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WHAT'S BEHIND AMYLOID DISEASES? Pores or ion channels made of amyloid proteins may cause degenerative age-related disease SOPHIE L. WILKINSON, C&EN WASHINGTON
don't know whether the pore is formed on the pathway to the fibril or if there's a fork in the road somewhere and the protofibrils have a choice to either form a pore or form afibril.We're trying to study that." In 1993, NelsonJ. Arispe, Eduardo Rojas, and Harvey B. Pollard of the Labora tory of Cell Biology & Genetics at the Na tional Institutes of Health were the first to propose that, in addition to forming fi brils and plaques, amyloid β protein might form ion channels in the membrane of neu rons [Proc. Natl Acad Sci USA, 9 0 , 567 (1993)}. The channels could allow excess calcium and other cations to flow into the neurons, killing the cells and ultimately re sulting in disease.
you get amyloid fibrils, and they all have difficult and protracted fight to very similar structures," Lansbury says. be accepted. But if their creators " % u would think, 'Similar structure, sim are stubborn and persistent, these ilar function, and they're all involved in dis hypotheses may survive long ease.' " But these high-visibility markers, enough to be refined and further evaluated which were first described in the 19th cen THE RESEARCHERS, who used planar as additional information accumulates. tury, may prove to be red herrings. lipid bilayers as models for cellular mem branes, bolstered their thesis by showing One such thesis concerns the identity of In Lansbury's case, one of the first find that both aluminum, which binds to amy the toxic agent in Alzheimer's, Parkinson's, ings that weakened his commitment to the loid β protein, and tromethamine, which and other degenerative age-related dis toxic-fibril theory was made by Christo eases. For years, most researchers have blocks channels, prevented calcium pas pher M. Dobson, professor of chemical backed the notion that clumps of long sage through the bilayer. and structural biology at the University of amyloid fibrils located in the brain and oth Cambridge, who showed that many pro Initially, the researchers' idea met with er organs are responsible for killing neu teins—including those that aren't associ a cool reception. In fact, their amyloid rons and other cells in patients who have ated with disease—"can form amyloid fichannel hypothesis was dismissed by many these conditions. In Alzheimer's, these clumps are termed amy loid plaques and consist primarily of the poly peptide amyloid β pro tein; in Parkinson's, they're called Lewy bod ies and contain the pro tein a-synuclein. It has taken a while, but the hypothesis that a precursor of the insol uble amyloid fibril may be the more dangerous entity is now gaining ground. And some re searchers further believe that the smaller, soluble STRENGTH IN NUMBERS Harvard's amyloid pore team includes (back row, from left) Jeanprecursor, which is often Christophe Rochet, Petre, Walz, Lansbury, Jeffrey Kessler, Ding, and (front row, from left) Junecalled an amyloid olig Young Park, Richard J. Nowak, Magdalena Anguiano, Hartley, Voiles, and Lashuel. Images at right omer or protofibril, may show pores formed by mutant versions of α-synuclein (top two images) and amyloid β protein. do its damage by creat Each electron micrograph image measures 30.5 nm on a side [Nature, 418, 291 (2002)]. ing holes—pores or ion channels—in cellular membranes. of their peers. "They didn't take it seri brils. And maybe an amyloid fibril is a "The whole history of neurodegenera ously," Rojas recalls. default, stable structure. So maybe the fact tive disease has revolved around the ob Despite this rejection, Rojas, who is that all these fibrils share a structure says servation of the fibrils," says Peter T. Lanscurrently a biophysics and physiology more about the stability of that structure buryjr., an associate professor of neurology professor on the faculty of medicine at than it does about a shared function," Lans at Harvard Medical School. "People are the University of Chile, Santiago, and bury says. fixated on them." Lansbury himself wasn't Arispe, who is now a research professor "It maybe that in the neurodegenerative immune. "When I was at MIT, my whole in the anatomy, physiology, and genetics diseases characterized by fibrillization— lab was focused on the fibril," he concedes. department at the Uniformed Services which is essentially all of them—the fi There's good reason for that fixation. University of t h e H e a l t h Sciences, brillization produces, as an alternative prod "In all these neurodegenerative diseases, Bethesda, Md., have doggedly continued uct, an amyloid pore," Lansbury says. iCWe
