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The mysteries of smells

Brazilian researcher helps unravel the neurological and genetic bases of the sense of smell

016-021_Cheiros_155You don’t have to stand in front of the oven to guess what the after-dinner dessert will be. Scent molecules detach themselves from the pie in the oven and spread through the air, penetrate the nostrils and reach a special group of cells located in the innermost part of the nose, next to the base of the skull, relaying chemical messages that allow the brain to decipher the flavor of the pie: apple, with a touch of cinnamon. There is no pleasure in eating without the smell; the tongue’s repertoire is limited to salty, sweet, bitter, sour and umami – the flavor of sodium monoglutamate. The ability to perceive scents is what gives meaning to seasonings and herbs and which allows a person to distinguish the difference between orange and pineapple juice. In the last few years, researchers have begun to learn more details about how the smell system deciphers smells and allows someone to distinguish a rose from jasmine or a glass of fresh milk from a glass of spoiled milk. Part of these discoveries can be credited to the work of biochemist Bettina Malnic, from the University of São Paulo’s Chemistry Institute/ IQ-USP.

In the last few years, Bettina deciphered what could be referred as the code of smells, that is, how different odors interact with  neurons and send off information that will be interpreted by the brain, thus allowing human beings to make distinctions in a repertoire with thousands of odors. She discovered that each smell molecule fits into more than one type of protein on the surface of the neurons in the innermost part of the nose, as if each smell molecule were a tiny star with five unequal points, each point having affinity with a receptor. Each receptor, in turn, can receive stars with different shapes, provided that at least one of the points has the necessary characteristics to fit into the receptor. This observation led the researcher to conclude that the nervous system recognizes each molecule through the group of specific receptors which it fits into, and not through a single receptor. The combinations code greatly increases the repertoire of the human smell. If each molecule connected itself to only one receptor, we would be able to identify approximately 400 odors – the approximate number of types of distinct receptors that exist in the human nose. By doing so, she unlocked the doors to unravel the code that governs the perception of smells and showed what lies behind the incredible human olfactory perception.

Bettina started working in this nascent line of research almost by chance. In 1996, she went to the US’s Harvard Medical School to do a post-graduate course. Her initial plan was to focus on another topic, but she became interested in the work of neuroscientist Linda Buck, who five years before had identified the genes of the odorant receptors, having estimated that there were a little over one thousand different kinds of these receptors in the noses of mammals. The importance of this discovery, made when Linda was working with Richard Axel at the Laboratory of New York’s Columbia University, was officially acknowledged in 2004, when the American research duo was granted the Nobel Prize for Medicine. Because of her contribution to knowledge on how animals identify odors, Bettina was invited by Linda to participate in the award-granting ceremony.

Molecular orphanage
With 400 different kinds of receptors, the olfactory receptor system is the biggest family of proteins in the human body. Nonetheless, it is small when compared to mammals that depend on the sense of smell to survive: the 400 human receptors correspond to one-third of the receptors in mice and to one half of the receptors in dogs. Until Bettina began her work in the United States, these receptors were still orphans – a term used by specialists to specify that they do not have any known partners. In the course of nearly four years, Bettina identified the odors that fit into 14 of these receptors. In addition to reducing the number of orphans and discovering that the brain recognizes combinations and not specific receptors, Bettina confirmed that each olfactory neuron only produces receptors of the same kind. Thousands of them.

The process to achieve these results was lengthy and difficult. In the lab, she would expose the olfactory neurons of mice to one specific odor molecule each time. Soon thereafter, she relied on the help of a group of Japanese who specialized in detecting, by using a pink dye, which cells were activated by a given molecule. In Japan, these specialists would clip each activated neuron and send it back to the United States so that Bettina could look for the part of the DNA with the formula for the receptor, to identify it.

Bettina is still the only Brazilian researcher specialized in the molecular functioning of olfactory neurons; she has trained a number of researchers in this field since 2000, when she opened the Molecular Neuroscience Laboratory at IQ-USP. She has recently developed a more efficient method to map the perception of smells, based on a protein she had identified in 2005, with the help of Luiz Eduardo von Dannecker and Adriana Mercadante. This is the Ric-8B protein, which exists only in olfactory neurons – and is always associated with the receptors discovered by Linda Buck. The group from USP spent three years investigating how this protein functions and now the researchers have a clearer idea of how it works. The latest findings, published in July 2008 in Molecular and Cellular Neuroscience and which are described in Daniel Kerr’s doctorate thesis, show that Ric-8B interacts with several sub-units of another protein – the Golf Olfactory protein – which until then had been considered as the protein responsible for activating the biochemical cascade that activates the olfactory neurons. “Ric-8B enhances the Golf’s action, making the activation of the neurons perceptive.” explains Bettina.

