Engineering professor works to limit stroke damage by turning down the volume on cells’ calcium signaling
Shinghua Ding, a University of Missouri assistant professor in biological engineering, has won a National Institutes of Health Research Project Grant (R01) worth about $1.6 million for his cellular research on strokes.
“The R01 grants are intended for established or mature research programs,” said Jinglu Tan, the biological engineering department chair. “As such, they are very competitive and prestigious. Early-stage assistant professors rarely succeed with them. This is a major recognition for Shinghua and his research program.”
Ding’s research focuses on astrocytes, the dominant kind of glial cell found throughout the central nervous system. Glia is Greek for glue, and until the 1990s, glial cells were thought to serve a merely structural role: surrounding neurons and holding them in place. The neurons, in turn, were thought to be the electrically excitable cells responsible for cell communication.
In the early ’90s, however, Philip G. Haydon, now Chair of the Department of Neuroscience at Tufts University in Medford, Mass., serendipitously discovered that glial cells also join in on cellular conversations. Astrocytes, he detected, release the neurotransmitter glutamate, which “excites” nerve cells and prepares them to receive signals from other cells. Researchers have since found that astrocytes release other neurotransmitters as well.
The discovery of astrocytes’ active role in cellular communication has opened up new avenues for neuroscience research. In addition to looking at what signals astrocytes send, scientists have also begun studying what prompts these cells to “speak up” and how other cells react to their messages. The goal is to understand astrocytes well enough to be able to manipulate their functions to treat diseases or disorders that involve these cells, such as epilepsy, sleep disorders and stroke.
Ding began his work with astrocytes as a postdoc fellow under Haydon at the University of Pennsylvania in Philadelphia. Ding was part of a team that looked at how astrocytes respond in an epileptic seizure. The team found that following such an event, the calcium signaling within astrocytes swells and triggers a forceful release of glutamate that’s too much for the neuronal receptors to handle. Overstimulated, the neurons die. This overstimulation is known as neuronal excitotoxicity.
Having traced this process, the University of Pennsylvania team decided to see whether disrupting the astrocytes’ calcium signaling — or making these cells hush after a seizure — would result in less neuronal damage. Experiments showed inhibiting the calcium release did lessen neuronal damage, and the team concluded that astrocytes offered a novel therapeutic target for limiting brain damage from seizures. Ding would take much of what he learned in these experiments and build on it for the research that has won the NIH grant.
After finishing his postdoc, Ding became a research associate at the University of Pennsylvania. In 2006, he began an independent research project and won a grant from the American Heart Association to study astrocytes in stroke disease. He came to MU in 2007 to be an assistant professor in the Department of Biological Engineering as well as an investigator at MU’s Dalton Cardiovascular Research Center, which brings together researchers from engineering, biological and health fields “to advance knowledge” of cardiovascular disease “through research and teaching that merges discipline and technologies.” Shortly after arriving, Ding won another AHA grant.
Ding chose to concentrate on stroke disease because of its prevalence: stroke is the third-leading cause of death in the U.S., behind heart disease and cancer. Ding is focusing on ischemic stroke, or stroke that results from a clot obstructing a blood vessel, rather than hemorrhagic stroke, which occurs when a weakened blood vessel ruptures. Ischemic strokes account for about 87 percent of all strokes, according to the American Stroke Association. Ding knows of no one else studying the role of calcium signaling in astrocytes in ischemic stroke.
To look at astrocytes in ischemia, Ding is using two-photon microscopy to do in vivo imaging of brains in mice, the same approach used in the epilepsy research at the University of Pennsylvania. Two-photon microscopy is a fluorescence imaging technique that is particularly helpful for looking at thick samples, such as living tissue. In vivo, Latin for within the living, indicates the imaging is done in living mice. Ding has been able to purchase a two-photon microscope for his lab with funds from the NIH grant and MU’s Program for Research Infrastructure and Matching Expenses, or PRIME, fund.
As Ding couldn’t exactly wait for his lab mice to have strokes, he had to have a method for inducing them. Ding chose the photothrombosis model, which uses photosensitive dyes to induce ischemia. The dyes are injected intravenously and then activated through irradiation to produce an occlusion, or simulated clot, in the irradiated vessels. Ding’s team demonstrated that adjusting the intensity of output light and the size of the irradiated area can, with high reproducibility, generate ischemic infarctions, or areas of tissue death, of various sizes in mouse brains. That finding was published in the American Journal of Biomedical Sciences in 2010.
After choosing a model, Ding’s next step was to answer the basic question of whether ischemia has an impact on astrocytic calcium signaling. He found that indeed, calcium signaling increases within 20 minutes of an ischemic stroke. Ding hypothesizes that the calcium increase in astrocytes after ischemia, like the calcium released after an epileptic seizure, triggers an excessive release of glutamate, which in turn leads to an overstimulation of neuronal receptors.
Identifying these connections between ischemia, astrocytes and neuronal death has encouraged Ding to pursue astrocytes as a novel target for stroke therapies. Using the NIH grant, he will explore a genetic manipulation of astrocytes to treat stroke. Ding’s plan is to use molecular genetics to inhibit calcium signaling in astrocytes in vivo; he will test two different methods for doing this and see which one works better. One method is to use knockout mice, or mice in which researchers have “knocked out” a gene. Ding is using knockout mice that have a disrupted calcium signaling pathway in their astrocytes; the mice were actually generated in another lab for another study.
The second method Ding is using is viral transduction. In viral transduction, researchers use viral vectors, or modified viruses that deliver new genetic material into cells. Ding has already succeeded in two big first steps for this method: he had identified genetic material—in this case a protein molecule — with the potential to inhibit calcium signaling in astrocytes, and he has found a successful viral vector. Two years of research went into these steps, and Ding has shown his chosen vector introduces the genetic material only into astrocytes, not neurons, an important distinction and one difficult to achieve.
Ding has also shown that this genetic approach does have potential for significantly reducing astrocytic calcium signaling in mice. He demonstrated this effect in a controlled experiment in which he exposed both astrocytes that had received the therapy and astrocytes that had not to a stimulant known to trigger calcium signaling. He then compared levels of calcium release across the two groups and found that the treated astrocytes released significantly less calcium. Those findings were published this summer online in the journal Neuroscience.
Now the questions to answer are: 1) Does the transduction lessen calcium signaling following ischemia, and 2) If so, is less calcium associated with reduced neuronal damage?
Ding emphasizes that answering these questions will not immediately lead to clinical treatments. As he said, “Drug development is very, very slow.” But if Ding reveals that astrocytes can be manipulated to limit ischemia’s effects — and identifies a process for that manipulation — it will be a significant contribution to the fight against stroke disease.
“So many, many people study stroke, but there is only one drug approved by the FDA to treat it,” Ding said, referring specifically to ischemic stroke. He adds later, “Our results from this project will provide new insights into the causes of neuro-degeneration [and might also] suggest novel therapeutic targets for limiting neuronal death and brain damage after ischemia.”