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New Protein Implicated in Ischemia Points Toward Potential Target for Preventing Stroke

Valeo, Tom

doi: 10.1097/01.NT.0000429680.24348.58
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Investigators found a protein within CA3 neurons that may play a role in neuroprotection against stroke.

Researchers at Oxford University have identified a protein that enables CA3 cells in the hippocampus to resist the ravages of ischemia — a discovery that could point toward new methods of protecting the brain from stroke and other disorders that result in reduced blood flow.

The protein, hamartin — a product of the tuberous sclerosis complex 1 gene (TSC1) — suppresses excessive protein synthesis through the mTOR pathway. A loss-of-function mutation in TSC1 or TSC2, which codes for tuberin, results in tuberous sclerosis, a disease that causes tubers and tumors of the heart, skin, eye, and brain.

Reporting in the Feb. 24 issue of Nature Medicine, the researchers said they found that within CA3 neurons, hamartin expression increased after 10 minutes of ischemia, peaking at about 24 hours after reperfusion, according to lead author Alastair Buchan, DSc.

How long upregulation would have to continue to confer protection, however, remains unknown.

“It may be like hypothermia,” said Dr. Buchan, head of the Medical Sciences Division and dean of the Medical School at Oxford University. “If, within six hours after global ischemia, you drop body temperature for 36-72 hours, that will give you long-term survival of CA1 out to two or three years. Hypothermia is a very potent way to stop CA1 death. Does the hamartin response have a similar time course? We're trying to answer that now.”

It has been known since 1926 that after cardiac arrest CA1 cells in the hippocampus die much more readily than CA3 cells. For the past 25 years Dr. Buchan has been investigating this phenomenon in collaboration with William Pulsinelli, PhD, MD, Semmes-Murphey professor and chairman of the department of neurology at the University of Tennessee Health Science Center.



“He showed that the loss of CA1 cells doesn't happen immediately,” said Dr. Buchan. “It begins after about 24 hours and matures over time. Even with brief ischemia CA1 cells die, but they take longer to die. They're exquisitely sensitive.”

While these efforts attempt to prevent brain cells from dying after ischemia, Dr. Buchan decided to work the other side of the problem and investigate why CA3 cells are more resistant to damage after ischemia. “Rather than looking at glutamate toxicity, AMPA and NMDA blockers, caspase activation, apoptosis, and so on, I decided to investigate what makes CA3 cells less likely to die,” he said.

To find the answer Dr. Buchan, along with Michalis Papadakis, PhD, the scientific director of his Laboratory of Cerebral Ischemia at Oxford and the first author of the Nature Medicine paper, engaged in what he called a “fishing expedition” designed to detect proteins involved in CA3 neurons that weren't involved in CA1 neurons.

“I had no idea which proteins in CA3 were not in CA1, but after a complete assessment of the proteomics involved we came up with hamartin, which is an upregulation of the PI3K-Akt pathway,” he said. “Essentially it was inhibiting the mTOR pathway, which activated autophagy.”

The findings appear to open a new avenue of research into neuroprotection.

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The findings could point the way toward significant improvements in the treatment of brain ischemia, according to Sean I. Savitz, MD, whose research focuses on developing therapies for reducing damage from stroke.

“The big question is how important hamartin is compared with other proteins that have been identified over 25 years,” said Dr. Savitz, professor of neurology at the University of Texas Health Science Center at Houston Medical School. “The field has attempted a number of therapeutic approaches to collectively upregulate one or another pathway, but so far we haven't found a neuroprotective approach for stroke. Hamartin appears to be a very important protein. Would selectively upregulating this one target be sufficient to protect neurons after a stroke? Could those neurons survive injury if this protein were manipulated? These are really elegant studies that make a novel contribution to the field. [Dr. Buchan is] linking it with a mechanism called autophagy. That is also a novel link. This is a great article.”



Translating these findings into human clinical studies, however, is years away, according to Jeffrey Saver, MD, who works on the translation of animal findings to human clinical trials.

“I think this is a very elegant preclinical study identifying a key pathway in the brain's self-protective mechanisms that helps to explain the long-known phenomenon of selective vulnerability where some cells are resistant to low blood flow and others are very susceptible,” said Dr. Saver, professor of neurology at the University of California, Los Angeles David Geffen School of Medicine and director of the Stroke and Vascular Neurology program at UCLA.