In addition to being essential to increase sensitivity to subtle scents, this enhancement also allows researchers to detect the activity of odorant receptors in the lab. To improve the efficiency with which she associates receptors and molecules, Bettina is developing an ingenious method which she presented last August at the Brazilian Congress of Pharmacology and Experimental Therapeutics, held in the city of Águas de Lindoia. The paper will be published shortly in the Annals of the New York Academy of Sciences. She intends to distribute cells through 96 openings of a plastic tray similar to a miniature ice cube tray, whereby she will be able to introduce 96 different kinds of odor molecules at the same time to cells with the same receptor, or test the reaction of different receptors to the same odor. The researchers will add a protein that produces a fluorescent substance, when the cells are activated, to the formula. Then the tray will go through a fluorescence scanner which will identify where activation occurred. This method should speed up the existing process which is very slow and which is difficult to apply on a large scale.

Photos Eduardo césar | Illustrations Laura daviña | origamis Letícia KonishiThis would be a simple experiment if only the above were necessary. In view of the fact that it is impossible to use one’s own olfactory neurons, and to know which receptors are found there, Bettina had to develop a technique to manufacture the experimental cells: she grew human kidney cells in which she inserted genetic instructions to produce a given olfactory receptor. Even though researchers know how to maintain and manipulate these cells in the lab, at first it was difficult to make them act like neurons. “The receptor would remain inside the cell and would not go to the membrane, where it has to stay to have contact with the odors in the air,” the biochemist explains.

This problem was solved by Japan’s Hiroaki Matsunami, who had been a colleague of Bettina’s at Linda Buck’s lab and nowadays is Duke University in the US. Like Bettina, he presented recent findings of his research work in July in San Francisco, during the International Olfactory and Taste Symposium (the biggest sponsor of which is Ajinomoto). Without Bettina’s participation, he discovered another protein which is essential for the perception of odors. This is the receptor transportation protein (RTP), which helps transport the receptor from the site where it is produced inside the neuron to the surface of the cell. In an article published in August in Nature Protocols journal, Matsunami reported that it is enough to implant the protein in cells together with the gene that codes the receptor and the receptor migrates to the cell’s surface.

Bettina inserted the RTP into the kidney cells. But she did not add the Ric-8B, which enhances the activation, and was thus unable to detect the experimental cell’s reaction to odor molecules. When the system is completed, she believes that next year she will be able to take a part of the odorant receptors out of their orphan state. “We have the gene sequencing of all the receptors in the data base of the Human Genome Project,” says the researcher, who is planning to begin this new phase of experiments evaluating human receptors that do not exist in mice or dogs – there are approximately 20 of such receptors – to see which substances they recognize.

Now that researchers know how the Ric-8B functions in in vitro cells, one of the next steps will be to investigate their function in live mice. To this end, Bettina contracted the services of the Center for the Development of Experimental Models for Medicine and Biology at the Federal University of São Paulo (Unifesp), which will produce genetically modified mice by modifying the gene responsible for producing the Ric-8B and verify if the animals maintain their same olfactory sensitivity without the enhancing protein.

Smell in evolution
Identifying odor molecules that fit into each receptor and understanding how the olfactory ways function can help clarify a mystery that has intrigued the researchers in this field: How has the ability to detect scents changed throughout the evolution of the species. A review article headed by Masatoshi Nei, a renowned expert on evolution theory who works at the US’s Pennsylvania State University, published in Nature Review Genetics in 2008, analyzes the influence of chance and necessity in relation to the evolution of smell and taste receptors. Nei points out that the smell repertoires of human beings and chimpanzees are similar, and that they have shrunk in the course of evolution: both species have approximately 800 genes to produce odorant receptors, but less than half are functional. The other half lost their original function – they are referred to as pseudo genes. In comparison to human beings, mice have three times more active genes, totaling approximately 1,200, in addition to approximately 400 genes that stopped functioning in the course of evolution. Researchers who study olfactory evolution believe that the species that depend less on the sense of smell accumulated mutations in the course of time and lost the function of specific genes. This is the case of men and chimpanzees, which rely on good sight, allowing them to see colors and depth to deal with daily challenges.

