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Uncovering the origin of Huntington's disease
One in 10,000 Americans suffer from Huntington's disease — a fatal hereditary disorder for which there is currently no treatment. Most with the disease begin to show symptoms in middle age, developing jerky movements from the increasing loss of brain neurons. Eventually, the continuing loss of neurons precipitates a slide into dementia.
But, new research suggests these symptoms are a late manifestation originating much earlier, in the first weeks of embryo development. With this new finding, scientists, led by Ali Brivanlou PhD of Rockefeller University, may be able to break the impasse in Huntington's research by having developed a Huntington model from human embryonic stem cells.
Published in the journal Development, Brivanlou describes abnormalities in Huntington's neurons not previously associated with the disease. Neurons formed larger and larger structures.
"Our research supports the idea that the first domino is pushed over soon after fertilization with consequences continually falling down the line. The final domino falls decades after birth, when symptoms are observable."
Genes are made of DNA, which is made up of four chemical building blocks referred to as A, C, G and T. These stand for adenine, cytosine, guanine and thymine. They are the ‘letters’ of our genetic code and used as instructions for making proteins, the molecular machines that make our cells function and interact. The huntingtin (HTT) gene can cause Huntington’s disease — if mutated. That mutation affects the letters near the beginning of the HTT gene, where C-A-G is repeated several times. Huntington’s disease happens to people who have too many of these CAG repeats on the HTT gene. Huntington's is one of the few diseases with a straightforward genetic 'bad guy.' One hundred percent of people with a mutated form of the HTT gene develop the disease. The mutation produces extra DNA repeats causing the gene to produce a longer-than-normal protein. A mutated HTT gene continues to make repeat CAG sequences and will increase the likelyhood of an earlier onset of the disease with higher numbers of repeats.
Research on Huntington's has so far relied heavily on animal models of the disease, and left many key questions unanswered. For example, scientists had not been able to resolve what function the HTT gene serves normally, or how its mutation creates problems in the brain. But Brivanlou and research associates Albert Ruzo and Gist Croft, suspected the disease works differently in humans as our brains are much bigger and more complex than lab animals. So they developed a human stem cell-based model. Using CRISPR gene editing technology, they engineered a series of human embryonic stem cell lines which are identical to human cell lines — apart from the number of CAG repeats occurring on HTT genes.
"We started seeing things that were completely unexpected," explained Brivanlou. "In cell lines with mutated HTT, we saw giant cells. It looked like a jungle of disorganization."
When cells divide, they typically each have one nucleus. However, some of these enlarged, mutated cells had up to 12 nuclei — suggesting that the generation of new neurons was being affected by the mutation. Nuclei disruption was directly proportional to how many CAG repeats were present in a mutated gene. The more repeats there were, the more multinucleated neurons. Most huntingtin genes have between 10 and 26 CAG repeats and this range never causes Huntington's disease. But people with over 40 or more repeats in the huntingtin gene will develop Huntington's disease at some time in their life.
"Our work adds to the evidence that there is an unrecognized developmental aspect to the [neuronal] pathology. Huntington's may not be just a neurodegenerative disease, but also a neurodevelopmental disease."
Toxic or essential?
Treatments for Huntington's have typically focused on blocking the activity of the mutant HTT protein, the assumption being that the altered protein was more active than normal protein and thus toxic to neurons. However, Brivanlou's work shows that the brain disruption may actually be due to a lack of HTT protein activity.
To test HTT protein function, the researchers created cell lines completely lacking the HTT protein. These cells turned out to be very similar to those with Huntington's pathology, corroborating the idea that a lack of the HTT protein — not an excess of it — is driving the disease.
The findings are significant as they indicate existing treatments designed to block HTT activity may actually do more harm than good.
Brivanlou: "We should rethink our approach to treating Huntington's. Both the role of the HTT protein and the timing of treatment need to be reconsidered. By the time a patient is displaying symptoms, it may be too late to medicate. We need to go back to the earliest events that trigger the chain reaction that ultimately results in disease. We need to focus new therapies on the cause, not the consequences."
Huntington's disease (HD) is a fatal neurodegenerative disease caused by expansion of CAG repeats in the Huntingtin gene (HTT). Neither its pathogenic mechanisms nor the normal functions of HTT are well understood. To model HD in humans, we engineered a genetic allelic series of isogenic human embryonic stem cell (hESC) lines with graded increases in CAG repeat length. Neural differentiation of these lines unveiled a novel developmental HD phenotype: the appearance of giant multinucleated telencephalic neurons at an abundance directly proportional to CAG repeat length, generated by a chromosomal instability and failed cytokinesis over multiple rounds of DNA replication. We conclude that disrupted neurogenesis during development is an important, unrecognized aspect of HD pathogenesis. To address the function of normal HTT protein we generated HTT+/- and HTT-/- lines. Surprisingly, the same phenotype emerged in HTT-/- but not HTT+/- lines. We conclude that HD is a developmental disorder characterized by chromosomal instability that impairs neurogenesis, and that HD represents a genetic dominant-negative loss of function, contrary to the prevalent gain-of-toxic-function hypothesis. The consequences of developmental alterations should be considered as a new target for HD therapies.
Authors: Albert Ruzo, Gist F. Croft, Jakob J. Metzger, Szilvia Galgoczi, Lauren J. Gerber, Cecilia Pellegrini, Hanbin Wang, Jr, Maria Fenner, Stephanie Tse, Adam Marks, Corbyn Nchako, Ali H. Brivanlou
Funding for this work came from: Cure Huntington's Disease Initiative, New York Stem Cell Foundation, Robertson Foundation, and The Rockefeller University.
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Huntington's neurons show signs of trouble with the appearance of multiple nuclei (blue) in a single cell, decaades before outward symptoms appear. Image credit: Department of Stem Cell Biology and Molecular Embryology, The Rockefeller University.