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As the Worm Turns: Discovering of a Life of Discovery Guy A. Caldwell, PhD1,2 1The University of Alabama; Department of Biological Sciences; Tuscaloosa, AL 2Washington and Lee University; Biology, ‘86
“Do or do not, there is no try” – Yoda
He turned to me and said, “You are.” So, there it was. As I sat in what most people would describe as a tiny, crammed, makeshift laboratory in a rooftop of an old science building - my life was defined. Such was the answer of my boss, my professor, my mentor, and my friend, Dr. John Knox, in response to my innocent query, “What is a Molecular Biologist?” on a typically quiet, yet very special summer day, in “Big Lex” in 1986.
Today, November 1, 2002....Tuscaloosa, Alabama......the boss, the professor, the mentor, and friend to his own students describes the structure of a gene, and with a giddy passionate fervor, pulls out of his pocket a token souvenir from an old experiment, a small vial with a faded label, and he proudly says, “was the first tube of DNA I ever saw!”. Like the double-helix itself, a legacy of another time, written in the code of codes...a memory of life, intertwined with a memory for a lifetime.
College is a time wherein small fleeting decisions, often made on the spur of a moment, can change one’s entire life. The decision to ask Dr. John Knox to be my faculty advisor was one of the best I have ever made. For some reason, still unbeknownst to me, Dr. Knox saw within me a greater potential than I did in myself after a couple of years of less than stellar performance at W&L. As Dr. Knox knew, like many W&L Biology majors, that my parents expected me to become an M.D. It was supposed to be a given, as the man I was named for, my grandfather - Dr. Guy A. Caldwell, was co-founder of the Ochsner Clinic in New Orleans and a world-famous orthopedic surgeon. While I performed well in all my biology classes, I found anatomy to be excruciatingly boring, I hated the dissection of animals, and I avoided physiology at all costs. I was clearly not being honest with myself (or my parents), as the things that define a medical education simply did not stimulate me.
In contrast, when I first heard the words “recombinant DNA” in a genetics lecture, my life forever changed. I instantly knew that molecular biology was to be my future, not to mention the future of nearly all of biology. Dr. Knox took me under his wing and gave me the opportunity to work with him in his research. He believed in me and that forced me to believe in myself. That first vial of DNA from his lab, perhaps one of the first ever used at W&L in a molecular biology experiment, proudly sits on a shelf in my office now, as a reflection of the excitement I found at that time in my life. It also serves to remind this professor the manner in which he was respected and nurtured by a true mentor that I can only hope to be to my own students someday. With Dr. Knox’s encouragement, I applied to graduate school at the University of Tennessee in Knoxville, another fleeting decision that paid off handsomely.
Toward the end of my senior year at W&L, Dr. Wielgus indicated to me that he had an opening in his research lab for the summer. It was there, as a Robert E. Lee Research Scholar at the top of Parmly Hall in the summer of 1986, that I found my “home”, my scientific home to be precise. It was a place where dreams became a reality; where I learned what it was to discover things that others will only memorize; where it was possible to become someone who will write those textbooks and not simply read them. To me, Knox's rooftop lab was the closest place to heaven on earth.
The first piece of useful data I generated has not revolutionized our view of biology as we know it. With the ever-wise guidance of Dr. Wielgus, I determined the isoelectric point (the optimal pH) of a protein important to the development of the tobacco horned worm, Manduca sexta. I can think of few prouder moments in my life, then when I learned this small contribution to the world of science was to be published and forever added to the knowledge base of humanity (Wielgus, 1990). I had discovered something. It wasn’t much, but it was something that no other person in the history of this planet had previous known. Both then and now, I can think no cooler way to spend one’s life.
