The mystery of Huntington disease (HD) is how a toxic protein that is expressed in all the cells in the body kills only a select subset of neurons in the brain. In the disease, the mutant huntingtin protein (mHtt) affects mainly neurons in the striatum, leading to the movement disorders characteristic of HD. To a lesser extent mHtt affects the cortex, leading to dementia. The secret to these limits may lie in the selective expression of a partner protein that cooperates with huntingtin to elicit toxicity, according to a paper out in the June 4 Science. The work, from Solomon Snyder’s lab at the Johns Hopkins University School of Medicine, suggests that a G protein that is expressed mainly in the striatum, and somewhat in the cortex, is required for the mutant huntingtin protein to kill cells, likely through its effects on protein solubility. The work supports the idea that soluble huntingtin, not aggregates, is responsible for cell death, and could lead to a new target for reducing the protein’s toxicity.
The G protein, Rhes (Ras homolog enriched in striatum) was discovered over a decade ago by differential cloning of striatal transcripts (Falk et al., 1999), and has mainly been studied for its role in dopamine neurotransmission. Using coimmunoprecipitation approaches, first author Srinivasa Subramaniam was able to demonstrate that mHtt, but not wild-type Htt, formed a complex with Rhes in cells overexpressing the two proteins. The researchers found the same complex in striatal cells from transgenic mice expressing mHtt. In cultured HEK293 or striatal cells, neither Rhes nor mHtt alone caused significant cell death, but the combination did. Finally, the researchers showed that shRNA knockdown of Rhes in PC12 cells prevented cell death induced by mHtt.
How does Rhes mediate huntingtin toxicity? mHtt forms aggregates in cells, but similar to the amyloid peptide and other disease-causing proteins, the toxic culprit seems to be soluble forms of the peptides. In the case of mHtt, post-translational modification with the small, ubiquitin-like modifier SUMO has been shown to decrease aggregation and increase neurotoxicity in a fly model of the disease (see ARF related news story on Steffan et al., 2004). Subramaniam and colleagues found that in HEK293 cells, expression of Rhes increased mHtt SUMOylation and reduced aggregation. As in the fly studies, SUMOylation of mHtt correlated with cell death: mutation of the mHtt lysine acceptor residues that link to the SUMO peptide enhanced aggregation and rendered mHtt non-toxic in cells that also expressed Rhes and the SUMOylating enzyme SUMO1.
The results suggest that Rhes controls SUMOylation of mHtt, and further studies showed that Rhes associates with the ubiquitin-conjugating enzyme Ubc9, a cellular E2 ligase known to function in SUMOylation. Furthermore, Rhes stimulated SUMOylation of mHtt in test tube reactions in the presence of SUMO1. Rhes was not exclusive to mHtt, and it could stimulate SUMOylation of other cellular substrates, though not wild-type Htt. Finally, the investigators showed that the SUMOylation, disaggregation, and enhancement of Htt toxicity by Rhes relied on the farnesylation of Rhes and its membrane localization, but not on its GTPase activity.
These in vitro results imply that the toxicity of huntingtin may be circumscribed by the expression pattern of Rhes. That fits with what is known of Rhes distribution. “Rhes is very selectively concentrated in the corpus striatum, but there is also a good amount in the cerebral cortex. There is no Rhes in the cerebellum. Interestingly in HD, the primary initial symptoms are all motor, and then you get dementia. You never ever get cerebellar symptoms. It all fits with the localization of Rhes,” Snyder told ARF. The caveat is that all of the studies looking at Rhes expression have been in rodents, although Snyder reports that they have preliminary evidence that the pattern holds up in human brain.
The closest relative of Rhes, the dexamethasone-induced Ras protein 1 (Dexras1) is widely expressed throughout the brain, and recently has been implicated in regulating the signaling pathways emanating from amyloid precursor protein (Lau et al., 2008). In fact, Snyder says his lab first became interested in Rhes because they had spent 10 years studying Dexras1 function. Despite the similarity of the two proteins, Dexras1 does not promote Htt toxicity, he says. That role is unique to Rhes.
“The report by Subramanian and colleagues presents evidence for a potentially important new mechanism elucidating a pathway for striatal neuronal degeneration in Huntington’s disease,” says Michael Levine of the University of California at Los Angeles, who was not involved with the study. “This mechanism has the potential for uncovering a novel therapeutic target but still needs validation in a mammalian organism in an in vivo preparation where neurons exist in an environment in which both cell autonomous and cell-cell interactions have important roles.”
Snyder reports that those in vivo experiments are underway. His lab is now crossing Rhes knockout mice (Spano et al., 2004) with Huntington mice, and if the model is correct, the loss of Rhes should alleviate symptoms. The researchers will have results within a year, and an affirmative would boost the idea that Rhes itself or the Rhes/huntingtin interaction could be a promising target for neuroprotection in HD.—Pat McCaffrey
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- Subramaniam S, Sixt KM, Barrow R, Snyder SH. Rhes, a striatal specific protein, mediates mutant-huntingtin cytotoxicity. Science. 2009 Jun 5;324(5932):1327-30. PubMed.