The Laboratory of Neurochemistry, now jointly led by Drs. Paul Whiteaker and Ronald J. Lukas, focuses on nicotinic acetylcholine receptors, which are critical to chemical signaling and electrical wiring throughout the brain and body. Nicotinic receptors normally respond to the natural chemical signaling agent acetylcholine, which is released from activated nerve (or other) cells. Nicotinic receptors also are targets of tobacco-based nicotine and are relevant to tobacco-related diseases and drug abuse.
They exist as a family of subtypes, each with unique properties and distributions, but all responding to nicotine and acetylcholine. These receptors are involved in mood and emotion, attention and cognition, autonomic homeostasis, and movements. Because they play such broad roles, it is not surprising that nicotinic receptor actions and deficits have been associated with a variety of neurological, psychiatric, and many other conditions affecting hundreds of millions of Americans (see Chart 1).
Science of Medicine, Spirit of Discovery
Turning back again to fundamental laboratory science, there are many lines of investigation in the laboratory. First and foremost, because there is not just one type of nicotinic acetylcholine receptor, fundamental work involves identification and classification of the diverse family of nicotinic receptors, each of which is defined by the building blocks or subunits that constitute them. These studies identify subunits and receptor subtypes in different tissues and organ systems, exploit tumor cell lines as factories naturally making nicotinic receptors like those found in normal tissue, or involve creation of genetically engineered cell lines fashioned to examine features of nicotinic receptors suspected or known to exist. Research tools are critical to advances, and we have created or exploited many, including natural products from frogs, snakes, harpoon-wielding sea snails, or mushrooms, and their synthetic analogues.
Molecular and cellular biology, immunology, protein chemistry, pharmacology, and electrophysiology converge in these studies to provide the most comprehensive description of nicotinic receptors under study that is possible. For example, identification of differences in the ability of different nicotinic receptor subtypes to interact with specific drugs is used not only to discriminate receptor subtypes, but also to discover new drugs that are selective or specific in their preference for a given receptor subtype. This creates the opportunity to find a nicotine-like drug that could elevate mood by acting at one receptor subtype without causing nicotine dependence through interactions with receptors in the pleasure-reward center of the brain.
Work is continuing to define which of the 17 subunits identified to date combine to make unique receptor subtypes, and features of every subtype identified then need to be characterized. Studies of the effects of chronic nicotine exposure on receptor numbers and function are being done to define how the brain changes in response. Fundamental features of nicotinic receptors in pleasure-reward, emotional, and attention centers are being studied, as well as in neurological and other medical conditions.
Studies extend beyond muscle and nerve cells. For example, there is evidence that nicotinic receptors are in blood vessels in many organs including the brain, where they contribute to formation of the blood-brain barrier and influence cytotoxic and vasogenic phases of edema during stroke. Other studies concern roles nicotinic receptors play in nervous system/immune system interactions and in lung or lung tumor growth. Bone formation and reproductive organ function also seem to be influenced by nicotine exposure, implying that nicotinic receptors are found on the relevant cell types.
Nicotinic receptors also can be used as models to test new techniques and to push back biotechnological horizons. For example, we are evaluating nicotinic receptors as models for the development of sophisticated tools for proteomics research. Genetically engineered cells and site-directed mutagenesis studies are being used not only to define structure-function relationships for the many interesting domains in nicotinic receptors, but also as models of genetically based neurological diseases and for studies to define the functional consequences of such mutations.
Our work has revealed nicotinic receptor subunit gene polymorphisms, which may prove to be indicators—assessable through gene array techniques—of susceptibility to neurological or psychiatric disease or to nicotine dependence and likelihood of success in smoking cessation therapy. Conversely, other studies have revealed changes in gene activity induced by nicotine exposure, possibly revealing how some effects of nicotine exposure can be long-lasting but also revealing important targets for normal signaling through nicotinic receptors.
