The Discovery of Amyloid

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Cai'ne W. Wong1

Dr. George G. Glenner died (too young) in 1995 due to complications of cardiac amy-loidosis. If he had survived, perhaps he and I would be writing this story together. Instead it will only be my personal recollections of the work we did in 1982-85 that led us to the discovery of the Alzheimer's disease "beta protein," (Glenner and Wong 1984a) now also known as A-beta and beta-A amyloid.

How it started

My introduction to George Glenner was in 1982, when I interviewed for a job in his new laboratory at the University of California at San Diego (UCSD). He had recently moved to UCSD after two and a half decades at the National Institutes of Health in Bethesda, MD, where he had made a career of investigating human systemic amyloids. Now at UCSD, he was still focused on amyloid but on those found in the human central nervous system. His research goal was to identify the critical protein(s) that made up the amyloid deposits found in the brains of Alzheimer's disease (AD) patients. During the interview, I learned his research program was just beginning. The actual bench biochemistry would start when I did. This was clearly apparent since the two rooms that comprised the laboratory were empty with the exception of two large ultra cold Revco chest freezers. These freezers contained donated AD brains. The year before, George Glenner had created one of the first AD brain banks and it was to be a source of diseased human brains for us and other researchers. The mission of the brain bank was made known to the National Alzheimer's Disease and Related Disorders Association. Through this network and other AD support groups, brain donations were made to the brain bank by relatives, who had legal authority, of individuals suspected to have died of AD. The family would receive a no-cost diagnosis based on a postmortem neuropathological examination. The postmortem diagnosis, although not always 100% accurate, it was still the most definitive assessment for AD. In the months that followed, I would grow to appreciate the genius of that part of George Glenner's research plan and to realize how pivotal the brain bank would be to our success. Having ready access to AD brains with exceptional amounts of amyloid deposits was a tremendous advantage to our research.

We were a small research group, composed of George Glenner, Karen Rasmussen and myself. Karen was responsible for maintaining the brain bank and the myriad tasks associated with it. My responsibilities were to order equipment and supplies, to set up and maintain the new laboratory and to perform the biochemical experiments.

1 Scarborough, Maine USA

In 1906, Alois Alzheimer, in a seminal presentation in Tübingen, Germany, made an association between abnormal amyloid deposits (which would later become known as neurofibrillary tangles, (NFTs) in the brain of Auguste D. and her dementia. This neurological malady would shortly become known as Alzheimer's disease. In time, two other forms of brain amyloid, neuritic plaques (NP) and cerebral vascular amyloid (CVA), would also be associated with the disease. In 1982, despite nearly eight decades that had lapsed since Alois Alzheimer introduced the disease to science, there was still nothing known of the amyloid etiology. Electron microscopy (Kidd 1963; Terry et al. 1964)) plus X-ray fiber diffraction studies (Eanes and Glenner 1968) showed that the amyloids were protein fibrils with a high content of beta-pleated sheet. The unknown etiology helped fuel a controversy over the role (if any) that amyloid played in the neuropathology of AD. To solve the ambiguity surrounding AD amyloid, George Glenner knew that it would be necessary that to identify the amyloid protein(s) identified. If nothing else, he speculated, there might be diagnostic potential in the identify.

Success, disappointment, then success and uncertainty

We began our investigation with CVA. In his histological examination and cataloguing of AD brains for the brain bank, George Glenner found that more than 90% of AD brains had amyloid deposits in blood vessel walls (Glenner 1983)). Of particular interest were the brains that had extensive deposition in the meninges. In these he saw the potential of obtaining enriched CVA preparations simply by stripping the meningeal membrane away from the cortex and thereby circumventing contaminants from that source. Once the meningeal membrane was stripped off and cleaned of residual cortex, it was finely minced and homogenized. The homogenate was centrifuged to collect a pellet. The pellet was composed ofa large white bottom layer and a thin tan top layer. We examined the layers to determine which contained the bulk of the amyloid. This was done by making thin smears on microscope slides of each layer and examining them using the same Congo red histological stain technique used for examining brain sections. We found that the top tan layer was enriched for amyloid but was still contaminated with a significant amount of connective tissue-like debris. In a previous research position, I had used collagenase to perfuse rat livers to obtain single hepatocytes (Hatoff et al. 1985)). After a prolonged collagenase perfusion, the once lobed liver would become a shapeless bag of disassociated cells. When I suggested we try to remove the presumptive connective tissue contaminant by incubating the tan layer with collagenase, George Glenner immediately recognized the merit ofthat idea. As it would turn out, thecollagenasewas very effectiveatremovingthe bulk of theconnectivetissue contaminants without affecting the CVA. One of the hallmark properties of amyloid is the acquisition of apple-green birefringence after being stained with Congo red dye and viewed under a polarized light microscope. The collagenase-treated sample slide showed an almost unreal field of pure apple-green birefringence. George Glenner was known by those who knew him personally for the twinkle he had in his eyes. His eyes were exceptionally twinkling bright that day.

