The Beginning of an Odyssey

Transcription

1 2 The Beginning of an Odyssey fter three years at the NIH, it was time to move on. I felt ready to build my own laboratory, but I somehow managed to grow up and take what in retrospect was a remarkably mature approach to gaining the education that I would need for studying the brain. I chose to undertake an abbreviated residency in neurology before setting up a laboratory. Most amazing, I still didn t have a clue about what I wanted to study. My goal was to identify, during my residency, a great problem for investigation. In early September 1972, I was performing a lumbar puncture (or spinal tap, as it is often called) when the hospital page operator summoned me: Dr. Prusiner, Dr. Stanley Prusiner... As soon as I finished the procedure, I called in and learned I had been assigned a patient on Dr. Donald Macrae s ser vice, admitted for evaluation of a rapidly progressive dementia. When I entered her room, my new patient, dressed in a print hospital gown, was sitting on the side of the bed. She was well- tanned, slim, with straight, graying brown hair cut just above the shoulders and deep blue eyes that glowed with enthusiasm. She seemed calm and somewhat detached. This beautiful sixty- year- old woman from Marin County, north of the city, was having difficulty with her memory and the fine movements of her fingers. She had problems finding words and was unable to describe the symptoms that had brought her to the hospital. Her husband recounted the initial difficulties that had alerted them, such as her 16

2 inability to insert her car key into the ignition or to unzip the ball compartment on her golf bag. During my mental- status examination, I found that her ability to remember recent events was poor; according to her husband, these memory deficits were new. While I was testing her gait, muscle strength, sensory modalities, and reflexes, she exhibited myoclonus jerky movements in her muscles. I had no idea what had caused her to worsen so quickly. Progressive dementia which involves a loss of intellect as well as memory combined with myoclonus prompts consideration of Creutzfeldt- Jakob disease (CJD). When the se nior resident proposed this diagnosis, I vaguely remembered an affliction I had read about in medical school called Jakob- Creutzfeldt disease, but this was my first year of residency, and I had had no clinical experience with it. (I later learned that the names were transposed, for the most idiotic of reasons, in the short interval between my medical school studies and my residency.) CJD is invariably fatal. It is one of a cluster of human and animal diseases now called prion diseases, the most familiar of which is bovine spongiform encephalopathy (BSE), or mad cow disease. Another is scrapie, which affects sheep and goats (the name arose from the tendency of infected animals to scrape off their wool or hair in an attempt to relieve severe itching). Typically, the brains of CJD patients, like those of livestock afflicted with scrapie or BSE, contain numerous holes ( vacuoles ), which give the brain a spongy ( spongiform ) appearance under the microscope (figure 4). Sometimes these illnesses are referred to as the transmissible spongiform encephalopathies, or TSEs. The etiology of CJD exactly what was transmitted from one patient to another was unknown, but a slow virus, one that causes disease only after many months or even years of incubation, was suspected. C. Joseph Gibbs, D. Carleton Gajdusek, and their colleagues at the NIH had shown the disease to be transmissible by injecting brain extract from a dead CJD patient into a chimpanzee, which developed symptoms of CJD thirteen months later. 1 These same researchers had also experimentally transmitted another TSE, known as kuru, a fatal neurological disease found in the Fore people of the Eastern Highlands of New Guinea, to chimpanzees. 2 Like CJD, kuru was thought to be caused by a slow virus. To a busy first- year neurology resident, the viral explanation seemed adequate. But as I watched my patient deteriorate rapidly over the next The Beginning of an Odyssey 17

