CLEBER A. TRUJILLO AND ALEXANDER HENNING ULRICH/IQ-USPLike an explosion: formation of neurons, in redCLEBER A. TRUJILLO AND ALEXANDER HENNING ULRICH/IQ-USP
The same molecule that Brazilian researchers introduced to the world 53 years ago as a powerful blood pressure regulator – that resulted in a class of anti- hypertension drugs – has once again surprised us with its versatility. More than five decades after its identification by the physicians Maurício Rocha e Silva, Wilson Teixeira Beraldo, and Gastão Rosenfeld, this molecule, called bradykinin, is now drawing attention due to hitherto unimaginable effects. A protein fragment (peptide), the molecule is found in blood and other body tissues and it is released in higher concentrations during inflammatory processes. Studies conducted in the last few years by Brazilian research teams have shown that bradykinin transforms stem cells into neurons and protects them from death in brain injuries. Another study suggests that in fatty tissue bradykinin regulates the release of the hormone that induces satiety and reduces the accumulation of fat. These findings do not have any clinical applications yet; however, they open up the way to understanding how the brain is formed and how certain neurological diseases, other than obesity, appear. These findings also renew expectations that perhaps a few years from now more efficient ways of dealing with these issues might be found.
The suspicion that bradykinin might do something other than lowering blood pressure and unleashing local inflammations – the body’s natural response to injuries – first came up in the mid-1990s when biochemist Alexander Henning Ulrich was doing his doctorate at Hamburg University in Germany. At the time, Ulrich was investigating the tumor proliferation mechanism of the neural tissue. He noticed that bradykinin activated certain signaling mechanisms in those cells, though the effect was milder or non-existent in other cells. From 2002 onwards, when he was already working as a professor at the Chemical Institute of the University of São Paulo (USP), he resumed his studies on the role of bradykinin.
During his doctoral studies at Ulrich’s laboratory, biomedical scientist Antonio Henrique Martins was investigating the transformation of immature stem cells into neurons – the brain cells that store and transmit information and that allow us to learn, remember, and even think about the ability to think – when he came across an unexpected result. Neurons cultivated in one of the plastic vials remained in a sleep-like state even after being bathed in acetylcholine, a neurotransmitter that is one of the chemical messengers that usually arouses the neurons.
Henrique called Ulrich: “I must be doing something wrong. Those cells aren’t responding to acetylcholine.” They repeated the tests, but the results remained unchanged. Once again they saw cells that resembled neurons – but did not behave like neurons – weeks after the stem cells had been placed in vials with a combination of nutrients. The cells were stimulated to take on specific functions in a process called cellular differentiation. Something was interfering with the maturing of the stem cells.
The researchers re-examined the ingredients in the cells’ culture medium. The only different component was a synthetic compound called HOE-140 that inhibits the activity of bradykinin, which at the time was not known to have any effect upon the brain. In a kind of molecular race, this synthetic component adheres to a protein on the surface of the cell –the B2 receptor – that the bradykinin should bind with. Thus, the HOE-140 keeps bradykinin from interacting with the cells.
When binding with the B2 receptor, bradykinin activates a chain of chemical reactions that modify the intracellular environment. Small pouches release the calcium ion into the cytoplasm, the gelatinous part of the cell that envelops the nucleus. In the cytoplasm, oscillations in calcium levels – which can increase by 10 to 100 times – act as a code that activates certain groups of genes in the nucleus and defines the destiny of the cell, i.e., whether it will continue multiplying and preserve its potential to originate different types of cells or whether it will become specialized in a specific function.
Henrique and Ulrich then conducted more trials with the same embryo cell strains from a mouse tumor. These cells can develop into fibroblasts, brain muscle, and tissue cells. For eight days – the time it takes for lab-created immature cells to become neurons – the researchers measured the quantity of B2 receptors and the release of bradykinin. They also compared the maturity level of cells treated with bradykinin with that of cells subjected to a combination of bradykinin and HOE-140, which annuls the effect of the peptide identified by Rocha e Silva and their colleagues in the 1940s. The researchers concluded that the transformation and the maturity cannot be completed without bradykinin. The neurons are imperfect.