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SCIENCE & TECHNOLOGY with their efforts to publish their ideas. Others who have kept the idea alive in clude Bruce L. Kagan, a senior research scientist at the University of California, Los Angeles, Neuropsychiatrie Institute, and Ratneshwar Lai, a senior research sci entist at UC Santa Barbara's Neuroscience Research Institute. The going hasn't been easy, particularly because funding has been tough to come by Federal granting agencies consider the ion-channel hypothesis to be "heretical," Lai explains. In addition, Kagan notes that his group and others have had trouble getting their papers into high-profile journals. Review er "comments have been things like: 'This can't be true,' or This might be true but it has nothing to do with how amyloid pro teins work,' " he says. "It's long been re garded as just not relevant." THE REASON for this skepticism, Lansbury suggests, is that channel proponents have not explained what distinguishes healthy amyloid proteins, which don't clump together, from those that aggregate into ion channels. "I myself was a skeptic because it was difficult to see how this ac tivity, ascribed to a normally expressed monomelic protein, could be specific to disease," he says. Lansbury and his team have taken a dif ferent approach than the ion-channel sup porters in their attempt to identify the pathogenic species. They began by exam ining the effect on amyloid fibrillization of the mutations that cause inherited cas es of Parkinson's and Alzheimer's. The group "found that the mutations promote the formation of amyloid protofibrils, and that led to the idea that the protofibrils, rather than the fibrils, could be pathogen ic," says Hilal A. Lashuel, neurology in structor at Harvard. But protofibrils can adopt many different sizes and shapes, so the researchers looked more closely to identify the conformation that was most harmful. "That's when we found that the mutations in b o t h Parkinson's and Alzheimer's promote the formation of porelike structures." Lansbury, along with Lashuel, assistant professor of cell biology Thomas Walz, Walz's research assistant Benjamin M.
Petre, and assistant professor of neurolo gy Dean M. Hartley, have just published a "Brief Communication" in Nature [418, 291 (2002)} that shows images of the amy loid pores. The large size of the holes seen in these electron microscopy images "sug gests that the amyloid proteins are more likely to form rather unspecific pores than channels that are specific for certain ions," Walz says. Lansbury's group hasfiverecent and up coming full papers in Biochemistry and the Journal ofMolecular Biology that provide further information on pores. For instance, graduate student MichaelJ. Voiles demon strated that "only the protofibrils are ca pable of disrupting membranes via a mech anism that is consistent with that mediated by a porelike structure," Lashuel says. This proposed mechanism resembles the lethal pore-formation activity of tox ins secreted by organisms such as Staphy lococcus aureus, which causes food poisoning, andBacillusanthracis, which causes anthrax. Atomic force microscopy images of the
HARMLESS? Lansbury's team suggests that amyloid fibrils (such as the α-synuclein fibrils seen here) could be nontoxic, in contrast to their smaller pathogenic precursors, the annular protofibrils (arrow) [Biochemistry, 41, 3855 (2002)]. α-synuclein amyloid pores in membranes by group member Tomas Τ Ding provide additional evidence. Christopher A. Ross, a professor of psy chiatry and neuroscience at Johns Hop kins University School of Medicine who
studies the basis for diseases such as Hunt ington's and Parkinson's, says the Lansbury group's "work on protofibrils and other oligomers as toxic species is groundbreak ing and important. The data in the Nature communication are beautiful and show that annular species can form in vitro. What remains to be shown is whether this is relevant in vivo." FOR NOWp Ross says, he remains "skepti cal" about the pore hypothesis. "I'm not sure about the biological plausibility," he explains. "In the Huntington's disease field, for instance, there is reasonable agreement that a major site of toxicity is the cell nu cleus, with alterations in gene transcrip tion. But it is not at all clear how a poreforming mechanism could be involved." However, the good news is that, "with such a clear visualization of these annular struc tures, the amyloid pore hypothesis is now testable," he adds. Lansbury himself describes the pore pic tures as "strong circumstantial evidence." But he is determined to collect more com pelling evidence or disprove the hypothe sis in the attempt. Working with a Drosophila model of Parkinson's developed by Mel B. Feany, an assistant pathology professor at Harvard Medical