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Pharmacology

Racing against time

Team identifies how an essential brain protein causes cellular death in Alzheimer patients

EDUARDO CESARIn only 24 hours, common proteins found in the brain form an anomalous structure and intertwine. They look like sea urchins fitting between the brain cells, which become bloated and twisted in response. One week later, these plates already reach their final size, as can be seen in patients with Alzheimer’s, the most common cause of memory loss and intellectual capacity in the elderly. The speed surprised US researchers, who used a special multiphoton microscope, capable of tracking these cells’ displacement in real time in live rats. The images, published in February in the journal Nature, further the idea that this protein, β amyloid, plays a key role in Alzheimers, but there are controversies. Some people die of Alzheimer symptoms yet have no brain plates, and many have brain plates but do not develop the disease.

“Surrounding the plates are other smaller, soluble β amyloid clusters that cannot be seen in most analytical techniques,” explains Sergio Teixeira Ferreira, a biochemist from the Federal University of Rio de Janeiro (UFRJ). He believes that these are the villains – the plates actually indicate too much protein near the brain. In order to unveil the mystery of this disease that has a new victim every seven seconds, according to 2005 WHO data, the group lead by pharmacologist Cristoforo Scavone, of the Biomedicine Sciences Institute of the University of São Paulo (USP), thoroughly researched Alzheimer’s biochemistry, to detect it before symptoms become manifest, and in particular to find a way to stop it – to date, medicine can only postpone cognitive loss.

Therefore, Elisa Kawamoto, from Scavone’s group, focused on studying how β amyloid changes cells’ biochemistry and eventually kills them. With the collaboration of Maria Christina Avellar, from the Federal University of São Paulo (Unifesp), she found that in concentrations below toxic levels, β amyloid stimulates a protein, the kappa B transition factor (NF-kB), to act.

US biologist Mark Mattson, one of the world’s leading researchers of senile dementia, believes that NF-kB protects the brain against neuron loss that is due to old age. Elisa did part of her work at Mattson’s lab at the National Ageing Institute, resulting in productive collaboration that helped the USP group show that the relationship is not that simple. In normal concentrations, the protein induces the brain to recruit protecting substances. But the imbalance caused by excess β amyloid eventually activates genes related to cellular death.

It was already known that β amyloid damages the brain, causing typical Alzheimer lapses, such as leaving home and forgetting the way back, or not recognizing close relatives. However, Elisa and Scavone wanted to understand the natural resistance mechanisms against these deleterious effects. So she cultivated the brain cells of rats  in the lab, from a region naturally resistant to degeneration from Alzheimer’s – the cerebellum, i.e., the center that controls balance and precise movements, such as flipping pages in a magazine. “We wanted to know why Alzheimer only attacks certain regions,” says Scavone.

By incubating β amyloid with this culture that was 90% neurons and only 10% other brain cells, Elisa showed that the protein in fact acts upon the neurons, the cells that transmit information and are a minority in the brain. The neuroglia, made up of cells that are ten times more abundant, provides structure for the brain and is not affected in the same way by the protein. The results of the USP group’s studies are in this month’s issue of the Journal of Neuroscience Research. “Proving that the action takes place in the neurons is a significant advance in this area,” celebrates Scavone.

Closing the doors
In order to communicate with the inside of the cells, β amyloid surrounding the neurons send signals through N- methyl D-aspartate receptors, or NMDA. The USP group discovered what happens next: the receptors activate the NF-kB, which migrates to the cell’s nucleus, influencing their genetic activity there. Discovering this was crucial, as it suggests a way to block the signals unleashed by β amyloid, which results in cell death: by blocking the receivers.

Sergio Ferreira and Fernanda De Felice, both from UFRJ, have already shown the role of the receptors in a paper published in the Journal of Biological Chemistry in 2007. The group from Rio de Janeiro is now trying to unravel how to block the receptors. One of the possibilities is the amino acid taurine, available in high concentrations in the young brain – a concentration that decreases with ageing and Alzheimer’s. Ferreira showed, in articles published in 2004 in the Faseb Journal and in 2005 in Neuropharmacology, that taurine, found in energy drinks, protects neurons from β amyloid’s toxicity. “It works as an antidote to the activation of the NMDAs,” explains the biochemist.

His group is currently investigating the possibility of using taurine to fight Alzheimer’s. Together with USP’s Brain Ageing Project, which keeps a bank of elderly people’s brains and makes this material available to associated researchers, the biochemist from UFRJ is comparing the levels of taurine in the brains of people who died aged 50 or older, with and without Alzheimer’s. The initial results show a difference, which, however, cannot be stated yet with statistical reliability.