CLEBER A. TRUJILLO AND ALEXANDER HENNING ULRICH/IQ-USPBradykinin in action: the neurosphere, on the lower right hand side, produces more neurons, in red…CLEBER A. TRUJILLO AND ALEXANDER HENNING ULRICH/IQ-USP
The researchers noticed that the number of bradykinin receptors increases gradually during the natural differentiation process. In addition, the cells expel part of the bradykinin they manufacture, which influences the functioning of their neighboring cells. This results in neurons that are sensitive to the acetylcholine neurotransmitter, the chemical messenger that transmits information from one brain cell to another, as described in detail in an article published in 2005 in the Journal of Biological Chemistry. However, this activity was interrupted under the action of the HOE–140 compound. At the conclusion of the tests, the cells did not respond to acetylcholine and did not show the elongations that are characteristic of neurons. “The neurons remained incomplete and only some neurons survived,” says Henrique, who is currently a professor at the Universidad Central del Caribe, in Puerto Rico.
“Bradykinin does not initiate the cellular differentiation process; it defines the path that the cells will follow,” explains Ulrich, who came to Brazil in 1999 to work at the University of São Paulo (USP) with physician Walter Colli – one of the world’s leading specialists in Chagas’ Disease – to search for molecules that could prevent the cause of this disease, which invades mammalian cells.
Biologist Cleber Trujillo, who was also enrolled in the doctoral program headed by Ulrich, tested stem cells extracted from the brain tissue of mouse embryos to evaluate the effect of bradykinin on differentiation and neural maturity in experimental models. Chemist Telma Tiemi Schwindt and biologist Priscilla Negraes helped him in this endeavor. Cleber placed isolated stem cells in a culture medium and waited for each one to produce the so-called neurospheres, groups of approximately 100 thousand progenitor cells of two types of brain cells – neurons and neuroglia. Then, he added bradykinin and waited to see what the outcome would be.
“When we added bradykinin to the culture medium, more progenitor cells travelled longer distances,” says Ulrich. This cell movement is directly connected with the formation and maturity of the neurons. The farther the cells move away from the neurospheres, the more neurons arise, with more ramifications, which are crucial for the formation of brain connections.
In the presence of bradykinin, up to 30% more neurons were formed than normal – while a smaller proportion of neuroglia arose. The production of neurons grew even more when Cleber added a compound called captopril to the stem cells in the process of differentiation. Captopril was the first anti-hypertension medication to act – albeit indirectly – on bradykinin, and maintained bradykinin in an active state for a longer period of time. Developed in the 1970s by U.S. researchers, captopril is based on a molecule that had been identified in the poison of the Bothrops jararaca pit viper by pharmacologist Sérgio Henrique Ferreira, when he was doing his doctorate with Maurício Rocha e Silva at the University of São Paulo (USP) in Ribeirão Preto.
The re-directing of the cellular destination determined by bradykinin was confirmed by tests conducted on transgenic mice provided by the team headed by João Bosco Pesquero, a molecular biologist from the Federal University of São Paulo (Unifesp). According to the data submitted for publication, brain stem cells of rodents genetically modified not to produce the B2 receptors when isolated and induced into differentiation did not produce a higher proportion of neurons.
As the findings were obtained with mice and rats, it was necessary to find out what happened with human cells. To this end, Cleber contacted the lab headed by Brazilian researcher Alysson Muotri at the University of California in San Diego. Here, he learned how to work with human stem cells obtained from the reprogramming of skin cells. Once again, bradykinin influenced the stem cells to turn into neurons.
People might wonder what the benefit of increasing the number of neurons in the brain in comparison with the neuroglia is. In healthy organisms, it is likely that these two cellular types could affect the brain architecture. The way in which the cells are organized and connected among themselves determines how the brain functions – at least, in the light of our current understanding of how the brain works.