School, Lansbury's group plans to create transgenic flies with mutations in the genetic sequence for α-synuclein. One set of mutations will accelerate synuclein fibril formation and prevent pore formation. The other set will promote pore formation but prevent fibril formation. Lansbury's team also wants to find small, organic, druglike molecules that will do the same thing. The researchers will then de termine which of the flies display Parkin son's symptoms and whether disease symp toms correlate, as expected, with the presence ofpores rather than fibrils. They also plan to do similar work with mice. For now, proponents of the membranehole hypothesis must make do with less définitive results. For instance, "people have measured channel activity by measuring currents or the flux of tracer ions across a membrane," Kagan says. "When you do that, you can fairly conclusively determine that something is forming a channel." Some of the strongest support
It has taken a while, but the hypothesis that a precursor of the insoluble amyloid fibril may be the more dangerous entity is now gaining ground. 32
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of this type comes from Lai's team, he says. For example, Lai's group showed that amyloid β protein can insert itself into the membrane of liposomes and form ion channels. Using radiolabeling, he found that the vesicles took up calcium, and that
by raising their cholesterol content de creases their susceptibility to killing by β amyloid, Kagan says. "In order to make a channel, the amyloid protein must in sert into the membrane. If you make the membrane stiffer, it's harder to insert a
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PARTNERS UC Santa Barbara's Lai (left), seen here in his atomic force microscopy lab, has worked closely with senior research associate Hai Lin on amyloid research. Atomic force microscopy shows that an ion channel formed in a planar lipid bilayer is composed of subunits that Lai says may consist of single peptides of amyloid β. Such channels would transport ions across the cytoplasmic membrane into a cell. The resulting disruption could lead to cell death and, presumably, Alzheimer's disease. Each image is 18 nm on a side and shows six subunits [FASEBJ., 15, 2433 (2001)].
a fair amount of evidence in favor of the membrane-hole hypothesis as an alterna tive to the fibril hypothesis. So why have the channel proponents in particular en countered such resistance? One reason, Kagan says, is that "cells, particularly neurons, are loaded with ion channels. So showing that your one amy loid channel is the one that's killing the cell is very difficult." Another problem: The results used to be nonreproducible. "Peo ple became very frustrated with trying to look for channel activity or even cell-killing activity because sometimes it would work and sometimes it wouldn't," Kagan says. "It would depend on whom you got the peptide from and how they made it and how you handled it." Researchers ultimately traced the cause of the erratic behavior. The explanation lies with the degree of aggregation of the peptides, which depends on their prepa ration history, including protein concen tration and the type of solvent, Lai says. These conditions determine whether the amyloid β protein remains as monomers, forms oligomers, or bands together into fibrils.
SEVEN OTHER amyloid proteins have been shown to form holes either in lipid bilayers or in cells since the 1993 Arispe paper, Kagan notes. They include a-synuclein, which comprises the Lewy bodies passage could be blocked by antibodies to new protein in there." Making the mem that are the hallmark of Parkinson's dis amyloid β protein or by zinc (known to be brane more fluid by depleting cholesterol ease; huntingtin, which is associated with an amyloid β protein channel blocker). makes the cells more sensitive to β amy Huntington's disease; the prion protein, Likewise, in work with both neuronal loid. Kagan has published a similar arti which is involved in conditions such as and other cells, Lai's group showed that "if cle, along with a review article on the mad cow disease; and islet amyloid you treat cells with the amyloid β protein, channel hypothesis, in July's special is they die," Kagan says. "And if you add sue of Peptides, which includes several au polypeptide, also called amylin, which is tied in with diabetes. things which block the channels in vitro— thors' papers on the subject of amyloid like zinc or the cation tromethamine—you Amylin is a hormone that may moder β protein. protect the cells from being killed." ate glucose metabolism. It is normally seTaken all together, there appears to be Lai adds that the experi m creted along with insulin by ment was designed so that < pancreatic islet