3 Figure 4 Brains of CJD patients, like those of livestock afflicted with scrapie or BSE, contain numerous holes ( vacuoles ), which give the brain a spongy ( spongiform ) appearance in the microscope. few weeks, I was perplexed. She exhibited no signs of an infectious disease: Her neck was supple, not stiff; she did not have a fever; and she had no increase in white blood cells in either her blood or her cerebrospinal fluid. Besides being seen by Macrae, an outstanding clinical neurologist and celebrated teacher, my patient was also examined by Robert Fishman, chairman of UCSF s Neurology Department (figure 5). Both agreed that she had CJD and was likely to die within a few months. When Fishman, a superb clinician and teacher but with no par ticu lar expertise in the dementias, CJD, or viral diseases, rotated off the ser vice, another senior neurologist, J. Richard Baringer, became the attending physician. Baringer was interested in viral infections of the brain; his research was focused on the persistence of herpes viruses in the ner vous system, and slow-acting viruses were thought to cause herpes infections as well as 18 The Beginning of an Odyssey

4 Figure 5 More teachers and mentors. From left: Robert Fishman, Ivan Diamond (right of unidentified woman), Donald Macrae. CJD. Our conversations about CJD grew provocative, and he referred me to papers by the radiation biologist Tikvah Alper, who had described the strange properties of the scrapie agent infecting sheep and goats, which was thought to be similar to the slow virus causing CJD. Alper, who trained in Berlin with the nuclear physicist Lise Meitner in the 1930s, was an outspoken, feisty woman who grew up in South Africa, where her Russian- Jewish parents had immigrated. She moved to London in 1951, her forthright opposition to apartheid probably prompting the South African government to facilitate her exit. There she became director of the Medical Research Council s Experimental Radiobiology Unit at the Hammersmith Hospital in 1962 and continued in that position until her retirement in It was during this period that she published her seminal work on the scrapie agent (figure 6). From her studies on the extreme re sis tance of the agent to inactivation by ultraviolet light and X-rays, Alper reasoned that it could not be a virus, since the high doses of ultraviolet light and X-rays would have The Beginning of an Odyssey 19

5 Figure 6 Tikvah Alper used ultraviolet light of different wavelengths to analyze the scrapie agent. killed any virus by damaging its nucleic acid genome. 3 I concluded that Alper was unlikely to be correct; after all, every known infectious pathogen, viruses included, had a nucleic acid core that directed the synthesis of its progeny. How else could a pathogen flourish? Either DNA or RNA had to be involved, since they are the ge ne tic materials of life. I thought a novel virus was the most reasonable explanation for Alper s results. What else could the scrapie agent be? There was nothing else. My patient s hospitalization lasted almost three weeks, so I was able to watch her neurological status decline daily. Eventually she could not walk even with assistance. She left the hospital for a nursing home but returned four weeks later, unable to sit up, feed herself, or speak she had become a neurological vegetable. The beautiful woman who had entered the hospital less than two months earlier was now mute and all but immobile, with no understanding of who she was or what had happened to her. I felt profoundly sorry for her and for her husband, who could only stand by and watch his wife being destroyed by a slow virus. Baringer and I were helpless there was not a single medicine that could modify her outcome. Three weeks later she died. At autopsy, Baringer removed her brain and, after examining small pieces of it under the microscope, confirmed 20 The Beginning of an Odyssey

6 the diagnosis of CJD, although the characteristic spongiform change was minimal. He sent some of her frozen brain tissue to Joseph Gibbs at the NIH, where it was homogenized and injected into the brain of a monkey that, sure enough, developed the disease two years later. Over the next eight months, I read everything I could find about scrapie and CJD. I reread Alper s work several times, but it didn t clarify the question of etiology. If anything, I became more confused and simultaneously more enthralled. The problem was beginning to capture my imagination. The agents causing CJD in people and scrapie in sheep and goats were mysterious. They resisted killing by boiling or exposure to chemicals such as alcohols and formaldehyde, the principal ingredient of formalin. Formalin is a common fixative, used to harden tissues before cutting sections for microscopic examination. It is also used to inactivate viruses during the preparation of vaccines, such as (for example) the Salk polio vaccine; it renders the poliovirus harmless but allows the vaccinated individual to produce antibodies to the virus. In the 1940s thousands of sheep in Scotland were vaccinated with formalin- treated louping- ill virus (LIV), which causes meningoencephalomyelitis in livestock only to develop scrapie a couple of years later. The crude preparations of LIV had been produced from a sheep that also harbored the scrapie agent. Though formalin had inactivated the LIV, it had not killed all of the scrapie agent. In contrast to viruses that could be readily identified in the electron microscope, the scrapie and CJD agents remained elusive. The medical literature was replete with unsubstantiated claims and counterclaims about electron- microscopic sightings. Some investigators argued, like Alper, that the scrapie agent was devoid of nucleic acid and therefore could not be a virus. Others thought that her findings were fallacious and that the scrapie agent s re sis tance to destruction by ultraviolet light and X-rays could be explained by some undiscovered viral attribute that the scrapie agent was an unusual virus, but a virus nonetheless. What ever the explanation, the scrapie agent was unlike any other infectious pathogen. I was baffled, my curiosity thoroughly aroused. Whenever others argued that the mysterious nature of the agent made it too difficult to study, I found myself more and more attracted to the challenge. What causes scrapie? I could not let it go. By the end of my first year of residency, I was obsessed by the inexplicable. As I discussed with The Beginning of an Odyssey 21

