Olfactory Cilia Use Extracellular Glucose to Fuel Odor Transduction

Material below summarizes the article, Energy Requirements of Odor Transduction in the Chemosensory Cilia of Olfactory Sensory Neurons Rely on Oxidative Phosphorylation and Glycolytic Processing of Extracellular Glucose, published on June 7, 2017, in JNeurosci and authored by Pablo S. Villar, Ricardo Delgado, Cecilia Vergara, Juan G. Reyes, and Juan Bacigalupo.

Organelles are specialized subcellular structures that serve specific functions in all eukaryotic cells.

Most of them, such as the mitochondria and the endoplasmic reticulum, are found in the cytoplasm surrounded by their own lipid membranes, which are not connected to the plasma membrane.

Other organelles, such as cilia, microvilli, and flagella, are finger-like structures that protrude from the plasma membrane of specialized cells and have a lumen opened to the cytoplasm. Some examples are the cilia of olfactory sensory neurons, photosensitive microvilli of insect photoreceptors, and microvilli of the cochlear hair cells involved in mechanotransduction, all of which transduce sensory stimuli into electrical signals.

These organelles are subcellular machines that are very well defined morphologically and fully dedicated to a function that is unique to each cell type. They generally confine all of the proteins that partake in their extremely sophisticated functions, usually assembling them into protein complexes that work coordinately. This allows for the notable proficiency with which they carry out their jobs. 

In some cases, such as in the chemosensory olfactory cilia and the sperm flagella, the respective molecular mechanisms consume important amounts of energy that needs to be efficiently supplied. In both cases, due to their very small diameters (approximately 0.2 micrometers), these elongated organelles (up to hundreds of micrometers) exclude the mitochondria (approximately one micrometer), the ubiquitous organelle that provides most all cells with ATP by oxidative phosphorylation.

How processes occurring in cilia and flagella are powered is a matter of general interest.

Generally, ATP diffuses approximately 10 micrometers in 0.5 seconds within a cytoplasm. Therefore, if these organelles relied on ATP from mitochondria that are located, at best, near their base, it is hard to imagine that ATP could move along their entire lumen in sufficient amounts to fuel their molecular mechanisms.

In our work, we investigated whether olfactory cilia make use of complementary energy sources besides or instead of mitochondrial ATP to sustain their responses to prolonged odor exposures, such as those normally occurring in nature. 

The odor sensory neurons of the olfactory epithelium extend their single dendrite up to the epithelial surface, where the dendrite swells into a tiny knob from which the chemosensory cilia project into a mucus layer that covers the epithelium.

Odor transduction, which takes place in the cilia, is triggered when an odorant molecule binds to a membrane receptor, which couples to a GTP-binding protein that activates adenylyl cyclase, an enzyme that uses ATP to generate cyclic AMP. This second messenger opens cyclic nucleotide-gated cation channels through which Ca2+ ions flow into the lumen and open Ca2+-activated Cl- channels. A Ca2+ pump, a Na+/K+ pump, and a diversity of kinases playing regulatory roles also consume ATP.

Because the dendritic knob can accommodate no more than two or three mitochondria, it is reasonable to think that ATP consumption would lead to a deep drop in the rate of ATP diffusion in the cilium when the transduction machinery is turned on along the entire cilium, threatening the availability of luminal ATP unless additional ATP sources are available.

With a combination of biochemical, immunochemical, and electrophysiological approaches, we tested the hypothesis that the cilia could generate ATP locally by glycolysis to fulfill the demands in their apical regions.

If this hypothesis were true, glucose would need to be delivered to the cilia. Where would the glucose come from?

Our hypothesis was based on a previous paper in which we reported a stereotyped cellular distribution of plasma membrane glucose transporters (GLUT1 and 3) in the olfactory mucosa.

Such a distribution made us think that glucose arriving to the mucosa through blood vessels could be transferred towards the apical region of the epithelium, perhaps through the sustentacular cells that surround the olfactory neurons. These cells would release it to the mucus embedding the epithelium, from where glucose would be taken by the cilia to generate ATP glycolytically.

Using a specific antibody that fluorescently labeled GLUT3, we first confirmed that the apical membranes of the cilia and the sustentacular cells microvilli express GLUT3. In addition, we found that the cilia can incorporate glucose by this transporter. To find this out, we applied a fluorescent glucose analog over the epithelium surface, observing that the cilia and knobs became fluorescently labeled. We confirmed this in individual olfactory neurons and sustentacular cells dissociated from this tissue. 

Importantly, in the presence of a GLUT inhibitor, the cells failed to uptake glucose, showing that GLUTs were necessary for this to occur. We also demonstrated the presence of key glycolytic enzymes in the cilia, consistent with the notion that the cilia can metabolize the incorporated sugar.

An absolutely crucial question was whether the mucus contained glucose. Strikingly, we detected substantial levels of glucose (approximately one millimeter) by means of a colorimetric assay. 

All of this evidence aligned with our idea that mucus glucose might serve to power transduction in the cilia. However, did this really happen?

To investigate, we used electrophysiological recordings both from intact olfactory epithelia and from individual dissociated olfactory sensory neurons.

Remarkably, we observed that the electrical responses to odor stimulation required extracellular glucose (1 mM), because when we removed it from the solution bathing the cells the response became significantly impaired, and it recovered if we restored the glucose. Also, inhibitors of glycolysis and of oxidative phosphorylation reduced the odor responses. 

Altogether, the evidence supports our hypothesis and let us to propose a model where the cilia obtain ATP both from the dendrite mitochondria of the knob and glycolysis in the ciliary lumen and use it to support chemotransduction.

Visit JNeurosci to read the original article and explore other content. Read other summaries of JNeurosci and eNeuro papers in the Neuronline collection, SfN Journals: Research Article Summaries.

Energy Requirements of Odor Transduction in the Chemosensory Cilia of Olfactory Sensory Neurons Rely on Oxidative Phosphorylation and Glycolytic Processing of Extracellular Glucose. Pablo S. Villar, Ricardo Delgado, Cecilia Vergara, Juan G. Reyes, and Juan Bacigalupo. JNeurosci May 2017, 2640-16; DOI: 10.1523/JNEUROSCI.2640-16.2017

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Pablo S. Villar
Pablo S. Villar is currently doing his PhD thesis on the intrinsic and extrinsic inhibition of the neuronal circuitry in the olfactory bulb in Ricardo C. Araneda’s lab at the University of Maryland. He received his bachelor’s degree from Universidad Católica de Valparaíso in Chile. The bulk of the results described in the JNeurosci paper on which this Neuronline summary is based is derived from his undergraduate thesis carried out in the laboratory of Juan Bacigalupo at the University of Chile.
Pablo S. Villar
Pablo S. Villar is currently doing his PhD thesis at the University of Maryland.

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