Study of an olfactory gene may indicate disease mechanisms

Mice without the RIC8B gene in the olfactory epithelium took longer to find food and were not put off by strongly unpleasant smells. Inactivation of the gene prevented embryo development.

By André Julião  |  Agência FAPESP – A study performed on mice in Brazil shows a direct link between a specific gene (RIC8B) and olfactory capacity. Furthermore, this gene, which is also present in humans, plays a key role in murine embryo development.

Analysis of the link between RIC8B and olfaction was made possible by use of the conditional knockout technique, which permits tissue-specific gene inactivation.

“When the gene RIC8B is inactivated specifically in the olfactory epithelium of mice, this tissue ceases to produce the protein RIC8B, as you would expect, but we also found that the olfactory epithelium ceases to express the protein Gαolf,” said Bettina Malnic, a professor at the University of São Paulo’s Chemistry Institute (IQ-USP) and principal investigator for the study. An article describing it was published in Journal of Neuroscience.

“The protein Gαolf is fundamental to the activation of olfactory neurons in this epithelium by odorants, and in its absence, animals display severe olfactory deficiencies,” Malnic told Agência FAPESP.

This technique and others used to produce transgenic animals were presented during the 33rd Annual Meeting of the Federation of Brazilian Societies for Experimental Biology (FeSBE), an event supported by FAPESP that took place in September in Campos do Jordão, São Paulo State, Brazil.

A second part of the study, as yet unpublished but presented to the conference, shows that the effect is different when the gene is inactivated in the entire organism instead of a specific tissue. In attempts to produce transgenic mice without RIC8B in all tissues, the embryos simply failed to develop.

“In these embryos, the neural tube in the region of the head doesn’t close as it normally should, showing the gene is associated with essential functions besides olfaction,” Malnic said. Embryos with the gene inactivated were also much smaller than those with the gene intact.

Malnic’s group also found that as a result of RIC8B inactivation, the mTORC2 signaling pathway was altered in these embryos. This pathway is involved in important functions of the organism and is still poorly understood in some respects.

For example, heightened activation of the mTORC2 pathway is known to correlate with some kinds of cancer. If altered, it can be associated with defects in the nervous system and contribute to neuropsychiatric disorders such as some forms of autism and schizophrenia.

Cage smells

To investigate the association between RIC8B inactivation and olfactory capacity, transgenic mice were produced by the conditional knockout technique, and their heterozygous and wild-type siblings were used as controls.

While the knockout mice had two inactivated copies of RIC8B, the heterozygotes had only one. Both copies were active in the wild-type mice.

Several behavioral tests were performed with these mice. In one test, they were deprived of food for 24 hours and then put in a cage containing food pellets buried under wood shavings. The controls found the food in two or three minutes. The knockout mice took almost twice as long.

In another test, the cage contained peanut butter and milk odorants as well as water. The controls spent a few seconds sniffing the water and much longer over the milk and peanut butter odorants. In other words, they preferred the smell of food to water, which was considered neutral. The mice with RIC8B inactivated spent roughly the same amount of time sniffing the food odorants and the water.

Lastly, the animals were each placed in a cage containing a piece of filter paper scented with butyric acid in one corner. This acid has a highly unpleasant smell. The controls avoided that side of the cage, especially the corner with the butyric acid, as much as they could, while the mice with the gene inactivated moved normally all over the cage.

The results of these behavioral tests were reinforced by analysis of the animals’ olfactory system. When subjected to different challenges, the olfactory epithelium in knockout mice was found to have a smaller number of active olfactory neurons than that in the controls.

“Olfactory neurons in knockout mice don’t respond to odorants and die more frequently,” Malnic said. Analysis of their brains also showed that the olfactory bulb was smaller than in the controls.

Another sign of the importance of RIC8B is its universal conservation in mammals. “The amino acid sequence of the protein RIC8B in mice is approximately 95% identical to the sequence of human RIC8B,” Malnic said.

This means that over the course of evolution, there has been selective pressure to maintain the amino acid sequence of this functional protein.

“This conservation shows that RIC8B also plays a key role in humans. We don’t yet know what that role is, but it may be linked to the functions observed in the constitutive knockout mouse, such as nervous system development, for example,” Malnic said.

The article “Conditional deletion of RIC8B in olfactory sensory neurons leads to olfactory impairment” (doi: https://doi.org/10.1523/JNEUROSCI.0943-17.2017) by Cleiton F. Machado, Maíra H. Nagai, Cassandra S. Lyra, Thiago M. Reis-Silva, André Machado Xavier, Isaias Glezer, Luciano F. Felicio and Bettina Malnic can be read at jneurosci.org/content/37/50/12202.