7 colleagues the evidence for and against the hypothesis that a slow virus causes CJD, I grew convinced that a biochemical research program devoted to isolating and characterizing the scrapie agent might be a way forward. Unlike most of my colleagues, who thought my desire to study scrapie was misguided, the eminent Stanford ge ne ticist Charles Yanofsky was enthusiastic (see figure 3). Charley and I had first met at a dinner he and his wife gave at their home in Palo Alto for some friends we had in common. A tall, handsome man with extraordinary intellect, he had a somewhat halting gait. His feet had been frostbitten when he was a U.S. infantryman at the Battle of the Bulge; the frostbite had undoubtedly saved his life, since it prevented him from participating in further combat. Charley understood my vision, felt my excitement, and thought I should get started. When we met again, in my last year of residency, he was disappointed that I hadn t yet begun experiments to determine what causes scrapie. I m still taking care of patients, I explained. I won t have time to begin those studies until next summer, when I finish. You need to get going, he roared. You re wasting time! It was easy for Charley to understand what I wanted to do. As the discoverer of colinearity, the direct relationship between the structure of genes and that of their encoded proteins, he was a legend in molecular biology. I, too, wanted to wake up in the morning excited by unknowns in my research. I wanted to go to bed at night energized by the thought of making a breakthrough that would improve people s lives. I wanted to experience again the excitement I had felt in medical school in my experiments with brown fat metabolism. Based on that work, I was sure I could construct another important set of investigations. Identification of the scrapie agent was a problem that just might be worth committing all my intellectual energy to. The clues from Tikvah Alper s work that the scrapie agent was different from all other infectious pathogens were tantalizing. Many scientists likened the scrapie agent to kryptonite. 4 I couldn t imagine a better problem to tackle. It had all the hallmarks of a new frontier in neurologic medicine a truly great scientific puzzle that cried out for solution. Moreover, deciphering the riddle of What causes scrapie? might well lead to an understanding of CJD. By now, the triad of scrapie, kuru, and CJD had been well established by transmission and neuropathologi- 22 The Beginning of an Odyssey

8 cal studies. Defining the nature of the infectious agents causing these diseases was clearly the next important step. Scrapie was no longer the exclusive province of veterinarians; it had been catapulted to the center of human medicine by the transmission of kuru and CJD to apes and monkeys. Every scientist must choose a problem; it is probably the single most important decision of his or her professional life. Yet few seem to do so with much forethought. Most choose a problem related in some way to their training. The argument for this is that granting agencies will not support you unless you have already worked in the chosen area. And why not work for an expert before setting out on your own? At first glance, it seems a reasonable way to proceed. Apprenticeships have been around for centuries; they work well in training doctors, lawyers, engineers, and other professionals but they may not be the best formula for scientists or other creative people engaged in forging new endeavors. Before beginning my neurology residency, I had decided for many reasons not to do any research during that time. First, no problem had strongly captivated me; second, any problem worth investigating would need my full- time attention; third, I was in no par tic u lar hurry to produce another paper, since I had already written more than two dozen and published most of them in reputable journals. Some of my UCSF colleagues thought my focus on scrapie was a passing fancy and that a more promising career could be found in studies of glutamate metabolism of the nervous system. Glutamate is the most abundant amino acid in the brain, I was a neurologist, and I had had considerable training in the study of enzymes that convert glutamate into glutamine and vice versa. But I could not be dissuaded. Evidently I believed that the scrapie problem was relatively easy before I started my investigations. Neurology chairman Bob Fishman s recollections provide an interesting assessment: He was in the residency finishing up, and I said, Stan, what do you want to do? He said, Well, you know, I spent a lot of time doing protein chemistry with Stadtman. To paraphrase what he said, in so many words, he wanted to work on the scrapie agent, because, number one, he thought it created these interesting degenerative diseases and, number two and I ve tweaked him about this he said, It The Beginning of an Odyssey 23