Today, I get the thrill of reliving the emotional rush of discovery through the eyes and work of my own Ph.D., M.S., and undergraduate research students here at the lovely main campus of The University of Alabama. At the place where legendary football coach Paul “Bear” Bryant once led the Crimson Tide to countless victories, I too am the coach of my own team - researchers dedicated to triumph over disease. Moreover, I spend each and every day with the love of my life, my beautiful and talented Ph.D. collaborator, Dr. Kim Caldwell (Figure 1). Kim and I met in graduate school, where we had a dream of being a research team someday. As professors here at Alabama, we are dedicated to a life of shared discovery, a shared passion for making a difference with our research, and to helping the students we are so privileged to instruct find their own path in life.
Figure 1. Drs. Guy and Kim Caldwell at work in their lab in The University of Alabama.
Kim and I feel like we get paid to play. We have a lab full of expensive, amazing toys, wonderful students, and the freedom to pursue whatever our intellectual whims allow us, as long as we can convince someone to fund those whims! So far, so good. Our lab is funded to examine the molecular basis of human neurological diseases like Parkinson’s, Huntington’s, dystonia, and epilepsy. Despite a career journey that has taken me from Lexington to Knoxville, then New York City and now Alabama, I am back where I started...working with worms! I must admit that I have forsaken my original wormy critter in the Weilgus lab for yet another type of worm, the tiny microscopic nematode Caenorhabditis elegans. You are probably wondering what does a microscopic worm have to do with brain diseases? Well, I hope I can convince you that the answer is...quite a bit.
Whereas the human brain contains over 100 billion neurons, C. elegans has exactly 302. Moreover, the complete neuronal connectivity of this animal has been determined and diagrammed by electron microscopy. Despite its simplicity, this nematode shares many of the hallmarks of human neuronal function including ion channels, neurotransmitters (dopamine, serotonin, acetylcholine) and axon pathfinding cues, among other molecules (Bargmann, 1998). The C. elegans genome contains many predicted proteins that exhibit a high degree of similarity with gene products implicated in human diseases. In fact, approximately 50% of all genes for which a human genetic cause has been defined have a counterpart in C. elegans. This model system has been exploited to gain insights into sensory and neurological mechanisms underlying a variety of disorders and is at the forefront of finding molecular answers (therapeutics) to molecular questions (Culetto, 2000; Dawe, 2001). If I have not convinced you, then perhaps a particular group of folks in Stockholm can, as they awarded the 2002 Nobel Prize in Medicine to researchers who started the C. elegans field in recognition of the contributions this tiny worm has made to science.
Torsins share amino acid sequence similarity with a diverse family of proteins that includes heat-shock proteins, proteosome subunits, proteases, and the molecular motor protein, dynein. Aberrant protein folding is a recurrent theme in neurological disease, as is evidenced by the presence of misfolded protein inclusions in the brains of Alzheimer’s, Huntington’s, and Parkinson’s patients (Satyal, 2000). In this regard, mutations in neuroprotective molecules, such as molecular chaperones, that monitor protein folding, may also be responsible for the symptomatic features of dystonia. We hypothesized that torsins may function in a capacity similar to molecular chaperone proteins, in facilitating the proper cellular management of misfolded proteins. To experimentally test this hypothesis, we utilized a set of genetically-engineered worms designed to allow us to examine states of differential intracellular protein aggregation in living nematodes (Taylor, 2002).
I first learned to work with C. elegans while training as a postdoctoral research scientist at Columbia University. A postdoc is like a medical resident: long hours; low pay; little respect. In short, it was paying the dues to earn one’s own lab someday. It happened that the actual day I applied for the position at Columbia, the lab within which I was to work landed a paper on the cover Science magazine (Chalfie, 1994). This paper described the first use of gene from jellyfish called Green Fluorescent Protein (GFP), the one that causes them to glow, as a marker for where genes turn on and off in living animals. There are few things more amazing than watching a living animal, that one has personally genetically engineered to glow, swim around a simple agar Petri dish. Transgenic worms, who’d have thought it, eh?