We then looked for a way to dissolve the CVA sample so that we could subsequently fractionate the components. Amyloids, as a rule, were known to be difficult to solu-bilize (Selkoe et al. 1982b). CVA would not be an exception to that rule. A number of strong biochemical detergents, including SDS, were systematically tried at increasing concentrations, elevated temperatures, varied pH and incubation times. None of those experiments were successful. We then turned to chaotrophic denaturing agents and eventually found that prolonged incubation with 6 M guanidine-HCl under reducing conditions plus EDTA worked moderately well. Denaturation of the CVA was monitored by the loss of Congo red dye-mediated birefringence. We hoped that the denaturation was the result of solubilization. After removing the guanidine from the CVA supernatant by dialysis and concentration by lyophilization, we were able to resolve CVA proteins on SDS-PAGE. Comparing the protein band profile of the CVA sample to the profile of normal control brains meninges, we found a unique low molecular weight band at the electrophoretic front of the CVA sample lanes. We repeated the experiment with a higher percentage SDS polyacrylamide gel augmented with urea. This was done to resolve the low molecular weight band away from the electrophoretic front so we could determine the approximate molecular weight. There were other differences in the SDS-PAGE protein profiles but the low molecular weight band was the most salient, and that is where we focused our attention.

Now that we had a protein (or peptide) of interest, we scaled up the purification protocol by using a calibrated preparative G-100 Sephadex sizing column to resolve guanidine supernatants of CVA. The unique low molecular weight protein (or peptide) was recovered in the fractions centered at 4,200 daltons (CVA4200). The protein (or peptide) could be recovered from different CVA samples but not from control samples. CVA4200 was submitted for automated protein sequence analysis at the UCSD Weingart Protein Sequence Core Facility. The results were disappointing. There were multiple amino acid signals at every sequencing step, which suggested the sample had multiple proteins (or peptides) and/or ragged amino termini. It was clear that our purification scheme needed augmentation with an additional fractionation method.

Fortune, opportunity, and perhaps coincidence would collide next. Rob Nicholas, a friend and housemate, was a biochemistry graduate student at UCSD in Jack Kyte's laboratory. Rob and his colleagues were studying the structure-function of the Na+-K+ ATPase, a membrane protein with multiple transmembrane domains. I told Rob about our disappointing sequencing results and that we were looking for another purification scheme for small peptides. Rob then told me his lab had had a recent success at resolving small proteolytic peptide fragments from the transmembrane domains of the Na+-K+ ATPase using reverse phase HPLC and a newly formulated liquid phase. Perhaps, he suggested, it would work for fractionating CVA4200 as well. I was invited to use the Kyte laboratory HPLC system and was tutored by Rob in its operation. The results were immediate. On the very first run, three elution peaks were resolved. The second and third overlapped, eluting at 35% and 36% acetonitrile. After confirming that the results were repeatable with different CVA samples, fractions from the three peaks were submitted for amino acid sequencing. The first peak was called alpha and yielded no sequence information. The second and third peaks, called beta and gamma, respectively, yielded sequence information to 24 residues and the sequences were identical up to that point. Because of that identity, we renamed the second and third peaks beta-1 andbeta-2. We expected approximately 38 residues based on a 4,200 dalton molecular weight. Amino acid analysis predicted 33. With only 24 amino acid residues, we knew we had only a partial sequence. With the partial sequence in hand, we enlisted the aid of Dr. Russell Doolittle, Research Professor of Biological Science at UCSD. He was assembling one of the early protein-nucleic acid sequence databases. His computer search found no homology of the beta protein with any protein in any database. Excitement tempered with trepidation followed. Had the beta protein sequence been homologous with a known protein, particularly a human protein, we would not have felt so uncertain of what we had found. Previously, studies of human systemic amyloid proteins showed them to be derived from endogenous human proteins. However, Russell Doolittle had counseled us not to be too disappointed and overly concerned about the lack of homology, since all sequence databases (at the time) contained only a miniscule percentage of the expected number of proteins predicted to exist.

Uncertainty abated

Soon after we obtained the beta protein sequence from AD CVA samples, we obtained a similar sequence from a CVA sample of an adult Down's syndrome (DS, aka Trisomy 21) brain (Glenner and Wong 1984b). It was known that virtually every case of adult DS resulted in an AD-like dementia after 40 years of age. Microscopic postmortem examination of adult DS brains showed they also contained amyloid lesions indistinguishable from those found in AD brains. The DS result was an important finding in that it provided the first biochemical relationship between AD and adult DS. In addition, it further supported the possibility that the etiology of AD was located on chromosome 21. However, it did not provide an independent verification that the beta protein was a component of the CVA.

