Researchers at Duke University have developed a rat corticostriatal co-culture assay platform that can be used to screen compounds to treat neurodegenerative diseases, such as Huntington's disease, the researchers said at a scientific conference last week.
The assay recapitulates as closely as possible the in vivo environment, while allowing the investigators to screen compound libraries at a higher throughput than if they had used a brain slice explant technique, according to the scientists.
Joseph Trask, head of cellular imaging technologies in the center for drug discovery at Duke University Medical Center, presented his group's work at Cambridge Healthtech Institute's Sixth Annual High-Content Analysis conference, held last week in San Francisco, Calif.
"I am actually the imaging-analysis guru, so to speak. The primary culture assay itself was developed by McLean Bolton and Linda Kaltenbach, two researchers here at Duke," Trask told CBA News this week.
Two years ago, Donald Lo, director of the center for drug discovery and an associate professor of neurobiology at Duke University Medical Center, discussed the rat brain tissue explant model for neurodegenerative diseases in an interview with CBA News (see CBA News, 1/20/06).
Comparatively, the new assay has a higher throughput "because we can look at more wells and extract more cells out of the animal and do more compound profiling," Trask said this week. The idea is that scientists can put a glial bed down that supports the growth of primary striatal and cortical neurons that have been dissected out of the brain of the embryonic rat.
When isolated and grown on their own, MSNs are actually aspiny in culture, because they lack the glutaminergic input which is normally prevalent in the striatum, according to Vahri Beaumont, director of neurobiology at the Cure Huntington's Disease Initiative, which is collaborating with Duke to screen compounds using the assay. The corticostriatal coculture tries to overcome some of that by culturing the MSNs in the presence of cortical neurons to maintain some of the glutaminergic input.
"One of the aspects of Huntington's disease is that we think that there are aberrations in the glutaminergic signaling between cortical neurons and MSNs," said Beaumont. By preserving those synapses in culture, the researchers can screen for compounds that may be relevant to that disease process, she added.
The Duke researchers are transfecting these cultured neurons with mutant huntingtin constructs which, once they are transfected into either MSNs or cortical neurons, will cause them to die, Trask said during his presentation last week.
What they are actually scoring is the amount of cell death and the time in cell culture of both cortical and striatal neurons, and they are looking at the rescue of cell death by the application of different compounds.
In their screening collaboration, CHDI is triaging compounds which then get "filtered through to our lab, and get evaluated and profiled using the [Cellomics ArrayScan] high-content imager to look at the survivability of the neurons," said Trask.
He went on to say that his lab is now running "roughly 40 96-well plates per week of this co-culture model to look at cell survival."
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CHDI has worked with Lo's group at Duke for about five years, trying to set up phenotypic screens to look at the potential to identify neuroprotective compounds in vitro, Beaumont said. She added that CHDI is "trying to bring a relatively high-throughput screen to some of our existing [compound] libraries.
"I believe this group is, although I am not entirely sure, the only group doing corticostriatal co-culture in relation to Huntington's disease," Beaumont said. She went on to say that she believes others are doing straight MSN cultures, but "one of the aspects of this technique that I like is the preservation of glutaminergic synapses, and the fact that MSNs are in a more 'physiologically relevant' condition."
Currently the Duke investigators are starting to evaluate "other, alternative phenotypic markers, including dendritic analysis," said Trask. However, these other markers have not yet been validated.
"We have identified some compounds that preserve cell survivability … that have been validated using other models," Trask said.
Trask's presentation of his work at the HCA meeting follows on the heels of last month's announcement by investigators at the University of Wisconsin in Madison that that they were able to generate induced pluripotent stem cells from skin fibroblasts taken from a child with spinal muscular atrophy — the first study to demonstrate that human iPS cells can be used to model the specific pathology seen in a genetically inherited disease (see CBA News, 1/2/09).
"In terms of the iPS approach, the advantage is that you can use humanized or human HD cells to actually create a more relevant model," said Beaumont. However, she said, "You still have to make your iPS cells into something that actually looks, feels, and is to all intents and purposes, an MSN in culture." This is difficult to achieve.
"I think that is one of the greatest challenges to the iPS-based technique," she said.
For his part, Trask said, "I am still out with the jury on this. I think that strides that have been made from the primary cells are quite extraordinary, but in terms of stem cells, I am still waiting."
Although stem cells are appropriate for some things, such as neurogenesis, "I think it's more challenging" for neurodegeneration, he said. "For degeneration, I think you need a fully validated cell, and I think that is where the problem is."
Trask said that the next step in this project for his lab involves getting more validated data — "How are the dendrites actually affected by HD and any other neurodegenerative disease? What are the genes doing to the cell's phenotypic characteristics or the morphology of the cell? How can we measure those using high-content imaging?"
Understanding the role of apoptosis and mitochondrial dysfunction is also very important, said Trask. "Mitochondrial dysfunction does occur in Huntington's disease, and mitochondrial trafficking is something that people are looking at," he said. Mitochondrial dysfunction also plays a role in Alzheimer's disease.
Trask said that his group is currently in the process of validating a mouse model of Huntington's disease that they will be using to profile compounds that rescue neurons from cell death. He said that the premise is the same in terms of the model involving cortical and striatal neurons co-cultured over a glial bed that serves as the growth matrix.