Altman and Fu were fortunate enough to get a behind-the-scenes look at where HD breakthroughs are being made. They attended a lab meeting, where several of MacDonald’s postdoctoral (post-PhD) research fellows and interns gave presentations about their individual research projects. “Presenting research and sharing results is an important part of science,” said MacDonald.
Research fellow Julie Woda discussed her work on huntingtin normal function. Past research has shown that the protein is quite large and contains 36 HEAT-like repeats. The acronym HEAT comes from four proteins in which these repeated sequences have been found. These HEAT repeat sequences, which fold up into a spiral structure (alpha helix-loop-alpha helix), may serve as docking sites for other proteins. Most huntingtin partner proteins (“binding partners”) bind to one of its end segments. Based on its binding partners, huntingtin is involved in a variety of processes in the cell, including signal transduction, regulation of transcription (a big step in the process of turning DNA code into a protein), trafficking of molecules and other materials within the cell, maintaining the function of the cell’s cytoskeleton, and gene splicing.
Woda believes that the normal huntingtin protein is essential for the development of the human embryo. Studies conducted in the past have revealed that inactivating the Huntington gene (which produces the huntingtin protein) in a mouse results in abnormal embryo development. Supplying the mouse with either the altered (mutant) or wild-type (normal) huntingtin protein can rescue the non-huntingtin phenotype, meaning that the mouse can be brought back to a normal state. This phenomenon occurs because altered huntingtin acts like wild-type huntingtin, but gains some kind of new functions, such as a kind of “stickiness” that might make it more likely to interact inappropriately with another protein.
While touring the MacDonald lab, Altman and Fu spoke with some other postdoctoral research fellows about their research projects. Each trainee approaches HD from a different angle. For example, research fellow Elisa Fossale and intern Sony Mysore work together to prepare tissue cultures (a technique used to grow body tissue outside the body on a culture medium, a liquid or gel-like substance containing nutrients). The tissues kept in the culture medium are from the striatum of the brains of mice that carry the Huntington gene homolog (the mouse version of the gene). This brain region is known to deteriorate during the course of HD. The researchers are currently looking at differences in energy metabolism in mutant and wild-type striatal nerve cellclones. Preparing the cells for experimentation is a laborious process, generally taking about four to six hours. After this preparation, they can operationalize the changes in energy metabolism as changes in levels of the cell’s major energy carrier, ATP. These researchers have observed that ATP levels decrease in mutant cells, suggesting that metabolism is sluggish. Therefore, the cells are weakened through decreased efficiency in producing energy for their own survival. This information may be important to other researchers who use these types of cells for screening drugs that have the potential to combat decreases in energy metabolism.
Intern Sony Mysore learns how to perform a tissue culture.
HOPES team member Shawn Fu looks at striatal nerve cells under a microscope.
Other researchers and technicians work on a type of tissue culture called cell lines. Cell lines represent generations of a primary or original culture, such as a bacterial colony that arose from a single cell. Cell lines are “immortalized” biochemically so that they continue to reproduce themselves and can subsequently be used to develop tests called screening assays for potential drugs that are available from biotechnology companies and academic laboratories under contract. Once researchers have figured out the appropriate target for a drug (such as mitochondria that make ATP or nerve cell transportation machinery slowed by the effects of mutated huntingtin), they can test these chemical compounds to see which one of them really goes after the target.
Cell lines in cold storage in the MacDonald lab.
Alex Lloret, another research fellow in the lab, studies sections of the brains of transgenic mice with different genetic backgrounds to observe the formation of huntingtin in the nucleus and later formation of neuronal inclusions (NI) in the nuclei of nerve cells. Neuronal inclusions are clumps of mutated huntingtin protein fragments that result from having HD. There is generally one inclusion per nucleus in mutant mice (those with excess CAG repeats).
Lloret also looks for proteins that interact with the normal huntingtin protein. He has found that huntingtin’s function is similar to that of a scaffold, or facilitator, because it organizes groups of proteins that play various roles in signal transduction in nerve cells. He is currently in the process of mapping one end fragment of the huntingtin protein in order to understand how other protein complexes bind to it. He is also looking at HEAT repeat sequences in huntingtin and trying to determine how these sequences interact with many protein complexes.
Researcher Alex Lloret prepares sections of transgenic mouse brains for experimentation.
Sections of transgenic mouse brains are mounted on slides for viewing under a microscope.
Other methods of investigating HD include the genomic, RNA interference (RNAi), and biochemical approaches. The genomic approach entails screening various genes in a human or mouse’s body for genetic markers, measuring the transcription level (amount of transcription) of each gene, and measuring the amount of RNA produced. The RNAi approach consists, in part, of describing the normal function of the huntingtin protein. Working with HD cell models, researcher Songshan Jiang uses short interference RNA (siRNA) to stop the translation of the huntingtin protein. Nerve cells treated with siRNA do not contain huntingtin at all. Jiang then examines the phenotype of the cells, looking at the way in which they are affected by the lack of huntingtin. This kind of observation can tell him what huntingtin’s regular function is within the nerve cells.
The biochemical approach involves understanding and describing how the structural and physical properties of biological molecules (such as proteins) influence the functions of those molecules. To study these molecules, they must first be purified. One of the biggest breakthroughs in biochemistry was the introduction of chromatography, which made it possible to separate and isolate different kinds of molecules quickly and efficiently. In the MacDonald lab, a technique called gravitation chromatography is used to compare the mass of the mutant and wild-type huntingtin protein. Researcher Ihn-Sik Seong has found that mutant huntingtin is in large complexes that are even bigger (have more mass) than the complexes with normal huntingtin. They also regularly use high pressure liquid chromatography (HPLC) when studying changes in the biochemical molecules that are due the presence of mutant huntingtin in cells. Generally, protein molecules are separated according to their physical properties such as their size, shape, and affinity for other molecules. HPLC facilitates the separation of molecules under high pressure in a stainless steel column filled with a special chemical substance called a matrix. A computer controls both a pump and a means of collecting data. By using HPLC, researchers can describe various properties of mutated and wild-type huntingtin. Knowing more about the characteristics of wild-type huntingtin will give the researchers a basis of comparison for mutant huntingtin since they each have different properties.
High pressure liquid chromatography (HPLC) machines in the MacDonald lab.
Last Modified: 05/22/2009
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