Naturally, many factors can influence nicotinic receptor levels and function, from the cytoskeleton and extracellular matrix, gene expression regulators, other types of receptors engaging in cross-talk, and cytoplasmic entities and the signaling cascades they trigger. Reciprocally, nicotinic receptor activity can affect all of those. Hormones and small peptides are among the other agents that have such effects. Some of our most exciting work concerns another large family of molecules initially revealed by studies of the immune system and that resemble in some ways natural product toxins that target nicotinic receptors. These molecules can have a wide range of effects, and combinations of them and receptor subtypes challenge us with a huge matrix of possible interactions of biological relevance. Indications are that genetic variation in some of these molecules can predispose individuals to disorders such as anxiety and can affect how the maturing brain is constructed.
We take great pride in our studies and contributions we have made. We are even more excited when we make new discoveries that overturn accepted science and dogma, even if we had a hand in formulating those initial but still-immature perspectives and interpretations. We invite inquiries about this, but now we are creeping deeply into esoteric matters. For example, it once was thought that one receptor subtype that is a major target of nicotine action at human smoker levels had one structure. We and others then did studies showing that that subtype actually existed in two “isoforms” that differed in the ratio of the two building blocks constituting the subtype. These isoforms responded differently to markedly different levels of acetylcholine or nicotine. We then developed tools to modify the receptors very subtly but by keeping control of ratios and arrangements of the building blocks. That led us to discover critical differences between interfaces between building blocks that influenced sensitivity to acetylcholine and nicotine. Further studies, even more esoteric, showed that activity dictated by what building blocks made up interfaces is further influenced by the next-door building blocks!
Picture this – nicotinic acetylcholine receptors at the atomic level are in constant motion, wiggling like leaves in the wind. Each leaf has a little different structure and wiggles differently in the same wind. These are the receptor subtypes. If a bug lands on a leaf, the leaf’s response to the wind changes, just as the receptor’s does when it encounters nicotine or acetylcholine. That is how delicate these entities are, and the likelihood that they are in the closed or open channel state is influenced by interactions with acetylcholine or nicotine. Just as a dry leaf or a water-soaked one will behave differently in the wind, other elements in and around cells where nicotinic receptors are found can loosen or restrict these movements. Just as an infestation can decimate a tree of its leaves, or hearty winds shaking the leaves can cause branches to crash to the earth, harmful processes can lead to loss of receptors, and receptor hyperactivity can be harmful to the cells that make them, in either case, perhaps setting disease wheels in motion.
- Demonstrating recognition for our work nationally and internationally, past or current funded studies or studies leading to published contributions involve dozens of collaborating scientists at the University of Arizona, Arizona State University, Banner Sun Health Research Institute, several biotechnology companies in the state, nationally, or internationally, and at many institutions in the country and worldwide. Co-workers are in Arizona cities including Phoenix, Tempe, Tucson, Scottsdale, and Sun City; other U.S. cities including Baltimore, Bethesda, Boston, Denver, Durham, Gainesville, Houston, Ithaca, Kalamazoo, Lubbock, Los Angeles, Memphis, Palo Alto, Philadelphia, Raleigh, Richmond, Salt Lake City, San Diego, San Juan, St. Louis, Tampa, and Winston-Salem; and non-US cities including Bahia Blanca, Bath, Edinburgh, Edmonton, Geneva, Heidelberg, Montpellier, Oxford, and Paris.
- Visiting foreign national scientists, faculty doing sabbatical work, and educators doing summer research projects have been active in our laboratory.
- Contributed to demonstration of nicotinic acetylcholine receptor diversity
- Identified or created natural or genetically engineered cell models for nicotinic receptor research
- Contributed to basic pharmacological and structural characterization of nicotinic receptors
- Definition, at the molecular level, of effects that occur with smoking and of chronic nicotine exposure on nicotinic receptor function
- Insights into medical conditions as diverse as Alzheimer’s, epilepsy, and multiple sclerosis
Nicotinic Acetylcholine Receptors in Health and Disease
Our studies have involved basic scientific research about structure and function of nicotinic acetylcholine receptors, which is essential if we are to know how they are involved in health and disease and how they might be targeted therapeutically. Much of that work has involved studies of drugs that affect nicotinic receptors in various ways and that are leads for therapeutic intervention. Because subtle changes in nervous system or receptor properties can have profound behavioral and health or disease impact, some of our work also illuminates how numbers and function of receptors are regulated normally or in disease conditions.