β-cells. But the concentration of amyloid | deposits formed from fibrils β protein was low and its in © of this amyloid peptide can cubation time with the cells be found in the islet cells of was short, conditions that patients with type 2 dia prevented fibrils from form betes. As with the other ing. That means fibrils can't types of amyloid proteins, be blamed for the cellular however, the researchers death that was observed. In who support the membranefact, Lansbury and Voiles hole hypothesis believe that have shown that amyloid fi these deposits aren't the brils are unable to form holes pathogenic entities that im in vesicles. pair and kill off the pancre BIG CHANGES Lai and Lin found that amyloid β protein atic cells. Other evidence for the induced morphological changes in cultured cells within minutes channel hypothesis comes of treatment and eventually led to cell degeneration. Arrows in Oligomeric protofibrils from work with cholesterol. the image on the right show where the cell has retracted formed by amylin can take Arispe will soon publish a pa following exposure to the protein. The researchers attribute the on a conformation rich in per showing that stiffening harm to the formation of ion channels in the cell membranes by β-pleated sheet content, a the membranes ofbrain cells the protein [FASEBJ., U , 1233 (2000)]. structure that helps them HTTP://PUBS.ACS.ORG/CEN
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SCIENCE & TECHNOLOGY insert themselves into cell membranes and form pores, Lansbury notes. He be lieves that the pores allow calcium to build up in the pancreatic cells, causing them to deteriorate and then die. Because these cells are responsible for secreting insulin, their death eventually leads to diabetes, the researchers propose. Rojas and Arispe take a similar stance in explain ing the formation of ion channels. "It is possible that the essential feature, common to all pathogen ic channel-forming pep tides, is the β-pleated sheet structure in combination with hydrophobic domains on the cell membrane," they wrote in an article in the Journal of Biological Chem istry {21S, 14077 (2000)}. They collabo rated on the paper with Masahiro Kawahara and Yoichiro Kuroda of t h e molecular and cellular neurobiology de partment of the Tokyo Metropolitan In stitute for Neuroscience.
"We propose that age-related diseases, such as Alzheimer's and type 2 diabetes mellitus, share in common this causal mechanism, which involves peptide inser-
The reason for skepticism is that channel proponents have not explained what distinguishes healthy amyloid proteins, which don't clump together, from those that aggregate into ion channels. tion in the cell membrane and further mo lecular rearrangements, leaving the βpleated sheet domains lining the pore of the putative amyloid cation channel," the team wrote. "The formation of unregulat ed cation channels in susceptible cells con tributes to metabolic stress and eventual cell death." Although several researchers believe that the holes —whether they are pores or
ion channels—kill cells by interfering with calcium flow, Lansbury offers some other possible mechanisms. In one scenario that he sketches for Parkinson's, protofibrillar a-synuclein binds to mitochondrial membranes in neu rons, forming pores that permeabilize the membranes, which then leak cytochrome C into the neuronal cytoplasm. It's also possible that permeabilization ofvesicular membranes permits dopamine, a cytotox ic neurotransmitter that neurons package in vesicles for safekeeping, to leak into the cytoplasm and kill the neurons, Lansbury says. In fact, he says, "there could be 10 things that happen that are bad. The process of a cell dying in these diseases is probably a mess, with lots of different things hap pening simultaneously" It is important to understand as many of those processes as possible, but they don't represent good independent drug targets. " % u don't want to try to individ ually treat the 10 things that an amyloid pore can do," Lansbury says. "Instead, it would be better to block formation of pores in the first place." •
University of Southern California Loker Hydrocarbon Research Institute and the Department of Chemistry Cordially Invites you to the Annual George A. Olah Lecture in Chemistry presented by
Professor F. Albert Cotton Department of Chemistry, Texas A & M University, Texas
'Intellectual and Aesthetic Charms of Molecules with Metal-Metal Bonds" Wednesday, October 2,2002 Refreshments: 3:30 p.m. Lecture: 4:00 p.m. Seeley G. Mudd Building 123 University Park, Los Angeles, California 90089-1661 For information: Phone: (213)740-5974; Fax: (213)740-5087; e-mail:
[email protected] The Scientific Community is Invited
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