10 March 2006. The appearance of clots of abnormal proteins inside neurons is characteristic of many neurodegenerative diseases. These intracellular inclusions were long presumed to be toxic manifestations of protein misfolding, and most research aimed in their direction seeks ways to prevent or dispel the aggregates. But what if some inclusions are protective, as suggested recently for the huntingtin protein (see ARF related news story)? If inclusions are, in fact, a cell’s attempt to quarantine offensive peptides, shouldn’t researchers be trying to help that effort?
Some are, according to a paper out this week in PNAS Early Edition. Using a high-throughput screen for inclusion formation in cells, Aleksey Kazantsev of the Massachusetts General Hospital in Boston and collaborators at MIT have zeroed in on small molecules capable of increasing the aggregation of a polyglutamine-expanded huntingtin-GFP fusion protein. The most potent hit, they found, also increases inclusion formation by the Parkinson disease protein α-synuclein. Even in the face of overall increased protein levels, the compound partially reversed proteasome dysfunction induced by huntingtin and prevented the toxicity of α-synuclein in cultured cells.
The results are consistent with the idea that intracellular aggregates of toxic proteins could serve as a sink to protect cells, though many questions remain about the therapeutic potential of the compounds themselves, or the approach. By using a novel strategy to find these small molecules, Kazantsev and colleagues have opened up a new opportunity to investigate the role of inclusions in Huntington and Parkinson disease. In addition, their new compounds will likely steer researchers to a better understanding of the regulation of inclusion development in these and possibly other diseases.
The new study builds on previous efforts by Kazantsev, MIT’s David Housman (who communicated the paper to PNAS), and colleagues to establish a cell-based assay for polyglutamine-induced protein aggregation (Apostol et al., 2003). That assay used PC12 cells engineered with a fusion protein that linked the N-terminus of huntingtin to the enhanced green fluorescent protein reporter to detect compounds that either enhance or inhibit protein expression. Last year, the group reported using the cells to hunt down compounds that prevent aggregation (Zhang et al., 2005). Now, after running through a library of 20,000 compounds, first author Ruth Bodner and coworkers report finding five compounds with the opposite effect of promoting protein accumulation and inclusion formation. The mechanism by which this happens is not clear, but it is specific for polyglutamine-expanded huntingtin, as the authors did not see it with normal N-terminus-EGFP fusion protein.
Since the huntingtin reporter gene was not toxic for the assay line, the researchers took another route to measure whether their lead compound, B2, could mitigate the pathologic effects of huntingtin. By enlisting a different reporter system, this one for proteasome-mediated clearance, they showed that expression of a polyglutamine-expanded form of huntingtin slowed the pace of proteasomal processing. Treatment with B2 resulted in normal clearance of the tagged protein, apparently reversing the proteasomal dysfunction associated with huntingtin expression.
Similarly, in α-synuclein expressing cells, B2 treatment caused a modest but statistically significant decrease in cell death, in the face of an increase in both α-synuclein expression and inclusion size and number.
The true test of B2 will come in models of HD that measure the effects of full-length huntingtin protein on neuron health. Kazantsev and colleagues have shown before that compounds they discovered to decrease huntingtin inclusions in their cell assay can block neurodegeneration in a Drosophila HD model. It will be critical to learn if increased inclusions in the same cell assay also predict positive results in Drosophila or other models.—Pat McCaffrey.
Bodner RA, Outeiro TF, Altmann S, Maxwell MM, Cho SH, Hyman BT, McLean PJ, Young AB, Housman DE, and Kazantsev AG. Pharmacological promotion of inclusion formation: A therapeutic approach for Huntington's and Parkinson's diseases. PNAS published March 6, 2006, 10.1073/pnas.0511256103.
Q&A with Aleksey Kazantsev
Q: What inspired this academic attempt at translational research?
A: It grew out of a sense of moral responsibility to contribute to drug discovery. It’s frustrating that there is a genetic diagnosis for Huntington’s disease but no good drugs. This disease is small enough to have “orphan” status; that is, its market is too small to provide strong financial incentives for drug companies to invest. We also found it difficult to obtain government funding for drug discovery in our academic setting. The major support for the published manuscript came from patient families.
Q: What is B2’s activity in other assays for htt toxicity, such as the Drosophila model or the hippocampal slice cultures you've used in the past to test aggregation inhibitors?
A: These experiments are on our list of things to do. Prior to testing in invertebrate and mammalian models, I'd like to optimize the lead compound for potency. That will increase our chances to detect effects in other systems.
Q: How might B2 and related compounds increase the levels of protein, or formation of inclusions of htt or α-synuclein in your cell assay?
A: To recapitulate disease features in both HD and PD assays, we dramatically overexpressed mutant polypeptides. These high mutant levels forced the formation of aggregates in a certain percentage of cells. Since the role of inclusions in pathogenesis has been disputed over the years as being either harmful, neutral, or protective, we asked this question: If a small molecule facilitates aggregation and increases inclusion-positive cells, what would be the translation of this event on the disease phenotype? We believe our data strongly imply a protective role of inclusions.
For proper evaluation of the experimental results, we were concerned that B2 might down-regulate levels of huntingtin and α-synuclein in our model systems. In this case, results would be trivial, since neurotoxicity was mediated by overexpression of huntingtin and α-synuclein. To observe some up-regulation in the level of α-synuclein with B2 was actually a relief.
We know that aggregated polypeptides have a dramatically different rate of degradation than soluble proteins; it is quite slow. Mutant protein synthesized de novo may join the aggregates without being degraded, and be preserved there, or remain in soluble form prior to degradation. That means that mutant polypeptides will slowly accumulate. However, since they get trapped in aggregates, they are harmless for the cells, in contrast to soluble neurotoxic species. Hence, facilitating aggregation can be therapeutic, since soluble pathogenic (i.e., neurotoxic) species end up trapped in inclusions. Therefore, we have to distinguish the increase of total protein level (which would be bad if the protein was soluble) and increased aggregated protein level (which is protective).
My favorite analogy is with kitchen trash: When sealed in garbage bags in the cabinet under the sink, it does not bother you too much. However, when spread over the entire space, it is nasty.
Q: How do you view the prospects of a compound that increases inclusions, but could also potentially increase soluble protein levels, as well?
A: That would be highly undesirable, and the issue has to be addressed. I think the probability of our compounds increasing endogenous protein levels is low. This statement is based on prior experience with general transcriptional activators, that is, the histone deacetylase (HDAC) inhibitors, which were effective in mouse models of Huntington disease. HDAC inhibitors greatly induced expression levels of exogenous polypeptides in our model systems, yet did not affect the level of endogenous huntingtin protein.
Q: Is the B2 compound a candidate for further development, or a proof-of-concept reagent?
A: Our major focus now is identifying B2’s molecular target. Knowing this drug target will stimulate further drug development for HD and PD. We are also looking for novel scaffolds, and other potential drug leads, using our cell-based assays. Abstract