Big Step in Understanding the Etiology of Huntington's Disease

Trushina et al from the Mayo Clinic have made a big advance in understanding the etiology of Huntington's disease.

Huntington's disease is a progressive and ultimately fatal disease that is characterized by uncontrollable limb movements and progressive dementia and psychosis. It is 100% penetrant and shows autosomal dominant inheritance in the causative gene called Huntingtin. We know that Huntingtin has a triplet repeat region -- the same three nucleotides over and over again -- that can be of variable size. Have a relatively short one and you are fine. Have long one and you are going to get Huntington's disease.

Unfortunately, no treatment has ever been found, largely because we have no idea why the defective gene causes the disease. Trushina et al have taken a big step in that understanding.

It was known that the Huntingtin protein normally interacts with elements of the cellular trafficking machinery. The trafficking machinery is responsible for endocytosing -- swallowing in membrane vesicles -- things on the outside that you want in and exocytosing -- spitting out in membrane vesicles -- things you want on the outside. It is a tightly regulated process involving many, many proteins. Basically if you want to endocytose something there are three pathways that are responsible. They are named for the protein that coats the vesicle -- the little membrane baggie pulled in:

• One of those pathways uses a protein called clathrin. Most times when a receptor binds to a protein outside the cell and brings it in, it uses the clathrin pathway.
• The other pathway uses a protein called calveolin. This protein is associated with things called calveolae. Calveolae are invaginations of the membrane that are highly lipid rich. They do a couple things, one of which is regulate signaling because some proteins tend to congregate in lipid rich domains. The other thing they do is regulate cholesterol metabolism -- which is the important aspect here.
• The final pathway is for uncoated vesicles. Not relevant here.

Anyway, it was known before that Huntingtin associates with members of the clathrin associated endocytic pathway. It was known that overexpression of mutant Huntingtin in cells prevents endocytosis. However, these researchers established that the method of endocytosis that Huntingtin impedes is the calveolin-related (that is a bit of an oversimplification...it probably stops both).

This is relevant because calveolin-related endocytosis is important in regulating cholesterol metabolism. The cholesterol metabolism in the brain is in many ways separated from the rest of the body. Under normal circumstances, the cholesterol does not cross the blood-brain-barrier (BBB) (whether it does under pathological circumstances is contraversial). Most of the cholesterol is synthesized in-house and it stays around for a long time (usually months), so the creation of it and more importantly where it is becomes important.

Cholesterol is a primary constituent of cell membranes. The level of cholesterol in membranes can also have consequences for cell signaling. For example, more cholesterol in membranes increases the rate of A-Beta formation (A-Beta is what forms the crud that accumulates in your brain in Alzheimer's disease). Dysregulated cholesterol is bad for the brain.

So we get to what the researchers found. When they overexpressed mutant Huntington in neurons they found that calveolin-related endocytosis decreased. Furthermore, they found that cholesterol began to accumulate in the membranes of the neurons. Interestingly, to prove that this was the correct pathway, they knocked out calveolin-1 in the neurons and showed that they effect went away.

Here is my favorite figure (click to enlarge):

Figure 4. Cholesterol accumulates in striatal neurons and tissue in vitro and in vivo. (A) Fluorescence images of filipin staining in primary striatal neurons from control (FVB/N) and HD72 mice. Control and HD72 neurons were plated side by side, cultured in cholesterol-free medium, fixed at the days indicated and stained with filipin. Images were taken on an Olympus fluorescence microscope using 100x oil objective. Scale bar, 10 µm. (B) Quantification of filipin staining in striatal neurons from control (FVB/N) and HD72 mice at the indicated days in vitro. Values represent relative fluorescence units and are the mean±SD of at least 30 cells in each of six independent experiments. *P<0.001. Black bars, control mice; gray bars, HD72. (C) Cholesterol accumulation in brain tissue of HD72 mice increases with age and correlates with progression of neurological abnormalities. (Right) Brain slices (30 µm) from control (FVB/N) and HD72 mice 40 weeks old stained with filipin. Box indicates the portion of striatum taken for quantitative analysis. HD72 mice display clasping phenotype (bottom left) absent in control mice (top left) that coincides with progressive accumulation of cholesterol in brain tissue [bottom right and (D)]. (D) Expression of mhtt causes accumulation of cholesterol in the striatum of HD72 mice in vivo. Striatal tissue was collected from control (FVB/N) and HD72 age-matched mice, cholesterol was extracted and measured using thin-layer chromatography (see Materials and Methods). Black bars, control mice; gray bars, HD72.

Translation to English: They took a mouse that is engineered to express the mutant Huntington (HD72). When they purified neurons from that mouse and compared them with normal mice, they found that the longer they spent in culture the more cholesterol they accumulated in their membranes -- as measured by a stain for cholesterol called filipin. They looked at the animals brains in the striatum -- the area most affected in Huntington's disease. They found progressive accumulation of cholesterol there as well.

They argue that this is a new mechanism for how the disease kills neurons. The mutant Huntingtin causes accumulations of cholesterol, which cause progressive neuronal degeneration in key areas of the brain:

Cholesterol is essential for promoting synapse formation and maintaining membrane integrity in CNS neurons (53,54). Moreover, defects in caveolae and/or perturbation of cholesterol homeostasis have been increasingly implicated in toxic mechanisms for Alzheimer's disease (AD), Parkinson's disease, Niemann-Pick Type C and other lipid storage diseases (55-60). Our discovery that mhtt inhibits caveolar-related endocytosis and causes accumulation of cholesterol potentially links HD and other neurodegenerative diseases. In rodent brain, depletion or accumulation of cholesterol can cause neurodegeneration with AD-like symptoms (55). In prion disease, glycosylphosphatidylinositol anchoring of prion protein (PrP) in the endoplasmic reticulum directs the PrP to the Golgi into cholesterol-rich caveolae-like domains. In caveolae, degradation of PrP slows and appears to be cholesterol sensitive (61). On the basis of these data, Prusiner and coworkers have raised the possibility that caveolae may be the sites of PrP misfolding facilitating its conversion to the aggregated, pathogenic form (61).

My suspicion is that there are many other aspects to the disease, but this may prove to be an important one. Nice work.

Hat-tip: Eurekalert.