Vito Quaranta, a molecular immunologist at Scripps Clinic and Research Foundation, would help formulate the next step in our research. Vito Quaranta suggested that anti-peptide antibodies raised against beta amyloid could be used to immunohisto-chemically stain the CVA in AD brain sections. If the anti-peptide antibodies localized to the CVA deposits, it would provide an independent demonstration that the beta protein was a component of the CVA. Moreover, he offered to provide the necessary scientific guidance and invited me to perform the antibody production work in his laboratory. Doing so would circumvent the lag time of setting up our own laboratory for antibody production. Furthermore, his laboratory was only a 10-minute trip from the UCSD campus. We designed overlapping synthetic peptides to span the beta protein sequence. The synthetic peptides were coupled to keyhole limpet hemocyanin and used to immunize both rabbits and mice.

The antibody response varied for the different peptides used. We found that a synthetic peptide corresponding to the first 10 amino acids of the beta protein sequence was especially immunogenic in BALB/c mice. A mouse antiserum (OP1MS1) with the highest titer and specificity in ELISA studies was selected for immunohistochemistry experiments. The results were dramatic. The OP1MS1 antiserum specifically stained CVA deposits in both AD and adult DS brains (Wong et al. 1985). This finding provided the much-needed independent evidence that the beta protein was an integral component of CVA. Prior to obtaining the antiserum, George Glenner and I had an ongoing "bragging rights" wager as to whether the NPs and NFTs would also be recognized by it. George Glenner, with his always upbeat optimism, wagered that the antiserum would recognize not only the CVA deposits but also the NPs and the NFTs. I was less optimistic and hedged my bet by restricting my choice to the CVA. As it turned out, we both lost that bet. The OP1MS1 antiserum recognized CVA and NP in AD and adult DS brains, but not NFTs. At face value, the results confirmed that the beta protein was an integral component of CVA and strongly suggested it was for NP amyloid as well.

When shown the results of the immunohistochemistry study, George Glenner had an uncharacteristically unrefined "HOLY SHIT" moment. Although George Glenner had wagered more, the clear and unambiguous results had surpassed his actual expectations. After carefully reviewing the experimental and control slides of the experiment, George Glenner remarked with his twinkling eyes and his widest grin, "Well, you can't win them all. Two out of three ain't bad. You had better get started on the writing." George Glenner did not say it specifically, but I took what he said to mean that we had a very good day. We had made progress towards teasing out the identity of two of the three amyloid deposits he had originally set out to find. We also learned something about the NFTs that we did not know before. The results suggested that NFTs were either composed of another peptide (or protein) or of the same peptide with the OP1MS1 epitope masked. It would be discovered later that NFTs are composed of hyperphos-phorylated tau, a microtubule associated protein (Brion et al. 1985; Iqbal et al. 1989). While the OP1MS1 manuscript was in press, we learned that the collaborating teams of Colin Masters and Konrad Beyreuther obtained a peptide sequence from AD and adult DS NPs similar to the sequences we had found from AD and adult DS CVA (Masters et al. 1985a). Our immunohistochemisty studies (Wong et al. 1985) dovetailed with their finding.

Reflections

While preparing the 1984 beta protein amino acid sequence manuscript, George Glenner and I debated whether or not it would be too presumptuous to use the name "beta protein" with the obvious connotation to beta-pleated sheet and amyloid. At that time, there was no independent confirmation that our beta protein was amyloid. In the end, George Glenner, with his usual optimistic view of things, said, "Let's let it stand." In the following months, the beta protein would be confirmed as the amyloid peptide and the name "beta protein" would morph into the more definitive "beta amyloid," "beta-A" and "A-beta" by the AD research field.

The (beta) amyloid precursor protein (APP) gene would be cloned by four laboratories (Goldgaber et al. 1987a; Kang et al. 1987; Robakis et al. 1987b; Tanzi et al. 1987) and would be found on chromosome 21. At least one laboratory, and possibly others, would be aided by the beta peptide amino acid sequence. The APP turned out to be a transmembrane protein and beta amyloid a peptide fragment from part of the transmembrane domain. Rob Nicholas' suggestion of using a HPLC protocol developed to isolate protelytic transmembrane peptides to resolve CVA4200 was more prophetic than we could ever have imagined.1

The discovery of beta amyloid raised many fundamental questions, as witnessed by the extraordinarynumber of publications concerningit since. The other contributors to

1 Rob Nicholas, now a professor of pharmacology at the University of North Carolina at Chapel Hill, was never publicly acknowledged for his crucial contribution to the discovery of beta amyloid. Hopefully, this will help correct that unfortunate oversight.

this volume have provided elegant and compelling answers to some of those questions and have, in turn, generated even more questions. Their work, along with that of many others, has extended, expanded and filled in the continuing beta amyloid story just as George Glenner had wanted.

I left George Glenner's laboratory in 1987 to attend graduate school at UCLA. In the years since, it has been a source of personal gratification to know that the work we did in 1982-85 opened a significant door for AD research and continues to be relevant to this day. It is rare to be part of something that has affected so many lives and I am grateful to have had the opportunity.

Konrad Beyreuther

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