Well, following my indentured postdoctoral servitude, I obtained my own laboratory at Alabama and had an idea about testing the function of torsin proteins. Students in my lab used modified forms of GFP to generate fusions of this inherently fluorescent protein with differing lengths of the amino acid glutamine (abbreviated, “Q”) to induce intracellular protein aggregation. Whereas a tract of 19 glutamines genetically fused to GFP (Q19::GFP) does not alter the normal diffuse fluorescence of this protein in cells, expansion of that glutamine tract to 82 residues (Q82::GFP) causes the GFP to misfold and form discrete punctate protein aggregates (Figure 2). This system allows us to identify factors that might suppress protein aggregation in living animals.
This was in fact the case, as co-expression of either a worm or human torsin gene in C. elegans cells that contain artificially induced protein aggregates of GFP (Q82::GFP) can suppress the formation of those inclusions (Figure 2). These data point to a role for torsin proteins in the management of protein folding in cells. Furthermore, we engineered the torsin gene to mimic the genetic deficit found in human patients who have dystonia (deletion of one amino acid at position 368 of the torsin protein). Expression of this mutated torsin gene product in worms containing polyglutamine-induced aggregates was incapable of suppressing protein aggregation (Figure 2). Therefore, we concluded that a natural activity of torsin proteins is to protect cells from misfolded proteins and that dystonia is perhaps a consequence of subtle changes in the molecular nature of torsin’s targets. This represents the first evidence of a function for this medically significant family of proteins and points to their potential use as a therapeutic for prevention of protein misfolding in a variety of disorders (U.S. Patent Pending, Caldwell, 2002; Caldwell, in press).
Huntington’s chorea is associated with expansion of the number of glutamine residues found in a specific gene in patients suffering from this disease. While healthy humans have about 17 repeats of glutamines in this gene, patients with Huntington’s have over 35 or more. In fact, as more of this molecular stuttering occurs in the genetic code of our cells, the sooner the time we will succumb to the disease (Ridley, 2000). Likewise, a cellular consequence of Parkinson’s disease is the formation of clumps of a protein called alpha-synuclein that form in the brains of patients. Alzheimer’s is associated with aggregated fibrils of beta-amyloid proteins. For other diseases, there are other aggregates (Taylor, 2002).
The equation is simple: misfolded protein = aggregates = disease; prevent misfolding, cure disease. Well, at least that is what we hope. There is much work to be done, but identifying molecules like torsins that can suppress formation of protein aggregates is a big start.
To
allow for my indulgent reminiscences, I will refrain from discussion of
ongoing work my laboratory has performed into the molecular basis of
childhood epilepsy. However, if you have the sudden uncontrollable urge
to visualize worms undergoing seizures, please visit our website (http://www.bama.ua.edu/~gcaldwel)
and know that the same gene in humans causes epilepsy when defective. The
stamp of evolution is all over our DNA, we just need to be smart enough to
decipher it.
Finally, during my junior year at W&L in 1985, I had the idea for a forum wherein undergraduate students and faculty could express their opinions on issues current to the scientific world, in addition to obtaining the experience of writing and publishing their original research. It is with sincere pride that, 17 years later, I am still able to contribute my research and ideas to the outstanding periodical that The Washington and Lee Journal of Science has become. I can proudly state that this month one of my own undergraduates will serve as the founder and editor of JOSHUA, The Journal of Health and Science of The University of Alabama. I wish to dedicate this story to all my fellow editors, contributors, and scientists from W&L, as well as my current students. It is my hope that just as Yoda would want - you have not simply tried, but you have done.
Works Cited
Bargmann CI. (1998) Neurobiology of the Caenorhabditis elegans Genome. Science 282:2028-2033.
Caldwell GA. and Caldwell KA. (2002) “Nucleotide Sequences that Code for Torsin Genes, Torsin Proteins, and Methods of Using the Same to Treat Protein-Aggregation” Patent Pending. U.S. Patent and Trademark Office.