CLEBER A. TRUJILLO AND ALEXANDER HENNING ULRICH/IQ-USP… than glial cells, in greenCLEBER A. TRUJILLO AND ALEXANDER HENNING ULRICH/IQ-USP
A recent study that is part of the research headed by Roberto Lent and Suzana Herculano-Houzel, from the Federal University of Rio de Janeiro, and by Wilson Jacob Filho, from the University of São Paulo (USP), suggests that the human brain contains practically the same proportion of neurons and of neuroglia. An adult man probably has some 86 billion neurons and 85 billion neuroglia (astrocytes, oligodendrocytes, and microglia). Glia is a word of Greek origin and means glue and until recently, neuroglia were seen merely as the physical support of neurons. However, neuroglia are becoming increasingly important as scientists discover that they have functions that are as important as those of neurons. More specifically, neuroglia aid nerve impulse transmission and help protect the central nervous system against invading microorganisms.
Researchers believe that the production of a higher quantity of neurons can be interesting in some circumstances, in addition to helping us to understand the brain’s ability to recover from injuries. By controlling the formation of neurons that arise from stem cells, it might be possible to replace dead cells that died due to neurodegenerative diseases, such as Parkinson’s Disease or ischemia (when the flow of nutrients and oxygen to the brain is interrupted because of clogged blood vessels).
“Based on experiments with animals, we already know that transplanting differentiated cells to the brain does not work because these cells die, as they are unable to remake the correct connections,” says Telma. “But it might be possible to implant immature cells and induce them to turn into neurons.” Telma and Enéas Ferrazoli are currently testing this hypothesis on a Parkinson model in rats. They are working on this jointly with Beatriz Longo, from the Federal University of São Paulo (Unifesp.) The initial findings are encouraging.
People afflicted with Parkinson’s Disease usually experience shaking, difficulties in some movements and in maintaining good posture. These symptoms are a consequence of the death of neurons in two regions of the brain – the substantia nigra and the striate nucleus, which produce dopamine, a neurotransmitter. As rats do not have these symptoms, Telma has resorted to another strategy to evaluate the progress of the disorder. By injecting specific chemical compounds, she induces the death of the neurons of the substantia nigra and of the striate nucleus in only one of the rats’ brain hemispheres. As a result, the animals start running around in circles after they receive a stimulating compound.
When conducting an initial test, Telma noticed that bradykinin, even when applied after the death of the neurons, led to the recovery of the two affected regions. Of the five animals used in the experiment, four stopped running in circles after treatment. “The rats’ brain stem cells may have migrated to the damaged regions and become neurons,” says the researcher.
The cell replacement observed in these experiments with animals were not the only beneficial effect of bradykinin on the central nervous system. Recent tests conducted by Henrique and biomedical scientist Janaina Alves, a doctoral student studying under Ulrich, indicate that bradykinin can avoid the death of neurons in the event of ischemia, the interruption of the flow of oxygen and nutrients provoked by clogged blood vessels. In a model that reproduces the damage caused by ischemia, Henrique and Janaina treated a region of the rats’ brains with N-methyl-D-aspartic acid. This compound, also known as NMDA, causes a stream of calcium to invade the cells – at levels sometimes one thousand higher than normal levels – to kill them. The measuring of neural activity showed that 80% of the cells of the hippocampus died after the administration of NMDA. The cell death rate, however, dropped to 20% when bradykinin was applied to the hippocampus in addition to NMDA, according to an article recently submitted to a science journal.
At the Universidad Central del Caribe, Henrique partnered with neuroscientists Pedro Ferchmin and Vesna Eterovic and with student Wilmare Torres, and observed that bradykinin avoids the death of neurons exposed to a compound that has the same effects as sarin gas, developed in Germany during the Second World War, and used in a terrorist attack in Tokyo in 1995. The compounds that mitigate the effects of these chemical weapons are not entirely efficient. “Soldiers that fought in the Gulf War and were exposed to chemical weapons were given an antidote and survived. However, they now have memory problems,” says Henrique.