Our work has contributed to the realization not only that there are a number of nicotinic receptor subtypes, which can be selectively targeted, but also a multitude of other biological entities that can have similar influences on receptor activity.
As just a few examples of the clinical relevance of these nicotinic receptors, their numbers are decreased in Parkinson’s and Alzheimer’s diseases, and we have mechanistic evidence for their involvement early and perhaps causally in these neurodegenerative disorders. This opens possibilities of targeting nicotinic receptors for drug therapies to slow or stop disease progression, especially given promising innovations that allow disease detection in the decades before memory loss or movement disorders become clinically evident rather than too late in disease progression for useful intervention.
Nicotinic receptors are involved in neuromuscular disorders, having been identified as targets for autoimmune responses or gene mutations causing myasthenia gravis and located in the dystrophin-related complex targeted in muscular dystrophy. Recent indications that mutations in nicotinic receptor building blocks rival those of other mutations implicated in amyotropic lateral sclerosis, Lou Gehrig’s disease.
Whether in these disorders or other neurodegenerative conditions such as Alzheimer’s disease, nicotinic receptor involvement in neurodegeneration may mimic developmentally relevant “programmed neuronal death” that occurs naturally to control, for example, numbers of motor neurons. Common to these phenomena is natural or aberrant overactivity in nicotinic receptors, which, when created in mutant mice or in human neuronal cell cultures, is lethal to neurons.
Mutations in nicotinic receptors are associated with certain inherited forms of epilepsy. We have identified reasons why these mutations might cause the excessive electrical activity seen in epilepsy and other seizure disorders. Nicotinic acetylcholine receptors act as ion channels, embedded in the neuronal cell exterior membrane surface. Electrical activity of these receptors occurs when they interact with acetylcholine, causing opening of the ion channel that is closed at rest. If channels stay open too much or for too long, electrical activity can become abnormally excessive and harmful in nerve cells.
We have shown that some epilepsy-related mutations in nicotinic receptors cause—even when measuring openings of single channels—prolongation of channel open times or open probabilities.
Our work has helped to show that nicotinic receptors are expressed widely throughout the body, with some receptors subtypes being richly expressed in immune system cells. The immune system evolved much earlier than did nervous systems, and so it should come as no surprise that the phylogenetically most-ancient nicotinic receptor family members are not in the healthy brain at all but are in the immune system.
In our studies of mouse models of some forms of multiple sclerosis, we have found that disease onset and severity involves harmful activity of one of these receptor subtypes. We think that we can target that family member to tamp down inflammatory and autoimmune responses that cause multiple sclerosis or other disorders. Another nicotinic receptor subtype seems to play roles protecting the brain and spinal cord in the multiple sclerosis model, offering another potential therapeutic target. Perhaps most exciting is our finding that there is no recovery from disease when another nicotinic receptor subtype is absent.
Diseases such as multiple sclerosis progress even if treatment or natural phenomena lower brain inflammation because of the loss of myelin, the material that creates “insulation” of neuronal “wires,” leading to short-circuiting in the disease. Myelin loss occurs because it and the specialized cells that make myelin are attacked by a misguided immune system. Recovery in multiple sclerosis requires restoration of those specialized cells so that new myelin can be made again. No cure for multiple sclerosis can occur unless there are these restorative processes. We think that nicotinic receptors are involved in replenishment of specialized myelin-producing cells, that their loss accounts for lack of recovery, and that we can target them therapeutically to effect useful treatment and perhaps recovery in multiple sclerosis.
Collaborations with Barrow investigators have allowed us to inform how inflammatory processes and nicotinic receptors are involved in hemorrhagic or ischemic strokes and in vascular malformations, sometimes involving nicotinic receptors on vascular complexes but also nicotinic receptors in the immune system.