However, the brain’s defense mechanisms against β amyloid are not limited to producing NF-kB. Elisa and Scavone found greater than expected gene activity in the cerebellum that produces the neurotrophic factor derived from the brain (BNDF) when compared to other areas of the brain. Pharmacologist Iván Izquierdo, from the Catholic University of Rio Grande do Sul (PUC-RS), had already shown that this protein is crucial for memory retention. The USP group is now building its expertise on Alzheimer’s further: they believe that the potential for producing more of this memory protein is precisely what protects cerebellum cells. In the regions of the brain that are sensitive to Alzheimer’s, such as the hippocampus and the prefrontal cortex, NF0kB inhibits the production of the neurotrophic factor. The opposite occurs in the cerebellum. “The cerebellum can produce it in other ways,” adds Scavone.

The discoveries favor the notion that the loss of the protein is related to the onset of Alzheimers. The areas of the brain in which NF-kB inhibits the production of the protective substance more effectively, i.e., the prefrontal cortex and the hippocampus, are responsible, respectively, for processing complex behaviors and memory storage – abilities that patients with the disease gradually lose. In the February issue of Proceedings of the National Academy of Sciences (PNAS), Izquierdo proved that injecting BNDF in the hippocampus is enough to restore the memory in rats whose deficiency in protein production resulted in memory loss.

However, it is still too early to celebrate and to look for memory pills based on the neurotrophic factor derived from the brain. “The protein has to be injected directly into the hippocampus, and that cannot be done in humans,” warns Izquierdo. Moreover, it acts by fostering the growth of synapses, i.e., the communication points between neurons. In high amounts, the protein could result in tumors.

There are substances in the brain that protect the cells and others that damage them, but Scavone and his team believe that even with  in-depth understanding of biochemical functions, interrupting them may have serious adverse effects. NF-kB, for instance, can cause or avoid cellular death, depending on the concentration and the brain region in which it is found. Scavone explains: “All of the compounds have positive effects in certain amounts and negative effects in others”. Therefore, Ferreira stresses, it is hard to predict what will happen when one interferes with chemical balance, whether using substances naturally produced by the body or using external ones.

In order to balance the biochemical paths, the safest answer does not appear to be medication that changes protein concentration, but diet. Restricting calorie intake, the only procedure proven to extend the life of animals in labs, seems to switch off the inflammation triggering genes. It also increases the level of the neurotrophic factor and the activity of the WNT protein, which, according to the study published by Ferreira’s group in the Journal of Biological Chemistry, is inhibited by the high levels of β amyloid. WNT is another of these substances that produce changeable effects: in small doses, it prevents the formation of β amyloid plaques but may trigger cancer in large doses.

Currently doing post-doctoral work at Scavone’s lab, Elisa tests the effects restricting calorie intake in rats submitted to strict diets: for one month, they alternate 24-hour periods with and 24-hour periods without food. The data is still preliminary – she only tested four animals aged 4 months plus four animals aged 24 months – but very exciting. Among the younger rats, which correspond to adults about 30 years old, the calorie restriction reduces the experimentally induced inflammation in the hippocampus and increases the concentration of the brain-derived neurotrophic factor, a protein that protects the neurons and aids memory formation.

Ageing well
However, at least among rodents, this effect is not always beneficial. Restricting the calorie intake of elderly rats that had not dieted while young caused oxidative stress. This, added to inflammation, may result in the death of brain cells.

Perhaps the restriction has to be constant throughout life, or perhaps one must find a level of calorie restriction that is appropriate for old age. After all, strict diets impose a physiological stress that may be too much for the old to bear. Yet among youngsters, stress may aid the body. “It’s like the story of the king that feared being poisoned, so he took a little bit of poison everyday,” compares Scavone.

For the time being, it seems that once a person is middle aged, it is too late to start dieting to avoid brain degeneration. “It is necessary to change the population’s eating habits,” says Scavone, who fills his children’s lunchboxes with fruit. For Scavone, pills are the last recourse rather than the first. It is crucial to take care of health from an early age, in an increasingly ageing population.

The Project
1. Changes in the nitric glutamate-nitric oxide path in NF-kB modulation by means of the peptide β amyloid (nº 02/09716-4); Type: Regular Research Awards; Coordinator: Cristoforo Scavone – USP; Investment: R$ 154,040.99 (FAPESP) and R$ 35,000 (CNPq).
2. Signals from protein WNT and neurotoxicity induced by peptide β amyloid in primary cell culture from neurons of the hippocampus. (nº 06/59722-1); Type: Regular Research Awards; Coordinator: Cristoforo Scavone – USP; Investment: R$ 153,271.19

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