Dr. Saver, who works on the translation of these types of animal findings to human clinical trials, acknowledged that genetic modulation of hamartin with the type of viral vectors used on animals in this research would not be practical in human stroke patients. “Ways to deliver this molecule and determine if it's beneficial in patients will have to be devised,” he said. “We'll have to look at how to get across the blood-brain barrier, and then get inside of cells. There are approaches using chaperone molecules that can help to convert these types of proteins into usable drugs, but work will have to be done on that.”



Dr. Buchan agreed that exploiting the neuroprotective properties of hamartin will require much more research, but may produce protection not only against ischemia but also other forms of neurodegeneration.

“I think ultimately what you want is something that will make brain more resistant to environmental stresses and strains,” he said. “I'm hoping this might open up something new that re-energizes the field.”

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Heng Zhao, PhD, assistant professor (research) of neurosurgery at Stanford University School of Medicine, has focused his research on finding ways to protect the brain from ischemic damage through postconditioning, which involves stimulating the body's endogenous protective mechanisms by inducing ischemia or hypoxia after a stroke.

Postconditioning appears to work by altering various cell pathways, but the precise mechanism remains obscure, so Dr. Zhao was intrigued by the role that hamartin appears to play in the protective process, but confused by the apparent conflict with previous research.

For example, the kinase mTOR (mammalian target of rapamycin) is known to contribute to autophagy, and Dr. Buchan suspects that the upregulation of hamartin after ischemia may inhibit the synthesis of proteins and thereby promote autophagy and enable it to recycle injured proteins and organelles. The results reported in the Feb. 24 Nature Medicine paper, however, seem to suggest the opposite to Dr. Zhao.

“A few previous studies have shown that mTOR activities promote neuronal survival after stroke,” Dr. Zhao said. “However this study suggests that hamartin, a protein that inhibits mTOR activity, offers protection.”

On top of that, the activity of Akt — a kinase involved in multiple cellular processes — is critical for the survival of neurons, and Akt phosphorylation contributes to preconditioning, according to Dr. Zhao. “However, Akt promotes mTOR activity, which further conflicts with the findings about hamartin in this study,” he said. “In my opinion, hamartin may play critical roles in preconditioning, as it is involved in both Akt and mTOR pathways, but whether it is detrimental or beneficial remains unknown.”

While the evidence in the Nature Medicine paper may seem to conflict with previous research, Dr. Buchan believes he and Dr. Zhao are actually “in complete agreement.” The apparent discrepancy, he suspects, results from the complexity of the cellular pathways involved.

“Our paper concentrates on hamartin,” Dr. Buchan said. “We don't make any statements about Akt. The upregulation of Akt he mentions is similar to what we find, which is an upregulation not just of hamartin, but of PI3K-Akt. Both of those work in complex ways to inhibit mTOR. Akt may well be protective via another mechanism. It doesn't just activate mTOR. Indeed, a negative feedback mechanism exists whereby increased levels of mTOR inhibit PI3K via the downstream molecule p70 S6 kinase (S6K). What we're seeing a slightly subtle difference in the way hamartin works to inhibit mTOR and induce productive autophagy.”

However, the role of autophagy in cerebral ischemia is hotly debated in the literature, according to Dr. Buchan, with some arguing that it is protective and some claiming it is detrimental.

“The answer may, however, lie in the modulation of the mTOR pathway via hamartin, and not simply in its inhibition or promotion,” he said. “For example, autophagy may well commence in cells, but it may be halted before completion, which means that the process cannot, by definition, be productive.”

This could lead to the erroneous conclusion that autophagy is detrimental when in fact it was the inhibition of autophagy that caused cell death.

“The robust demonstration that hamartin confers neuroprotection against cerebral ischemia by inducing productive autophagy suggests that it may have an important role in treating a variety of neurological diseases,” Dr. Buchan said.

Dr. Zhao is open to that possibility. “If Akt overexpression inhibits mTOR activity, as Dr. Buchan states, it might be in another category,” he said. In any event, he considers the Nature Medicine paper “an elegant study.”

—Tom Valeo

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