American biologist Barbara Trask, from the Fred Hutchinson Cancer Research Center in Seattle, found indications to explain how variations in relation to sensitivity to odors surface. Her group scanned the human gene, searching for alterations in the group of genes related to odorant receptors. The group found a significant variation in the number of copies; this variation appears when parts of genetic material are duplicated and remain in the genome. The gene for a given olfactory receptor can be copied many times and each copy can undergo modifications. The process can lead to the creation of new receptors or it can cause so many changes that the gene becomes unfeasible. In an article published in August in the American Journal of Human Genetics, Barbara’s group reported that it had found a number of variable copies in 16 of the functional genes of odorant receptors. In extreme cases, the mutations in olfactory receptor genes can cause people to become insensitive to certain odors – a condition referred to as anosmia. But when it causes less drastic changes, this variation generates differences in how people perceive smells. Thus, genetic research might be able to reveal if two people that smell the scent of a cup of coffee or eat the same piece of cake have identical sensations. As Bettina writes in her book O cheiro das coisas, published in 2008 by Vieira & Lent publishers, two people can be different in the way they smell the world.

From DNA to the brain
The biochemist from USP is also interested in genome mysteries. She is trying to understand what regulates the activity of the genes that produce the smell receptors. All olfactory neurons have the same set of genes inside their nuclei, but each one of them only produces one kind of receptor – in human beings, one choice from among 400 options. How each cell chooses which receptor to present to the nose is still unknown, but in an article published in 2006 in Genome Research, Bettina indicates where to start looking for the answer. With the help of doctorate students Jussara Michaloski and Pedro Galante, she analyzed 198 olfactory receptor genes of mice and showed that all of them contain similar stretches of DNA. In the researcher’s opinion, the characteristics and the location of these segments indicate that they function as targets for the molecules that switch on or silence each gene. Just like electric switches which turn on lights must have similar characteristics so that a visitor recognizes their function, genetic switches also need to have elements in common.

There are many more elements, besides genes and receptors, which need to be understood to obtain the brain’s complete olfactory map. The research work done by Linda Buck and Richard Axel has shown that endings of olfactory neurons with identical receptors come together in nerves before they reach the olfactory bulb, an elongated structure on the lower side of the brain. The human bulb is divided into approximately 400 regions – the glomerules -, each one activated by a single kind of odor receptor. The poster produced by the Nobel Foundation in 2004 has an illustration of a head with the olfactory nervous system, showing how the numerous endings that form a web in the nose join with the nerves before reaching the olfactory bulb. But this illustration does not show what happens behind the bulb, as if the pathway ended there. “To discover how information goes to the brain, it would be necessary to follow a substance that passes from one neuron to another,” says Bettina. The winner of the Nobel Prize, now working at the Fred Hutchinson Center for Cancer Research, is working on developing this technique.

What researchers do know is that the olfactory neurons activate different regions of the brain such as the olfactory cortex, responsible for identifying the odors, the hypothalamus, which influences behaviors such as appetite and sexual drive, the amygdale, involved in emotions, and the hippocampus, which forms olfactory memories. This complex anatomy is the reason why a perfume brings back childhood memories, why the smell of a freshly baked cake stirs up the appetite and why women who live together have synchronized menstrual cycles without being aware of the hormone smells that fill the air.

The code of scents
Odor molecules are like stars with colored extremities. Each extremity connects itself to only one specific olfactory receptor – yellow with yellow, red with red – inside the nose. Multicolor stars can fit themselves into several receptors, and distinct stars can go to each kind of receptor, provided that at least one extension is of an adequate color.

The Project
Receptors attached to the G protein and chemical feeling (07/50743-9); Type: Thematic Project; Coordinator: Bettina Malnic – IQ-USP; Investment: R$ 367,190.09

Scientific articles
KERR, D.S. et al. Ric-8B interacts with Gαolf and Gγ13 and co-localizes with Gαolf, Gβ1 and Gγ13 in the cilia of olfactory sensory neuronsMolecular and Cellular Neuroscience, v. 38, n. 3, p. 341-348, July 2008.
VON DANNECKER, L.E.C. et al. Ric-8B promotes functional expression of odorant receptors. PNAS, v. 103, no. 24, p. 9 310-9 314, June 2006.
MALNIC, B. Searching for the ligands of odorant receptorsMolecular Neurobiology, v. 35, no. 2, p. 175-181, April 2007.
MALNIC, B. et al. Combinatorial receptor codes for odorsCell, v. 96, no. 5, p. 713-723, March 1999.