The accumulation of insoluble protein deposits is the hallmark of many neurodegenerative diseases, including Alzheimer disease. Now, a new study suggests that age-related protein aggregation might be widespread even in healthy organisms. In a paper in the August 10 PLoS Biology, researchers led by Cynthia Kenyon at the University of California in San Francisco show that over the lifespan of normal C. elegans worms, hundreds of proteins form insoluble deposits. Mutant worms with extended lifespans, however, develop fewer protein clumps, indicating that this phenomenon is closely tied to biological aging, and is not just a function of time. Intriguingly, Kenyon and colleagues found that some of the proteins that form deposits in aging healthy worms are also present as minor components in amyloid plaques and other pathological protein tangles in people, implying that accumulation of these proteins could play a role in human neurodegenerative diseases.
“There have been a number of demonstrations of specific proteins whose aggregation increases with age, but to my knowledge this is the first example of a global measurement of protein aggregation,” said Chris Link of the University of Colorado at Boulder, who has studied various pathological aggregates in roundworms. He was not involved in the study. “The paper establishes something that I had assumed for a long time, but that no one had actually shown directly.”
Scientists already knew that the cellular machinery responsible for the proper folding of proteins, and the machinery that degrades old or misfolded proteins, becomes less efficient as an organism ages. Over time, oxidative stress also causes proteins to become oxidized and difficult to degrade. These factors are thought to contribute to the accumulation of harmful proteins in neurodegenerative disease, but much less research has been directed at what happens in the normal aging brain.
First author Della David sought to answer this question by comparing insoluble proteins in young and aged worms. After removing soluble and membrane-bound proteins with high-salt and detergent buffers, she solubilized the remainder with formic acid and used quantitative mass spectrometry to identify the proteins. Certain kinds of proteins were insoluble in young and old worms alike, such as cytoskeletal and collagen proteins, but David and colleagues found a set of 461 proteins whose insolubility consistently increased with age, with a mean increase of 2.5-fold. These proteins began to accumulate quite early in adulthood, by day 3 of the worms’ two- to three-week lifespan.
The authors ruled out that the increase in insoluble proteins was due to a simple increase in total protein by showing that protein levels did not increase with age. Using transgenic proteins tagged with fluorescent markers, David and colleagues also showed that protein aggregates occurred in different tissues throughout the worm body, and in diverse cellular locations. Although the authors did not directly examine whether these aggregates were amorphous or structured, an analysis of the amino acid composition and expected shape of the aggregating proteins predicted they were likely to form β-sheets, a conformation that could promote amyloid fibril formation.
The authors wondered how these normal protein aggregates might interact with disease-related ones. To investigate this question, David and colleagues expressed a fluorescently tagged aggregation-prone protein, KIN-19 (a homolog of casein kinase 1 isoform α), in the muscle cells of the worm body wall. By itself, the overexpressed KIN-19 formed rock-like deposits, but did not cause the worms any apparent problems. The authors then overexpressed KIN-19 in a worm that also overexpresses a protein containing 35 polyglutamine repeats (Q35) in the muscle cells of the body wall. Polyglutamine-repeat proteins aggregate in several human diseases, including Huntington disease. The Q35-expressing worm becomes paralyzed as it ages, starting at day 6 of life. When tagged KIN-19 and Q35 were expressed together, the researchers found that paralysis struck the worms earlier, with twice as many being paralyzed by day 6.
The results showed that the aggregation of non-disease-causing proteins can worsen a disease phenotype, but the mechanism is mysterious, Kenyon said. The polyglutamine clumps did not increase in the double-mutant worm, nor did the aggregates of KIN-19 and the polyglutamine-repeat protein physically touch. “They’re acting at a distance,” Kenyon said. “That’s really interesting because it suggests that there’s some third factor that we don’t know about.” One possibility, David suggested, is that the aggregates of KIN-19 might be enhancing polyglutamine oligomer formation, an idea she plans to follow up on.
Most intriguingly for human disease, many of the proteins that form deposits with age in C. elegans are overrepresented in human neurodegenerative disease aggregates. About half the minor components of amyloid plaques and neurofibrillary tangles, and about one-third of the minor proteins in Lewy bodies, are homologs of proteins that aggregate in aged worms. These minor proteins include such things as heat-shock proteins, ATPases, clathrin, and vimentin, to name a few. This finding raises the possibility that normal protein aggregation could contribute to pathological aggregation in people.
“Before, we thought that these minor components of Aβ plaques or neurofibrillary tangles were just randomly sticking to Aβ or tau,” David said. “Now we have evidence that these proteins themselves could be aggregating with age.”
Among those that become insoluble with age, the authors found numerous proteins that, when knocked down by RNA interference, increase lifespan. This raises the possibility that it might be the aggregation of these proteins that limits longevity. Conversely, the authors wondered whether a mutation that slows aging would affect protein aggregation. Mutation of the worms’ insulin/insulin-like growth factor 1 (IGF1) receptor is known to double C. elegans lifespan (see Kenyon et al., 1993). To mimic this, David and colleagues knocked down the worm insulin/IGF1 receptor with RNAi and found that the resulting long-lived worms showed far fewer insoluble protein deposits with age.
Knockdown of insulin signaling “has an amazing effect on these aggregates,” Kenyon said. Not only are there less of them, she said, but when they are present, “instead of being rock-hard, they’re soft and flexible.” Reduced insulin signaling has been shown to improve disease models of pathological protein aggregation, including amyloid-β models (see Morley et al., 2002 and ARF related news story on Cohen et al., 2009). Kenyon said the new finding “adds to the argument that we should be looking into how to modulate AD by trying to change the activity of this hormone pathway.”
One of the questions that remains is whether proteins accumulate in mammals during normal aging as they do in worms. To answer this, David said, the authors plan to examine insoluble proteins in aged mice and see if the same pattern holds. David said she is also interested in examining the mechanisms behind inherent protein aggregation. One possibility is that protein accumulates because of problems in the protein degradation machinery. David will look for specific factors that alter protein accumulation in worms to try to pin down the pathways involved.
The finding that inherent and pathological protein aggregates can influence each other dovetails with previous work in the neurodegeneration field demonstrating a synergistic effect among deposits of α-synuclein, mutant APP, and tau in double- and triple-transgenic mice (see ARF related news story and ARF news story). Taken together, the data suggest that any additional protein aggregates may tip the balance and make an animal sicker.
In the long term, Kenyon suggested, researchers might use both worm and mouse models of AD to investigate the interaction between inherent and pathological protein accumulation, by overexpressing normal aggregating proteins to see if that speeds up the disease process. If so, it might imply that reducing normal protein aggregation could be beneficial in delaying disease. Kenyon also suggested that aggregates of normal protein might have potential as early biomarkers for neurodegenerative disease in people, if indeed these aggregates can be shown to predict disease onset.
“[Protein aggregates] are a new aspect of aging, like wrinkles or gray hair,” Kenyon said. “They’re a new player in the aging scene, and they could have a big impact on neurodegenerative disease.”
David DC, Ollikainen N, Trinidad JC, Cary MP, Burlingame AL, Kenyon C. Widespread protein aggregation as an inherent part of aging in C. elegans. PLoS Biology 2010 Aug 10;8(8):e1000450.
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