Henrique, Janaina and Ulrich have proposed a new explanation for the neuroprotective effect of bradykinin. It seems that bradykinin prevents the death of cells thanks to glutamate, a neurotransmitter that is toxic when used in high dosages; it is widely believed that glutamate improves the flow of oxygen and nutrients because it provokes vasodilatation. According to Henrique, experiments indicate that bradykinin activates proteins that prevent cell death.
By means of a separate mechanism, Pesquero, from Unifesp, observed that bradykinin influences the consumption of energy in the organism. “We identified bradykinin’s direct action on the energy metabolism,” he says. Years ago, Pesquero noticed that the genetically modified mice that he produced during an internship at Germany’s Max Delbrück Institute had gained less weight than ordinary mice when submitted to a high-calory diet. The difference between the two groups of mice is that the genetically modified ones did not have the B1 receptor in the cells; this is the receptor that a sub-product of bradykinin binds with and triggers the typical phenomena that characterize inflammation.
The mice without the B1 receptor were more sensitive to the leptin hormone, as revealed by tests conducted by bioscientist Marcelo Mori and by veterinarian Ronaldo da Silva Araújo, both of whom were members of Pesquero’s research team and are now Unifesp professors. The findings of the tests were published in the Diabetes journal in 2008. Leptin, which is manufactured in the adipose tissue, inhibits the appetite and increases the body’s energy consumption. According to Pesquero, the elimination of the B1 receptor apparently causes the cells to produce more B2 receptors, which bradykinin binds with. “This suggests that bradykinin regulates the sensitivity to leptin,” says the researcher.
He also observed that in genetically modified mice the production of B1 in the adipose tissue only is sufficient to make these mice gain weight, just like ordinary mice. Pesquero believes it is possible to create a compound that can block the B1 receptor’s activity and help control obesity. He has tested an antagonistic molecule for the B1 receptor that a pharmaceutical company was developing to deal with the pain associated with inflammation. The molecule proved to be efficient in controlling the animals’ weight gain, but the company stopped the development of this molecule because of its undesirable side effects.
In spite of the promising results, it remains most unlikely that someday bradykinin will be used to treat ischemia or obesity. Although synthetic bradykinin has been around for nearly fifty years, it has not been approved for human use. Some studies suggest that the administration of bradykinin causes serious side effects, such as brain edema and a severe drop in blood pressure. “In vitro, bradykinin proved to be a neuroprotector, but in vivo this gets more complicated because there are many unforeseeable interactions that can occur,” says Henrique. Scientists hope to obtain a molecule similar to bradykinin but with fewer side effects and also providing neuroprotection. Cleber Trujillo points out that for the time being, “understanding how bradykinin acts on the adipose tissue and on the central nervous system is significant enough.”
The Projects
1. Artificial modulation of neuronal differentiation and function of receptors by synthetic oligonucleotides acting on gene and protein levels (nº 2001/08827-4); Modality Young Researcher; Coordinator Alexander Henning Ulrich – IQ/USP; Investment R$ 1,419,510.07 (FAPESP).
2. Molecular bases of stem cell differentiation and neuronal progenitors (nº 2006/61285-9); Modality Theme Project; Coordinator Alexander Henning Ulrich – IQ/USP; Investment R$ 1,038,469.28 (FAPESP).
3. Double transplant of neuronal microspheres and stem cells as therapy for Parkinson’s Disease (nº 2009/50540-6); Modality Young Researcher; Coordinator Telma Tiemi Schwindt – IQ/USP; Investment R$ 193,442.57 (FAPESP)
Scientific articles
MARTINS, A. H. et al. Neuronal differentiation of P19 embryonal carcinoma cells modulates kinin B2 receptor gene expression and function. Journal of Biological Chemistry. v. 280, p. 19576-86. May 20, 2005.
MORI, M.A. et al. Kinin B1 receptor deficiency leads to leptin hypersensitivity and resistance to obesity. Diabetes. v. 57, p. 1491-1500. Jun. 2008.
