11 May 2007. Two papers this week feature inventive strategies for uncovering new targets and potential therapies to treat neurodegenerative disease. Both deal directly with Huntington disease, and particularly with the polyglutamine-expanded huntingtin protein, but at the same time they present possible applications to Alzheimer disease and other neurodegenerative conditions.
The first study reports the discovery of small molecules capable of ameliorating the toxicity of aggregated huntingtin protein by stimulating its clearance via autophagy. The work, from David Rubinsztein and Cahir O’Kane from the University of Cambridge, England, and Stuart Schreiber at the Broad Institute of Harvard/MIT in the other Cambridge (Massachusetts), provides a first step toward the development of novel autophagy enhancers. Such compounds could provide a new therapeutic approach beyond polyglutamine diseases, to a range of neurodegenerative conditions where protein aggregation plays a role. The study appeared May 6 in the online edition of Nature Chemical Biology.
The second story presents a new approach for winnowing the wheat from the chaff of interactome data to come up with likely drug targets. Researchers from Prolexys Pharmaceuticals, Salt Lake City, Utah, and the Baylor College of Medicine in Houston, Texas, used yeast two-hybrid and affinity purification assays to assemble a list of more than 200 huntingtin-interacting suspects. Then, the scientists measured functional interactions by testing a subset of those proteins in a genetics screen for their ability to modify neurodegeneration in a fly model of HD. Published in the May 11 PLoS Genetics, this approach identified 17 genes whose loss resulted in a suppression of neurodegeneration, making them prime candidates for drug targets. The method should be applicable to many other diseases to ferret out likely targets from long lists of interacting proteins.
Eating for Good Health
Autophagy (“self-eating”) allows cells to destroy protein aggregates that are too large to enter the proteasome by incorporating them into cytoplasmic vesicles, which then fuse with lysosomes. Substrates for autophagy include many of the protein aggregates that form in neurodegenerative diseases. In normal neurons, autophagy seems to serve a vital housekeeping function, and recent work showed that mice deficient in the pathway develop widespread neurodegeneration (see ARF related news story). Work from the Rubinsztein lab and others has shown that the immunosuppressive drug rapamycin increases the activity of this housecleaning pathway and mitigates the toxicity of several disease proteins, including polyQ-expanded proteins such as huntingtin, but also α-synuclein, the protein that causes familial Parkinson disease, and the AD-related protein tau (for review, see Ravikumar et al., 2006).
However, rapamycin is a powerful drug that affects many cell processes besides autophagy, and this prompted Rubinsztein and colleagues to hunt for other small molecules with more restricted actions. To that end, first authors Sovan Sarkar and Ethan Perlstein took an ingenious chemical biology approach, in which they used yeast to screen for potential stimulators of autophagy in mammalian cells. They reasoned that if they could identify compounds that enhanced rapamycin’s growth-suppressive effects on yeast, there might be in that group some that affected only autophagy in mammals. When they tested a library of some 50,000 compounds, they found 12 enhancers of rapamycin’s antiproliferative effect. Of those, three proved to enhance autophagy in mammalian cells, as indicated by augmented clearance of A53T α-synuclein. The same compounds reduced the aggregation and toxicity of mutant huntingtin in mammalian cells, while increasing the accumulation of autophagic vesicles.
To ask whether the compounds affected a disease process in vivo, the investigators turned to a Drosophila Huntington’s model. Expression of a polyglutamine-expanded fragment of huntingtin causes neurodegeneration in the fly eye, which is blocked by rapamycin. Likewise, treatment with any of the three new compounds also prevented neurodegeneration in this model.
The target of the new compounds is unknown, and is a question of great interest, Rubinsztein told ARF. The three structurally distinct compounds did not act via rapamycin’s target, the mTOR protein, to stimulate clearance of proteins, and their effects were additive with rapamycin. They did not affect levels of several regulators of autophagy including the Atg-5, -6, -7, or -12 proteins. The compounds did not affect the proteasome, either. “The obvious targets are not hit, but this field is moving very fast and there are already other targets we are thinking about,” Rubinsztein said. Part of the problem is that the pathways regulating autophagy, and especially its stimulation by rapamycin, are poorly understood.
Could autophagy stimulators find use in AD? That is a hard question to answer for now, Rubinsztein says, because the interaction among autophagy, APP processing, and amyloid formation is complicated. “Autophagy is very relevant to tau degradation, as we’ve seen previously in fly models expressing wild-type or mutant tau,” he said. That makes autophagy a good candidate target for tauopathies, as well as for hereditary Parkinson disease and a range of polyglutamine diseases, he said. (For more on this topic, see comment below from Ralph Nixon.)
Partners in Pathology
The chemical biology approach does not depend on knowing exact targets for intervention. Complementary approaches, like the one from Robert Hughes of the Buck Institute of Age Research in Novato, California, and his former coworkers at Prolexys aim at identifying new proteins with a potential to be drug targets. The work, headed up by co-first authors Linda Kaltenbach of Prolexys and Eliana Romero from the lab of Juan Botas at Baylor, shows how a combination of approaches can significantly raise the chances of finding targets based on protein-protein interaction data.
In the beginning, the researchers used both a yeast two-hybrid screen and affinity purification and mass spectrometry identification to find proteins that interact with huntingtin. After extensive vetting of the results (their selection criteria disqualified more than 80 percent of the initial yeast two-hybrid hits), they assembled a list of 234 high-confidence interacting proteins.
To validate these proteins, the scientists chose 60 genes for which fly mutants were available, and tested them in a genetic model of Drosophila HD. They found that 27 of 60 were bona fide genetic modifiers, where either exacerbation or amelioration of degeneration occurred with more than one allele and/or in different genetic backgrounds. “Since the collection of genes tested in the fly assay represented an arbitrary sample of the protein interaction collection, this result indicates that as much as half of the proteins in our dataset may be modifiers of mutant Htt toxicity,” the authors write. The dataset seemed particularly target-rich, since 17 of the group were genes whose loss of function suppressed neurodegeneration, making them targets for small-molecule inhibitors.
Among the list of genes that modify huntingtin toxicity are several broadly related to autophagy, and to vesicular transport in general, but much more work remains to be done to elucidate the function of these genes in Huntington pathology. Rubinsztein offered ARF a “thumbs up” assessment of the Hughes work. “This is clearly a good strategy for target identification. The work shows a proof of principle for finding genetic modifiers, and those are what we want to target. This paper should have a big impact, not just for Huntington’s but for other diseases and situations where it is important and desirable to confirm biochemical interactions with functional readouts.”—Pat McCaffrey.
Sarkar S, Perlstein EO, Imarisio S, Pineau S, Cordenier A, Maglathlin RL, Webster JA, Lewis TA, O'kane CJ, Schreiber SL, Rubinsztein DC. Small molecules enhance autophagy and reduce toxicity in Huntington's disease models. Nat Chem Biol. 2007 May 7; [Epub ahead of print]
Kaltenbach LS, Romero E, Becklin RR, Chettier R, Bell R, Phansalkar A, Strand A, Torcassi C, Savage J, Hurlburt A, Cha G, Ukani L, Chepanoske CL, Zhen Y, Sahasrabudhe S, Olson J, Kurschner C, Ellerby LM, Peltier JM, Botas J, Hughes RE. Huntingtin Interacting Proteins Are Genetic Modifiers of Neurodegeneration. PLoS Genetics 2007 May 11; 3:e82 doi:10.1371/journal.pgen.0030082 Abstract