Feature: ‘Gatekeeper’ protein protects injured brain

This feature appeared in the September/October 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.

In the brain, two pluses can sometimes be a minus. The brain needs large amounts of iron, copper and zinc to function normally but, in a crisis, these divalent metal ions flood into neurons, triggering toxic oxidation reactions that can cause cells to die en masse.

Several years ago, Professor Seong-Seng Tan, head of the Brain Development Laboratory of Melbourne’s Howard Florey Institute, went looking for genes that are up-regulated in brain cells in response to stroke, traumatic injury or physiological stress.

Neurologists treating patients who have suffered a stroke, or surgeons dealing with brain injuries after car accidents, are all too familiar with the penumbra effect: a wave of programmed cell death that spreads from the site of the injury or blood clot, killing off healthy neurons.

In the first few days after a stroke or brain injury, the penumbra effect can greatly magnify the effect of the primary injury, leaving patients physically debilitated or with serious cognitive deficits.

Tan says that after traumatic injury or stroke, injured neurons produce the excitatory neurotransmitter glutamate, which over-stimulates healthy neurons, causing them to open membrane ion channels.

Metal ions flood into the cells, reaching toxic concentrations that cause oxidative damage to proteins and DNA. Beyond a certain threshold the damage becomes irreparable triggering cell death by apoptosis.

Iron, copper and zinc ions are also implicated in chronic neurodegenerative diseases. The beta amyloid plaques that clog the brains of Alzheimer’s patients are pinned together by zinc and copper ions, while iron binds alpha-synuclein molecules to form Lewy bodies in the brains of Parkinson’s disease patients.

“At the moment, there’s no effective treatment to prevent apoptosis in brain-injured patients, and clot-busting drugs are the only available treatment for stroke,” says Tan.

The rolling wave of programmed cell death propagated by distress signals from dying cells eventually slows and halts. If it didn’t, even relatively minor brain injuries could be lethal. Reasoning that natural selection would have created a circuit-breaker to constrain the penumbra effect, Tan used microarray technology to screen for changes in gene expression after brain injury in a laboratory mouse model.

Tan’s group identified a protein, Ndfip1 (Nedd4 family-interacting protein), that is sharply up-regulated six to 48 hours after brain injury. They needed to understand how Ndfip1 protected neurons before searching for a drug to increase its expression in the brain without unwanted side-effects.

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Tan’s group describes one of the key mechanisms in a paper in the Proceedings of the National Academy of Science. The collaborative study involved researchers at the Florey Institute, the Centre for Cancer Biology in Adelaide, La Trobe University’s Department of Biochemistry, the University of Iowa and the Department of Anatomy and Histology at the University of NSW.

The paper describes how Ndfip1 acts as a recruiting agent to promote protein ubiquitination – the process that attaches small molecules of ubiquitin to mark protein molecules for breakup and disposal. Ndfip1 shuts off the internal transport of toxic metals by marking a metal transporter protein (DMT1) for degradation.

Once the period of stress has passed, normal DMT1 levels are restored, facilitating the normal entry of metal ions into brain cells. Ndfip1 levels subsequently decline. “Ndfip1 does not directly ubiquitinate protein targets itself,” says Tan. “It recruits an E3-type ligase, Nedd4-2, which attaches ubiquitin to the DMT1 transporters, marking them for degradation.”

Tan and colleagues have moved on from cultured neurons into the whole animal by knocking out the Ndfip1 gene in mice. As they expected, the knock-out mouse proved to be hypersensitive to divalent metal ions, and post-mortem analysis of mice exposed to iron showed high concentrations of iron in their brain tissues. By contrast, another transgenic mouse engineered to over-express Ndfip1 was resistant to what would normally be toxic levels of iron.

“If we could make every cell in the human brain resistant to metal toxicity at times of stress, we might be able to limit the damaging effects of stroke,” Tan said.

“In our studies, we found that only a small proportion of brain cells are capable of protecting themselves from death, about five per cent at most. If we can find a safe drug to increase Ndfip1 expression we could make the other 95 per cent of brain cells resistant to stress and increase survival rates in stroke patients.”

Tan says that after a stroke or traumatic injury neurons remain vulnerable to metal-ion toxicity and apoptosis for around 48 hours, so it would be necessary to maintain protection only during this period.

“You wouldn’t want to maintain resistance to metal toxicity much longer than 48 hours because, at normal concentrations, these metals are essential for brain function,” he said.

In collaboration with scientists at the Harvard Stem Cell Centre, Tan and his colleagues have been screening a library of thousands of compounds, looking for drug candidates capable of transiently increasing expression of Ndfpi.

Tan says that, in the short-term, doctors would welcome a drug that would prevent the mass die-off of healthy neurons that can leave patients severely debilitated, with huge downstream costs for long-term care and rehabilitation.

This feature appeared in the September/October 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.