The HD protein is toxic in the cytoplasm

HD Lighthouse Contributing Editor's Comment: 

The HD version of the huntingtin's protein is sticky and it has a tendency to clump up. The discovery of protein aggregates in the brains of transgenic mice and then in brain tissue from HD patients caused enormous excitement in 1997. The immediate suspicion was that the aggregates were the cause of the disease especially since aggregates were found in the nucleus of the cell even though the huntingtin's protein is a cytoplasmic protein.

Research has shown that it is the nuclear location and accumulation of the HD protein and its toxic fragments which is a major source of disease pathology, not the nuclear aggregates in and of themselves. And, as Lighthouse readers know, there are other things that go wrong in Huntington's Disease.

Whether the HD protein in the cytoplasm might have a role in disease pathology has not received much attention until now. Clearly the cell is not able to clear it away properly in the cytoplasm either or the aggregates wouldn't be present.

Emory University researchers have generated an intracellular antibody which acts against the HD protein in the cytoplasm but not in the nucleus of the cell. They delivered an intrabody gene to the striatum of HD mice through an adenoviral vector.

The intrabody reduced the toxicity of the HD protein, the number and size of the aggregates in the cytoplasm, and the distribution of the the HD protein in neuronal processes. The result was that movement symptoms were less severe in the intrabody group than in untreated animals. However, survival time was not prolonged.

It's not surprising that life span wasn't extended given all we know about the damage the HD protein causes in the nucleus of the cell, including dysregulating the transcription of other needed genes. However, the improvement in symptoms does show that the HD protein is toxic in the cytoplasm too, and this is important to know for planning treatments.

Could an intrabody be used to treat the disease? There seems to be real potential here but there are a number of issues to be worked out, especially delivery. AAV delivery is not as efficient as it would need to be, although improvements are in the works (see http://www.hdlighthouse.org/showUpdate.php?p_articleNumber=569 ). It's also intrusive because it doesn't cross the blood brain barrier and has to be delivered directly to the brain through surgery. However, as Dr. Li points out, it may be possible to develop a drug which will act in the same way as the intrabody.

Reference

Anne Messer and Julie McLear. "The therapeutic potential of intrabodies in neurologic disorders:focus on Huntington and Parkinson diseases." BioDrugs 2006;20(6):327-33.

-- Marsha L. Miller, Ph.D.
Posted to the HDL: 06-14-2008

Xiao-Jiang Li, M.D., Ph.D.
Distinguished Professor of Human Genetics
Emory University

Suppression of neuropil aggregates and neurological symptoms by an intracellular antibody implicates the cytoplasmic toxicity of mutant huntingtin

Chuan-En Wang, Hui Zhou, John R. McGuire, Vincenzo Cerullo, Brendan Lee, Shi-Hua Li, and Xiao-Jiang Li

press release: 'Intrabody' can mop up mutant protein in Huntington's disease model

Scientists have created a tool for mopping up the clumps of mutant protein that drive neurodegeneration in Huntington's disease. Emory University researchers engineered a virus to make an intracellular antibody or "intrabody" against huntingtin, the protein whose mutant forms poison the brain cells of people with Huntington's.

Injecting the virus into the brains of mice that make mutant huntingtin improves their ability to move their limbs, although it does not prolong their lives.

The results will be published online and are scheduled for publication in the May/June issue of the Journal of Cell Biology.

Although other researchers have shown that various intrabodies can protect cells from mutant huntingtin, the Emory team was the first to examine the effects of an intrabody in living mice, says senior author Xiao-Jiang Li, PhD, professor of human genetics at Emory University School of Medicine.

Delivering the intrabody to brain tissues in people would be a formidable challenge, because it would require some form of gene therapy. However, it may be possible to use information about the intrabody's structure to find drugs that mimic its effects, Li says.

Huntington's affects about 30,000 people in the United States and usually begins in young- to mid-adulthood with the slow destruction of brain cells, leading to involuntary movements, cognitive impairment and sometimes depression or paranoia. Another 150,000 people are believed to have mutations that cause the disease, but have not begun to show clinical symptoms. The disease is fatal and currently there is no way to slow its development, although some medications can alleviate symptoms.

Disease-causing mutations involve a lengthening of part of the gene for huntingtin, so that it repeats three letters (CAG) of the genetic code dozens of times. Mutant proteins have a region consisting of the same amino acid (glutamine) many times, called poly-glutamine, which makes the proteins clump together inside brain cells.

Li says scientists who work on Huntington's have been studying where inside the cell the clumps have their toxic effect: brain cells' nuclei or in their axons and dendrites.

"Our goal here was to create a tool that could distinguish between the accumulation of mutant proteins in the nucleus and the cytoplasm," he says. "The intrabody binds huntingtin proteins with expanded poly-glutamine regions and it only works in the cytoplasm, not the nucleus."

Li and his colleagues showed that cultured cells that make both the intrabody and mutant huntingtin are able to get rid of the mutant protein faster and have fewer clumps of huntingtin.

Even though the intrabody only travels within the cytoplasm, it still alleviated the motor problems of mice that make mutant huntingtin when injected into the striatum, the scientists found. The striatum is the part of the brain most affected by Huntington's disease.

Li says finding an antibody that prefers to bind mutant, aggregated protein could also prove useful in the study of other neurodegenerative disorders, such as Alzheimer's disease or Creutzfeldt-Jakob disease.

"Several neurodegenerative diseases appear to involve defects in protein folding and metabolism, leading to the accumulation of protein aggregates inside cells," he says. "Our study suggests a strategy for dissecting the harmful effects of these protein aggregates in other diseases."

journal abstract

Mutant huntingtin accumulates in the neuronal nuclei and processes, which suggests that its subcellular localization is critical for the pathology of Huntington's disease (HD). However, the contribution of cytoplasmic mutant huntingtin and its aggregates in neuronal processes (neuropil aggregates) has not been rigorously explored. We generated an intracellular antibody (intrabody) whose binding to a unique epitope of human huntingtin is enhanced by polyglutamine expansion. This intrabody decreases the cytotoxicity of mutant huntingtin and its distribution in neuronal processes. When expressed in the striatum of HD mice via adenoviral infection, the intrabody reduces neuropil aggregate formation and ameliorates neurological symptoms. Interaction of the intrabody with mutant huntingtin increases the ubiquitination of cytoplasmic huntingtin and its degradation. These findings suggest that the intrabody reduces the specific neurotoxicity of cytoplasmic mutant huntingtin and its associated neurological symptoms by preventing the accumulation of mutant huntingtin in neuronal processes and promoting its clearance in the cytoplasm.

Source: Journal of Cell Biology 2008 May 26. [Epub ahead of print]