The group gave five macaques daily injections of cocaine for a year. (The researchers were able to use the expensive monkeys by sharing them with a group conducting another study.) Then, cDNAs copied from RNA in the nucleus accumbens were hybridized to a macroarray representing 588 human genes, which are very similar to monkey genes. In what Vrana describes as the first published study to use DNA arrays to chart cocaine's effects, his team found significant upregulation of four genes.4 These encoded the * catalytic subunit of protein kinase A (which is part of the signal transduction pathway studied by Greengard); protein tyrosine kinase 2; mitogen-activated protein kinase 1; and ß-catenin. Immunoblotting demonstrated that higher amounts of these proteins were also present. Vrana suspects that cocaine induced these changes long before the year was up, and he and Freeman have already performed time-course microarray studies on rats. "The key now is to go on to show functional implications," Vrana says. "That involves perturbing the activity of these enzymes and transcription factors or interfering with their synthesis through [gene] knockouts, for instance." Other groups are already exploring the functions of various proteins, the levels of which are known or suspected to be changed by cocaine use: Jay M. Baraban, a neuroscience professor at Johns Hopkins School of Medicine, specializes in the Early Growth Response (EGR) family of transcription factors, including zif268. In 1992, he reported that chronic cocaine administration suppresses mRNA levels of zif268.5 Now he is applying nerve growth factor to cultured cells to induce EGR proteins and thereby learn how they act and which genes they affect. Marc G. Caron, a cell biology professor at Duke University, studies G protein receptor kinases (GRKs). He hypothesizes that addiction might occur when overactive receptors can't turn off, a situation in which he thinks GRKs might play a role. Caron is also investigating PICK1, which helps the dopamine transporter traffic to the cell membrane.6 In response to cocaine, neurons might alter their production of PICK1. Scott A. Mackler, an assistant professor of medicine and pharmacology at the University of Pennsylvania, is looking at the transcription factor NAC-1.7 He says that cell-culture experiments have identified NAC-1 as a repressor. It increases in the first two months after cocaine exposure but only in the nucleus accumbens. Paul F. Worley, a professor of neuroscience and neurology at Johns Hopkins, examines two intracellular proteins upregulated by cocaine: Homer 1A helps control intracellular calcium, and Arc appears to be part of the glutamate-receptor signaling complex. Narp is another cocaine-responsive protein studied by Worley. Thought to be important in synaptogenesis and stabilizing active synapses, this secreted protein is suspected of helping intensify drug craving. Morphologies and Therapies Researchers acknowledge that cocaine-induced protein changes, even if they last for months, can't by themselves explain long-term addiction. The current theory is that "transient molecular adaptations can physically remodel the nervous system for a lifetime," explains NIMH's Hyman, citing as an example long-term memory mechanisms in the hippocampus. The question is then, Does cocaine use result in remodeling? Yes, according to Terry E. Robinson and Bryan Kolb, psychology professors at the University of Michigan and the University of Lethbridge in Alberta, Canada, respectively. They let rats self-administer cocaine for a month, waited a month, and then took a close look at the animals' brains. In March, they reported that the nucleus accumbens, which is thought to contribute to motivation, exhibited increased dendritic branching and dendritic-spine density.8 Strange-looking dendrites also appeared in the prefrontal cortex, which controls judgment. A more recent study found that these features were still present more than three months after cocaine use ended. Changes in the two brain regions "are pretty bad news for addicts," says Robinson. "You want the drug, and you lack the ability to judge the consequences of continued drug use." Ongoing experiments by the labs that collaborated on the March Nature paper indicate that Cdk5 plays a role in these morphological changes, Nestler disclosed two months ago at a Rockefeller University symposium on drug abuse and addiction. As for the therapeutical implications of this research, Nestler remarks, "All these proteins we're talking about, and a hundred others that regulate these proteins, each become a potential target for a new medication." The biological complexity of addiction doesn't preclude pharmaceutical intervention, he adds, noting that "depression is every bit as complicated as addiction, but many antidepressant drugs work really well." The National Institute on Drug Abuse is already conducting clinical trials of 60 compounds targeting cocaine abuse, according to director Alan I. Leshner. These compounds "work on a wide variety of targets, none of which are deep into the [signal transduction] cascade," he says. "They're either modifying various receptor functions or modulating neurotransmitter release." Leshner nevertheless describes signal transduction as an "accelerating area of interest" among drug-abuse researchers. Douglas Steinberg (dougste@attglobal.net) is a freelance writer in New York. References 1. J.A. Bibb et al., "Effects of chronic exposure to cocaine are regulated by the neuronal protein Cdk5," Nature, 410:376-80, March 15, 2001. 2. Chen, J. et al., "Chronic fos-related antigens: stable variants of DfosB induced in brain by chronic treatments," Journal of Neuroscience, 17:4933-41, 1997. 3. See also, A. Gupta, L.-H. Tsai, "A kinase to dampen the effects of cocaine?" Science, 292:236-7, April 13, 2001. 4. W.M. Freeman et al., "Chronic cocaine-mediated changes in non-human primate nucleus accumbens gene expression," Journal of Neurochemistry, 77:542-9, April 2001. 5. R.V. Bhat et al., "Chronic cocaine treatment suppresses basal expression of zif268 in rat forebrain: in situ hybridization studies," Journal of Pharmacology and Experimental Therapeutics, 263:343-9, 1992. 6. G.E. Torres et al., "Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1," Neuron, 30:121-34, April 2001. 7. S.A. Mackler et al., "NAC-1 is a brain POZ/BTB protein that can prevent cocaine-induced sensitization in the rat," Journal of Neuroscience, 20:6210-7, 2000. 8. T.E. Robinson et al., "Cocaine self-administration alters the morphology of dendrites and dendritic spines in the nucleus accumbens and neocortex," Synapse, 39:257-66, March 1, 2001.

Why Can't the Brain Shake Cocaine?

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