Huntington’s disease, a rare, inherited neurodegenerative disorder, presents a particularly vexing problem for researchers. Though the genetic mutation that causes the disease was discovered in 1993, it offers no clear strategy for a cure. That has not deterred Steven Hersch, HMS associate professor of neurology at Massachusetts General Hospital. In the Oct. 16 Proceedings of the National Academy of Sciences, he and colleagues report that a small molecule called C2-8 reduces pathology in a mouse model of the disease and has all the hallmarks of a drug. The compound could lead to a new treat`ment for Huntington’s disease, for which there is currently no known cure, and it may help researchers identify new drug targets to exploit.
Yeast Cells Point to Compound
The Huntington mutation, an expansion of a CAG trinucleotide repeat, lengthens a glutamine tract in the huntingtin protein. How the mutated huntingtin sickens neurons is not entirely clear—nor is the normal function of the protein. “The overall issue in Huntington’s disease, compared to a lot of other drug discovery problems, is that while we know the nature of the genetic mutation, that doesn’t give us a conventional drug target, such as an enzyme or a receptor, to block,” explained Hersch. But since huntingtin with an expanded polyglutamine (polyQ) tract is susceptible to misfolding and aggregation, Hersch’s colleague Aleksey Kazantsev, an HMS assistant professor of neurology at MGH, used this propensity as a measure of toxicity and began screening for small molecules that might reduce the pathology of the disease.
The researchers developed a screening method based on yeast cells even though they do not seem to suffer from any toxic effects of accumulating polyQ huntingtin aggregates. “You can have aggregates without necessarily having sick cells, but since aggregates are clearly a hallmark of the disease, yeast offers a way to rapidly screen compound libraries,” said Hersch.
C2-8 surfaced from a screen of more than 15,000 compounds. In addition to reducing polyQ huntingtin aggregation in yeast, the small molecule limits aggregation in cultured neurons, and it rescued neurodegeneration in a fruit fly model of Huntington’s. The researchers reported these findings in PNAS in 2005. “At that point in the story, we knew C2-8 suppressed aggregation, but it had not yet been tested in any model where the disease plays itself out,” said Hersch.
In the Hersch lab, research fellow Vanita Chopra, instructor in neurology Jonathan Fox, and colleagues have now taken C2-8 that further step, testing it in mice. First, they probed the compound to see if it has druglike properties. They found that though it is poorly soluble in water, it can be given as an oral suspension, and that it passes the blood–brain barrier and reaches relatively high concentrations in the brain. They also ran a series of toxicology tests to see if it harmed any major organs, such as the heart or the brain. The compound seemed well tolerated by the mice. “The results suggest that the compound can be taken by mouth and get into the brain, like a medicine will need to, and that it has a preclinical safety profile compatible with moving it into the clinic,” said Hersch. But does it help?
To answer that question, Chopra and colleagues gave C2-8 to R6/2 transgenic mice. These animals produce an N terminal fragment of human huntingtin carrying as many as 150 glutamine residues. R6/2 mice show many Huntington-like symptoms, including involuntary movements, tremor, and seizures. They also accumulate intraneuronal nuclear inclusions composed mainly of mutant huntingtin. The effects of the mutation are apparent as early as 9 weeks old, and the animals have a shortened life span, rarely living longer than four months.
The researchers began administering C2-8 to the transgenic mice beginning at 24 days old. By eight and eleven weeks, the treated mice appeared to be doing better than untreated controls. C2-8 mice fared much better on the “rotarod,” a rotating perch that tests agility, balance, and strength. While 8-week-old control mice typically lasted about 148 seconds before falling off the rod, C2-8–treated mice lasted 235 seconds, or 37 percent longer. At 11 weeks old, they improved even more, outlasting controls by 43 percent. The results indicated that the compound helped reduce loss of motor function.
To confirm that the improvements in motor function were related to disease pathology, Chopra and colleagues examined the brains of treated and control mice. R6/2 mice show a substantial reduction in the volume of the striatum, a region of the brain that sits beneath the cerebral cortex and which is involved in regulating motor and cognitive function. The researchers found that at the highest dose of drug given, striatal neurons in R6/2 mice were about 20 percent larger and less atrophied than control neurons at 13 weeks. The drug did not completely protect the mice, however, since neurons in treated animals were still about 20 percent smaller than in wild-type animals and the mice did not live any longer than untreated controls.
Histological examination also showed that C2-8 helped reduce the total volume of intranuclear huntingtin inclusions by about 35 percent (see figure). Interestingly, the number of aggregates did not seem to change, but they were significantly smaller than in untreated mice. “This may mean that the compound does not affect nucleation of aggregates as much as their growth or turnover,” suggested Hersch. That might also explain why the compound has no effect on survival. R6/2 animals already have aggregates at birth, explained Hersch. “It may well mean that if you gave the compound early enough, or used a different model where aggregates formed later, you might have fewer and smaller aggregates and a more potent benefit.” This is something the researchers are planning to investigate.
Though these results are promising, Hersch concedes that there is much more work to be done. The researchers do not know, for example, how C2-8 works. They know that it does not affect aggregation directly, because when added to a solution of polyQ huntingtin in a test tube, it does not prevent the protein from aggregating. And while Hersch says it is not entirely unreasonable to test this compound in humans given its druglike properties, he thinks it makes more sense to create something more potent before moving on to human trials. Currently, the researchers are focusing on building structural analogs of C2-8, but they also plan to pursue the C2-8 target itself. “That can be very challenging, but right now we have to focus on structural analogs, whereas if we know the target, then we could begin to design totally different compounds that may be more potent inhibitors of the same target,” said Hersch. Identifying what C2-8 binds to could also lead to some important new insights into the disease pathology.
