Several neurodegenerative diseases involve the slow accumulation of a misfolded protein in neurons over many years. The proteins involved in these diseases might differ, but the result is similar — eventually, the neurons die from the build-up of toxic misfolded proteins. Scientists have long been searching for ways to reduce the levels of the disease-driving proteins without also clearing their wild-type counterparts, which typically have myriad crucial functions. Writing in Nature, Li et al.1 show that this can be accomplished using compounds that interact specifically with both the misfolded part of the protein and the neuron’s protein-clearance machinery.

Read the paper: Allele-selective lowering of mutant HTT protein by HTT–LC3 linker compounds

Li and colleagues chose to focus on Huntington’s disease, which is caused by an abnormally long stretch of glutamine amino-acid residues in the huntingtin (HTT) protein. This expanded polyglutamine tract causes HTT to misfold. Affected individuals typically carry one copy (one allele) of the HTT gene that encodes the mutant protein, and one allele that encodes a protein with the normal-length glutamine tract.

Cells are able to degrade the mutant huntingtin (mHTT) through autophagy2 — a clearance mechanism that involves engulfment of proteins by a vesicle called the autophagosome. Li et al. hypothesized that compounds that bind to both the mutant polyglutamine tract and the protein LC3B, which resides in the autophagosome, would lead to engulfment and enhanced clearance of mHTT (Fig. 1). But no such compounds had been reported. The authors therefore conducted small-molecule screens to identify candidate compounds, and used wild-type HTT in a counter-screen to rule out compounds that bind to the normal version of the protein.

Figure 1 | Lowering levels of mutant huntingtin protein. a, Healthy neurons typically carry two copies of the gene that encodes the wild-type version of huntingtin protein (wtHTT). Only two proteins are shown, for simplicity, although many are produced from each gene copy. b, Huntington’s disease involves the expansion of a tract of glutamine amino-acid residues in one copy of HTT protein, producing a mutant version (mHTT) that accumulates in neurons, causing them to shrink and eventually die. Any strategy to decrease levels of mHTT in these cells must not affect wtHTT, which has key functions in the brain. c, Li et al.1 have identified four linker compounds that fulfil this role. Treatment with these compounds inhibits neuronal degeneration in various models of Huntington’s disease. The compounds bind to both mHTT and the protein LC3B — a key component of a protein-clearance pathway called autophagy. This enables selective engulfment of mHTT by a vesicle called the autophagosome, leading to the mutant protein’s degradation.

Li and colleagues initially identified two candidates, dubbed 10O5 and 8F20. These compounds had been shown3,4 to inhibit, respectively, the activity of the cancer-associated protein c-Raf and kinesin spindle protein (KSP), which has a key role in the cell cycle. The team found that 10O5 and 8F20 were able to clear mHTT independently of their effects on these other proteins.

The researchers showed that the regions of 10O5 and 8F20 that interacted with mHTT and LC3B in the screen shared structural similarities. Next, they screened for compounds that shared these structural properties but were structurally distinct from other c-Raf and KSP inhibitors (a compound that acts on mHTT without altering these proteins would be desirable for clinical treatment). This led them to discover two more compounds, AN1 and AN2, that link mHTT to LC3B and thereby selectively reduce levels of mHTT.

Li and colleagues validated their exciting discovery by showing that the four compounds reduced levels of the full-length mHTT protein (not just the protein fragment used in the screen). The compounds lowered levels of mHTT both in vitro — in mouse neurons and neurons derived from the biopsied skin cells of people with Huntington’s disease — and in vivo, in mouse and fly models of the disease.

Role of repeats in protein clearance

A key strength of the compounds identified by Li and co-workers is that they leave levels of wild-type HTT unchanged. This is crucial because HTT has multiple neuronal functions, both during embryonic development and after birth5. Existing mHTT-lowering strategies typically affect both HTT alleles6,7, which is not ideal. Equally, the compounds found by Li et al. did not affect other proteins that contain polyglutamine tracts of variable, but not disease-causing, length. These proteins often have many roles in the brain.

One question that naturally arises is whether treating cells with the compounds led to enhanced autophagic clearance of proteins other than mHTT. Li et al. assessed the levels of the repertoire of proteins in the cortices of mice that carried an mHtt allele. They found changes in the abundance of a small percentage of proteins in mice treated with the compounds, compared with untreated animals. What remains unclear is whether the levels of some proteins decreased because mHTT levels were diminished, or because of autophagy. Modest changes in protein-expression level (in the 20–30% range for some wild-type proteins) can cause neurological deficits8, so pinpointing any off-target effects of the compounds will be a crucial next step. Even effects that initially seem inconsequential might build up over the course of long-term therapy, becoming as problematic decades later as the original toxic protein.

Despite these concerns, the authors found encouraging evidence that the compounds could produce functional improvements in models of Huntington’s disease across three species. First, patient-derived neurons treated with each of the compounds showed significantly less shrinkage, degeneration of neuronal projections and cell death than was seen in untreated neurons. Second, flies that model Huntington’s disease and were treated with the compounds recovered climbing ability and survived longer than did untreated counterparts. Third, treated mice that model Huntington’s disease showed improvements in three motor tests, compared with untreated mice. That said, preclinical trials in mice will be necessary to ascertain that the benefit is sustained and robust over the course of long-term therapy.

Finally, Li et al. analysed mutant ataxin-3, a protein that is involved in a neurodegenerative disorder called spino-cerebellar ataxia type 3. The researchers found that the compounds targeted the long polyglutamine tract of mutant ataxin-3 and lowered protein levels. We already know that small reductions in the levels of mutant ataxin-1, ataxin-2 and ataxin-3 can reduce the severity of spino-cerebellar ataxia types 1, 2 and 3, respectively, in mouse models9–11. Thus, this therapeutic strategy might be useful not only for Huntington’s disease, but also for other diseases involving expanded polyglutamine tracts.

Moving forwards, there are three major research paths to pursue. The first involves establishing the mechanism by which Li and colleagues’ compounds recognize proteins with expanded polyglutamine tracts but spare normal proteins. Perhaps the compounds recognize a particular structural conformation that arises only after the polyglutamine tract exceeds a specific length. The second involves testing the compounds in other models of polyglutamine disorders and assessing their effects.

The third path involves conducting similar small-molecule screens for compounds that can clear polyglutamine proteins using other types of protein-clearance machinery. For instance, small molecules dubbed proteolysis-targeting chimaeras (PROTACs) link a ubiquitin ligase enzyme to a protein of interest. The enzyme tags the protein with ubiquitin groups, leading to the protein’s degradation by a cellular machine called the proteasome12. PROTACs have yet to be applied to a polyglutamine-expanded protein. But given that some of these proteins are degraded by the proteasome, the strategy could well prove viable — as long as it targets only the abnormally long polyglutamine tract.