9 ought to be an easy problem to solve. It s just a problem in protein chemistry. 5 Looking back at this turning point in my career, I m baffled by my determination on the one hand and rather impressed on the other. I was sufficiently insecure that I hungered for success; I was motivated, ambitious, driven. I had grown up in a family with very little financial means or social status, so what ever I wanted from life I had to make happen. My parents abundant unconditional love was surely instrumental in creating a personality that did not buckle under criticism which, as it turned out, would come from many corners as the astonishing properties of the prion were discovered. Interlude: Viruses, Genes, and Proteins Viruses are tiny infectious agents that can be seen only in an electron microscope. Viruses are one- tenth to one-one-hundredth the size of bacterial cells such as Staph and Strep, both of which can be seen in the light microscope. Every virus contains an inner core of genes. A gene is the smallest unit of ge ne tic material most genes encode proteins, which are the action molecules of life. Genes are composed of the nucleic acid DNA, except for the genes of some viruses. Such viruses possess RNA genes instead of DNA; both polio and the AIDS virus HIV are RNA viruses, while herpes is a DNA virus. The RNA or DNA genomes of viruses reside within the tightly packed core of the virus, which also contains proteins encoded by the viral genome. These viral proteins participate in the production of new copies of the virus. Viral nucleic acid contains all the instructions necessary for the production of nascent viruses. The exterior surface of a virus is composed of viral proteins and, sometimes, of proteins and lipids co- opted from the surface of the host cells. DNA is often referred to as the ge ne tic material of life. Most genomes consist of double helices of DNA, forming twisting ladders whose rungs are composed of four different bases adenine (A), thymine (T), guanine (G), and cytosine (C) arranged in varying sequence to form genes. In going from the DNA gene to protein, there is an intermediate step called messenger RNA. After a messenger ribonucleic acid (or mrna, as it s usually called) is assembled, it is used to instruct the protein synthesis machinery precisely how protein should be created. The order of the bases, which is specified by the mrna, creates a code that directs the assembly of twenty different amino acids into chains called pro- 24 The Beginning of an Odyssey

10 teins. Different combinations of three bases specify which amino acid should be added next in a growing chain. For example, the sequence CCA encodes the amino acid proline. Deciphering the ge ne tic code as well as the colinearity between nucleic acids and proteins was a major step in unraveling biological information that is passed from parent to offspring. The chains of amino acids that form proteins are assembled inside cells beginning from one end called the N-terminus. When the last amino acid is added, that end is designated the C-terminus. By convention, when scientists describe the beginning of a protein, they place the N-terminus on the left and the C-terminus on the right. Proteins are the essence of life; they perform many different functions and are the major components of all cells. Some proteins called enzymes speed up chemical reactions that are required for life, while others act as receptors and still others function as scaffolds. The biological activity of a protein requires that the amino acid chain fold into a compact shape called a conformation. Shape is everything to a protein denaturing a protein stimulates its unfolding into an inactive chain of amino acids. As the shape of a protein disappears, so does its biological activity. Sometimes, a protein can be refolded to regain its biological activity. The Beginning of an Odyssey 25