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McMaster Lab Protein Study Targets Huntington's

Photonics.com
Feb 2007
Kipp Lynch, PhD

HAMILTON, Ontario, Feb. 28, 2007 -- A new bioimaging facility at McMaster University is shedding light on how kinase inhibitors might prevent the protein that causes Huntington's disease from accumulating in the nucleus of brain cells, which could ultimately lead to a treatment for the disorder.

Researchers at Dr. Ray Truant’s Laboratory of Protein Dynamics and Polyglutamine Expansion Diseases Research at McMaster study how proteins move throughout the cell’s organelles. Using Förster resonance energy transfer (FRET) and, in particular, by detecting FRET by fluorescence lifetime intensity measure (FLIM), they have created novel techniques that enable them to analyze protein-protein interactions in vivo.
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Three proteins viewed in a live cell (Photo: raytruantlab.com)
Truant’s bioimaging facility is equipped with a series of scopes that are tuned for live cell imaging and for fluorescent proteins; these will eventually allow the team to develop a robust assay inside of live cells that they can then take into their Evotek Opera high-content screening scope, which is being retrofitted in Germany with multiphoton (MP) laser and FLIM. Soon the lab will be able to do high-throughput screening, using 384 well plates, at the rate of 100,000 wells per day in live cells at the level of FLIM.

Truant said the scope also works at 63x magnification using a special system to pump in water immersion at each position and pump that out when the plate comes back.

“In that respect, it is a unique system in that you don’t have the magnification problems that you have with other systems of high-content screening,” he said.

The lab has a spinning disk with multiple lasers as a standard Nipkow disk scope and a Leica SP5; it is probably the only one in North America that has an additional two-photon MP laser and spectral FLIM output in two channels, Truant said. 

His team is researching polyglutamine expansion illnesses such as Huntington’s, spinocerebellar ataxias, Kennedy’s disease and dentatorubropallidoluysian atrophy (DRPLA), all of which share one fundamental biological defect: a CAG DNA triplet-repeat expansion in the gene’s open reading frame. Recently, Truant has turned his attention to the huntingtin and ataxin-7 proteins. While huntingtin is required for normal function and is present throughout all cells in the body, in the disease state it results in Huntington's disease, which affects about one in 25,000 individuals.

Before coming to McMaster University, Truant was a postdoctoral researcher working with Dr. Brian Cullen at Howard Hughes Medical Research Institute at Duke University in North Carolina.

“We were looking at nucleocytoplasmic transport of HIV accessory proteins,” Truant said. “I developed a good toolbox to analyze nucleocytoplasmic transport, and after three years, I thought the research was complete with HIV.”

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Practical three-color live cell imaging of TAP/NXF1 (blue), ASF (green) and huntingtin (red) in a live cell (from Xia J, Kim SH, Macmillan S, Truant R, Biol Proced Online. 2006;8:63-8)
His interests led him to look for other disease situations that seemed to involve a mislocalization of the protein between the nucleus and the cytoplasm.

“I found that it looked like mislocalization was the case with not only Huntington’s, but the other ployglutamine diseases such as Kennedy’s and spinal and bulbar muscular atrophy (SBMA),” he said.

When Truant arrived at McMaster in 1999, he originally took a comparative biology approach to examine any similarities among the polyglutamine diseases at the level of cell biology.

“We found that there was no commonality at the level of the gene structure and the sequence; they are all completely different proteins doing completely different things,” Truant said. “But a lot of them yield a very similar pathology. For example, ataxia -- the protein responsible for Spinocerebellar -- is very different from huntingtin, yet both of them have the common theme of polyglutamine in the protein.”

Truant and his colleagues have observed that all of the proteins for these polyglutamine disorders, such as ataxin-1 and ataxin-7 as well as huntingtin, are using different mechanisms to shuttle in and out of the nucleus. But, as a result of polyglutamine expansion, nuclear export is inhibited. Other scientists have shown that the same is true for the androgen receptor in Kennedy’s disease and atrophin-1 in DRPLA.

“It’s striking that of the nine known polyglutamine disease proteins, it looks like seven out of the nine are shuttling, and that shuttling is somehow interrupted -- the export is inhibited by the presence of the polyglutamine tract,” Truant said. “The net consequence of that is going to be very different for each one of these diseases. In some cases, it could mean that the protein should be exporting the nucleus along with other factors and is not. And instead, it is potentially retaining the others factors in the nucleus.”

Truant is looking at the possibility of RNA being retained in the nucleus for huntingtin. One increasingly popular idea is that in many of the neurologic diseases, there is an entire series of abnormalities where normal nuclear export or facilitated nuclear export of a family of RNAs is being inhibited. Truant said that in Huntington’s disease, there may be a subset of RNAs that is being retained inside of the nucleus that is not being properly exported.

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Ray Truant (Photo courtesy of Ray Truant)
“My longer-term thinking is that these diseases end up being neurologic, due to the fact that a lot of these proteins may be involved in RNA localization within the cytoplasm, which is something that would be particularly important to a neuron as opposed to any other cell type within the body,” he said.

Neurons have a unique problem in that RNA export from the nucleus in most cells can generally rely on diffusion to get the RNA out to its subcellular localization or allow translation and translocation of the protein to its site of interest. But in neuronal cells, due to the relatively large distances, additional mechanisms for transport are needed.

“Neurons may be a specific set of cells that rely on these mechanisms more than any other cell type in the body,” Truant said. “That could be one of the things that explains the conundrum as to why a person may have Huntington’s disease and has a polyglutamine expansion in their huntingtin in every single cell in their body but it only manifests as a neurologic disease. They really don’t have any other pathology.”

Treatment of Huntington’s disease and other polyglutamine expansion disorders involves researching ways to prevent nuclear translocation or nuclear signaling of these proteins. Truant is building on similar research on Kennedy’s disease and collaborates with Dr. Albert R. La Spada’s laboratory at The University of Washington. Scientists have shown that the androgen receptor in Kennedy’s disease binds to testosterone; instead of coming back out of the nucleus as part of the off-switch regulation, it becomes trapped and toxic to the nucleus.

“If the problem is that the protein is being signaled to the nucleus, then let’s prevent signaling to the nucleus,” Truant said. “They used testosterone release inhibitors such as the compound leuprorelin to dramatically reduce the level of circulating testosterone. In both mouse models and in clinical trials, they’ve found this to be an effective therapy against Kennedy’s disease. So the theme is to stop the mutant protein from getting signaled into the nucleus.”

For huntingtin, understanding the signaling pathways that allow it to enter the nucleus will allow Truant and his colleagues to determine ways to inhibit those pathways and prevent nuclear entry of the mutant protein.

“The exciting thing about all of this is that we’ve hit upon a kinase pathway that we think may be able to modulate huntingtin toxicity,” Truant said. “My hope is that within the next year we’ll be able to find this kinase and the pathway and there will be some pharmaceutical company out there that already has the inhibitor and most of the pharmacology done.”

For more information, visit: www.mcmaster.ca


GLOSSARY
photonics
The technology of generating and harnessing light and other forms of radiant energy whose quantum unit is the photon. The science includes light emission, transmission, deflection, amplification and detection by optical components and instruments, lasers and other light sources, fiber optics, electro-optical instrumentation, related hardware and electronics, and sophisticated systems. The range of applications of photonics extends from energy generation to detection to communications and...
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