Aging
Investigating the connection between cell aging and the onset of proteotoxicity
By Scripps Research Institute
Aug 14, 2006 - 11:37:00 AM

Alzheimer's disease now strikes more than one in 30 Americans, and about half the population that lives past 85 acquires Alzheimer's. Approximately one million Americans have Parkinson's disease, including three out of every 100 people over age 60. Aging is the most important risk factor for both of these diseases.

The new study-conducted in a C. elegans model, a roundworm that expresses a protein whose aggregation appears to cause Alzheimer's disease-showed that toxicity from protein aggregation is "drastically reduced" when aging is slowed by modulating the insulin growth factor (IGF) signaling pathway.

Moreover, the researchers found two novel independent activities promoting this cellular survival. The first protective mechanism disassembles and cuts up protein aggregates. Surprisingly, the second protective mechanism enables the formation of larger aggregates from smaller ones that appear to be more toxic.

Kelly, who is the Lita Annenberg Hazen Professor of Chemistry at The Scripps Research Institute, a member of its Skaggs Institute of Chemical Biology, and dean of graduate and postgraduate studies, stresses that this novel work was a synergetic collaboration between the research groups at the two institutions.

The Dillin lab at Salk was interested in investigating the connection between cell aging and the onset of proteotoxicity. So, the group set out to determine if the aging process in the worm could be slowed by using RNA interference (RNAi), a naturally occurring process known to suppress certain gene activity in living cells, to lower the activity of the IGF signaling pathway. Indeed, this approach worked and the researchers discovered that if aging was delayed, the onset of proteotoxicity was also postponed.

"In switching this pathway on and off, we also found that we altered the high molecular weight aggregates-the plaque buildup in the animals," explains Dillin.

But the researchers also noticed an unexpected phenomenon. "Curiously," Dillin continues, "some animals were totally protected from proteotoxicity despite having high molecular weight aggregation buildup, while other animals were extremely sensitive to proteotoxicity even though they had no detectable high molecular weight aggregates."

Dillin was eager to further investigate these unexpected findings, so he contacted Kelly at Scripps Research, whose lab had the expertise to examine and analyze these aggregates.

Intrigued, Kelly's colleague Jan Bieschke, in collaboration with Ehud Cohen of the Dillin laboratory, did additional experiments perturbing individual components of the IGF signaling pathway. They focused on two downstream transcription factors, heat shock factor-1 (HSF-1) and DAF 16, to see what effect they had on aggregation. The results were surprising.

"When we inhibited only HSF-1, the result was a tremendous amount of aggregate buildup; when we selectively inhibited DAF 16, there were almost no aggregates observed," Kelly says. "That clued us in to the fact that these two transcription factors must be controlling effectively opposite activities."

His group had been using aggregation assays for a long time, and it occurred to him and his colleagues to add samples of the worm's ground-up tissue to these assays as a way of even more sensitively detecting how these transcription factors were controlling aggregation in the animal. The Scripps Research team subjected the worm contents to aggregation assays, using fluorescent dyes that emit light when amyloid is present to read out the extent of aggregation. "It turned out that this worked really well," Kelly says.

When Cohen and Bieschke examined the results, they expected to see less aggregation in the worms when insulin signaling was inhibited. In fact, what they saw was more.

Teasing apart the results, the researchers concluded that two mechanisms were protecting the worms against protein-aggregation-associated proteotoxicity. One mechanism was taking the aggregates apart and degrading them into small pieces; the second mechanism was taking smaller, lower-molecular-weight aggregates and transforming them into high molecular weight aggregates of lower toxicity.

Kelly says that this second finding is "quite surprising" in that heavier aggregates seem to be protective for the cell, albeit in a transient fashion-until the cell can "re-group" to dispose of the aggregates. "What we expected was that the amount of aggregates would correlate with toxicity in these worms, but there was no correlation."

"This second finding is clearly a shift in paradigm," says Dillin. "For nearly a year in this work, we assumed that large aggregates were the toxic species; however, our data proved otherwise. These results further support a shift in thinking for this field regarding the toxicity of small aggregates and lays the framework for new avenues to combat age-onset protein aggregation diseases, such as AD, Parkinson's, Huntington's, and ALS, owing to the protective biological activities discovered."

"Now, we want to use this mechanistic information to discover the macromolecular basis for these activities and to discover small molecules that will delay the aging program and thus delay the onset of proteotoxicity associated with these diseases by modulating aggregation and disaggregation activities," Kelly states. "The hope is that, by manipulating the protective mechanism inherent in cells, we can find a single entity-a single drug-that would be useful for a variety of neurodegenerative diseases where protein aggregation leads to neurodegeneration."

In addition to Kelly and Bieschke at Scripps Research and Dillin and Cohen at Salk, Technician Rhonda Perciavalle of the Salk Institute also made significant contributions to the Science study, titled "Opposing Activities Protect from Age Onset Proteotoxicity."

"This has been a wonderful, synergetic collaboration," says Kelly. "This work could not have been done solely in their lab or in ours. Fortunately, scientists at Salk are just down the road and have always been great neighbors."

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