Caldwell GA, Cao S, Sexton EG, Gelwix CC, Bevel JP and Caldwell KA. Suppression of Polyglutamine-induced Protein Aggregation by Torsin Proteins in C. elegans. Human Molecular Genetics, accepted for publication.
Chalfie M , Tu, Y, Euskirchen G, Ward WW, Prasher DC. (1994) Green fluorescent protein as a marker for gene expression. Science 263:802-5.
Culetto E, Sattelle DB. (2000) A role for Caenorhabditis elegans in understanding the function and interactions of human disease genes. Human Molecular Genetics 9:869-877.
Dawe AL, Caldwell KA, Harris PM, Morris NR, and Caldwell GA (2001) Evolutionarily Conserved Nuclear Migration Genes Required for Early Embryonic Development in Caenorhabditis elegans. Development, Genes, and Evolution 211:434-441.
Fahn S, Bressman SB, Marsden CD. (1998) Classification of dystonia. In Fahn S, Marsden CD, DeLong M, eds. Dystonia 3: Advances in Neurology. Philadelphia: Lippincott-Raven Publishers, pp. 1-10.
Nietzsche, Friedrich (1844-1900) Also sprach Zarathustra.
Ozelius LJ, Hewett JW, Page CE, et al. (1997) The early-onset torsion dystonia gene (DYT1) encodes an ATP-binding protein. Nature Genet 17:40-48.
Ridley, M. (2000) Genome. New York: HarperCollins, pp. 54-56.
Satyal S, Schmidt E, Kitagaya K, Sondheimer N, Lindquist ST, Kramer J, Morimoto R. (2000) Polyglutamine aggregates alter protein folding homeostasis in Caenorhabditis elegans. Proceedings of the National Academy of Science USA 97:5750-5755.
Taylor JP, Hardy J, Fischbeck, KH. (2002) Toxic proteins in neurodegenerative disease. Science 296:1991-1995.
Wielgus JJ, Caldwell GA, Nichols L and White CF. (1990) "The purification, characterization and fate of a Haemolymph Trophic Factor from the last larval instar of Manduca sexta. Insect Biochemistry 20:65-72.
About the Author… Dr. Guy A. Caldwell, a native of the New York City area, received his B.S. in Biology from Washington & Lee University in 1986. He was the founder and first editor of The Washington and Lee Journal of Science. He received his Ph.D. in Cell, Molecular, and Developmental Biology at the University of Tennessee in 1993. Following receipt of his doctorate, he moved to Columbia University in New York, where he was a recipient of fellowships from the National Institute of Neurological Disease and Stroke. He is currently an Assistant Professor in the Department of Biological Sciences at the University of Alabama, where he holds an undergraduate research appointment from the Howard Hughes Medical Institute. In 2001, he was named a Basil O'Connor Scholar of The March of Dimes Birth Defects Foundation for his research into the molecular basis of childhood birth defects of the brain. Previous recipients of this award include Dr. Francis Collins, the Director of the Human Genome Project, and Nobel laureate Dr. Joseph Goldstein (W&L ’62). Dr. Caldwell is the author of two editions of a widely adopted textbook in biotechnology, sold world-wide in 3 languages by Harcourt. His laboratory is funded by grants from The March of Dimes, Dystonia Medical Research Foundation, American Parkinson’s Disease Association, Parkinson's Disease Foundation and National Parkinson Foundation. He teaches courses in Molecular Genomics, General Biology, Signal Transduction in Neurobiology, and a seminar on bioethical issues surrounding the Human Genome Project. Dr. Caldwell welcomes inquiries from W&L students for summer research opportunities and graduate school. gcaldwel@bama.ua.edu; www.bama.ua.edu/~gcaldwel
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As a molecular biologist, it is my choice to apply the
tools of genetics, genomics, and recombinant DNA technology toward solving
problems pertaining to biomedical research. Dystonia is one such problem,
especially for the unfortunate ~400,000 people in North America suffering
from variations of this neurological disease. Our lab is studying the
most severe early-onset form called Oppenheim’s dystonia, 