Metastatic brain tumors occur more frequently than tumors that start in the brain, but some intriguing recent findings indicate that some nicotinic receptor building blocks not found in the healthy brain appear at high levels in certain forms of brain-specific tumors. At a minimum, this affords possible use of those receptors as biomarkers indicating the presence of tumor and responses to therapy, but these observations suggest that much more work is needed to understand why these changes occur and what they mean for tumor development.
Repeating a theme mentioned above, roles of immune system conversation with the brain in development or treatment of brain tumors, and actions of nicotinic receptor signaling across the immune and nervous systems in that conversation, might afford therapeutic options.
Affecting 10 times more individuals in the United States than those afflicted, for example, with Alzheimer’s disease (see Chart 1), is nicotine dependence. Nicotine dependence is the ultimate cause of all tobacco-related diseases. Our work has helped to advance hypotheses that tobacco use represents a form of nicotine self-medication, which develops in some individuals to correct chemical and electrical signaling deficits associated with emotional difficulties, cognitive difficulties, or both.
Indeed, individuals with mental health problems, including depression, anxiety, and attention deficit disorder, are at higher risk of developing nicotine dependence. This has been underscored by studies showing that children having those issues have such elevated risk, not that use of tobacco products causes those issues, as was thought to be the case 70 years ago. About 40% of the mentally ill, including as many as 90% of schizophrenics, are smokers.
More recently, individual genetic variations in some receptors have been associated with heightened susceptibility to nicotine dependence and to lung cancer. Our work has helped to inspire clinicians to provide true “nicotine replacement” in smoking cessation programs and to recognize that they need to identify and treat not just nicotine dependence, but also the underlying neuropsychiatric conditions that lead to nicotine self-medication, if we are to end use of tobacco products. Note that roles for nicotine itself, aside from dependence on it, in tobacco-related diseases remains unclear. This is in sharp contrast the clearly harmful effects of many of the other estimated 4,000 compounds in tobacco, which nearly double when tobacco is combusted, including known carcinogens, let alone inhalation of carbon monoxide. Serious and rational scientific evidence also is required to ensure that alternative products for nicotine delivery also are not harmful, as revealed by the observation that other constituents in nicotine vaping devices are culprits causing deadly respiratory consequences.
Our studies have helped to identify what we call “a neurochemical endpoint” for nicotine dependence. It is a reduction in function of certain receptor subtypes upon sustained exposure to human smoker levels of nicotine. Our work also has shown that useful aids to smoking cessation also have the same effect, taking the brain to the same neurochemical endpoint. Those observations have allowed us to help identify, sometimes in work supported via National Drug Discovery and Development Cooperative Group awards or industry collaborations, novel drugs that have superior preclinical features than smoking cessation products in the marketplace. Not by coincidence, we also have found that some drugs that are antidepressants target nicotinic receptors, and we have identified new compounds designed to act at nicotinic receptors that are superior in preclinical studies to antidepressants in the marketplace.
Drug codependence also is evident, in that upwards of 90% of alcoholics smoke. Nicotinic receptors are targets of much pharmaceutical research, in part because nicotine itself has the ability to improve attention and cognition in Parkinson’s, Alzheimer’s, or attention deficit disorder subjects; improve mood in depressed individuals; and reduce the frequency of ticks in Tourette’s patients. However, there are times in life, especially in perinatal periods and during adolescence and young adulthood, when abnormal signaling through nicotinic receptors mediated by nicotine can have harmful influences on many of the body’s organ systems.
Nicotinic acetylcholine receptors also have been implicated in a host of other medical conditions (see Chart 1). Already mentioned are anxiety, depression, alcoholism, and less common disorders such as Alzheimer’s disease, epilepsy, stroke, etc. But arthritis (afflicting over 100 million Americans), nearly as prevalent obesity, sleep and GI disorders, lung disorders, and chronic pain (nicotine was employed since ancient times as a potent analgesic) are even more common than nicotine dependence (and likely overlap with it).
Diabetes and even tinnitus appear to involve nicotinic receptors, pathogenically and perhaps as therapeutic targets, perhaps in part because nicotinic receptors play important roles in the birth of neurons, in their ability to form synaptic connections between nerve cells and nerve or other cell targets in virtually all organ systems, and in their roles in continued maintenance of those connections.