Claudia Colina

The incidence of obesity has markedly increased globally over the last several decades and is believed to be associated with the easier availability of energy-dense foods, including high-fat foods. The reinforcing hedonic properties of high-fat foods, including olfactory cues, activate reward centers in the brain, motivating eating behavior. Thus, there is a growing interest in the understanding of the genetic changes that occur in the brain that are associated with obesity and eating behavior. This growing interest has paralleled advances in genomic methods that enable transcriptomic-wide analyses. Here, we examined the transcriptomic-level differences in the olfactory bulb and striatum, regions of the brain associated with olfaction and hedonic food-seeking, respectively, in high-fat-diet (HFD)-fed obese mice. To isolate the dietary effects from obesity, we also examined transcriptomic changes in normal-chow-fed and limited-HFD-fed groups, with the latter being pair-fed with an HFD isocaloric to the consumption of the normal-chow-fed mice. Using RNA sequencing, we identified 274 differentially expressed genes (DEGs) in the striatum and 11 in the olfactory bulb of ad libitum HFD-fed mice compared to the chow-fed group, and thirty-eight DEGs in the striatum between the ad libitum HFD and limited-HFD-fed groups. The DEGs in both tissues were associated with inflammation and immune-related pathways, including oxidative stress and immune function, and with mitochondrial dysfunction and reward pathways in the striatum. These results shed light on potential obesity-associated genes in these regions of the brain.

Activation of short-chain free fatty acid receptors 3 (FFAR3) has been suggested to promote sympathetic outflow in postganglionic sympathetic neurons or hamper it by a negative coupling to N-type calcium (Ca _V 2.2) channels. Heterogeneity of FFAR3 expression in sympathetic neurons, however, renders single neurons studies extremely time-consuming in wild-type mice. Previous studies demonstrated large variability of the degree of Ca _V 2.2 channel inhibition by FFAR3 in a global population of rat sympathetic neurons. Therefore, we focused on a small subpopulation of mouse sympathetic neurons using an FFAR3 antibody and an Ffar3 reporter mouse to perform immunofluorescent and electrophysiological studies. Whole-cell patch-clamp recordings of identified FFAR3-expressing neurons from reporter mice revealed a 2.5-fold decrease in the Ca _V 2.2-FFAR3 inhibitory coupling variability and 1.5-fold increase in the mean I _Ca ²⁺ inhibition, when compared with unlabeled neurons from wild-type mice. Further, we found that the ablation of Ffar3 gene expression in two knockout mouse models led to a complete loss-of-function. Subpopulations of sympathetic neurons are associated with discrete functional pathways. However, little is known about the neural pathways of the FFAR3-expressing subpopulation. Our data indicate that FFAR3 is expressed primarily in neurons with a vasoconstrictor phenotype. Thus, fine-tuning of chemically-coded neurotransmitters may accomplish an adequate outcome.

We have previously demonstrated that rat type-1 cerebellar astrocytes express a very active Na⁺/Ca²⁺ exchanger which accounts for most of the total plasma membrane Ca²⁺ fluxes and for the clearance of Ca _i ²⁺ induced by physiological agonist. In this chapter, we have explored the mechanism by which the reverse Na⁺/Ca²⁺ exchange is involved in agonist-induced Ca²⁺ signalling in rat cerebellar astrocytes. Laser-scanning confocal microscopy experiments using immunofluorescence labelling of Na⁺/Ca²⁺ exchanger and RyRs demonstrated that they are highly co-localized. The most important finding presented in this chapter is that L-glutamate activates the reverse mode of the Na⁺/Ca²⁺ exchange by inducing a Na⁺ entry through the electrogenic Na⁺-glutamate co-transporter and not through the ionophoric L-glutamate receptors as confirmed by pharmacological experiments with specific blockers of ionophoric L-glutamate receptors, electrogenic glutamate transporters and the Na/Ca exchange.

Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na⁺/K⁺ pumps in order to maintain ion homeostasis. In this study we show that Na⁺/K⁺ pump activity is tightly regulated by a novel process, RNA editing. Three codons within the squid Na⁺/K⁺ ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues. At one site, a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine, a change that shifts the pump's intrinsic voltage dependence. Mechanistically, the removal of a single methyl group specifically targets the process of Na⁺ release to the extracellular solution, causing a higher turnover rate at the resting membrane potential. In order for excitable cells like neurons and muscles to generate electrical signals, they require ion gradients across their plasma membranes. For example, sodium concentrations are much lower inside a cell than outside, and for potassium it is the opposite case. The job of maintaining these ion gradients falls squarely on a single protein: the Na⁺/K⁺ pump. During each transport cycle, this enzyme moves three sodium ions out of the cell and imports two of potassium. Because this process is the foundation for so many physiological processes, the Na⁺/K⁺ pump is costly to operate, using ∼30% of the ATP generated by an organism. Proper regulation of its turnover rate is vital. In this work, we use the giant nerve cell of squid as a model to show that the Na⁺/K⁺ pump can be regulated by an unsuspected mechanism. Although the gene that codes for this enzyme can make a perfectly functional pump, sometimes its information changes as it passes through the messenger RNA. This is achieved by editing RNA and as a result subtly different versions of the pump can be made, differing at only three amino acids out of more than a thousand. We demonstrate that RNA editing modulates the Na⁺/K⁺ pump's turnover rate and sodium release.

Throughout evolution, enzymes have adapted to perform in different environments. The Na⁺/K⁺ pump, an enzyme crucial for maintaining ionic gradients across cell membranes, is strongly influenced by the ionic environment. In vertebrates, the pump sees much less external Na⁺ (100–160 mM) than it does in osmoconformers such as squid (450 mM), which live in seawater. If the extracellular architecture of the squid pump were identical to that of vertebrates, then at the resting potential, the pump's function would be severely compromised because the negative voltage would drive Na⁺ ions back to their binding sites, practically abolishing forward transport. Here we show that four amino acids that ring the external mouth of the ion translocation pathway are more positive in squid, thereby reducing the pump's sensitivity to external Na⁺ and explaining how it can perform optimally in the marine environment.

We have previously demonstrated that rat cerebellar Type‐1 astrocytes express a very active genistein sensitive Na⁺/Ca²⁺exchanger, which accounts for most of the total plasma membrane Ca²⁺fluxes and for the clearance ofloads induced by physiological agonists. In this work, we have explored the mechanism by which the reverse Na⁺/Ca²⁺exchange is involved in agonist‐induced Ca²⁺signaling in rat cerebellar astrocytes. Microspectrofluorometric measurements ofwith Fluo‐3 demonstrate that thesignals associated long (> 20 s) periods of reverse operation of the Na⁺/Ca²⁺exchange are amplified by a mechanism compatible with calcium‐calcium release, while those associated with short (< 20 s) pulses are not amplified. This was confirmed by pharmacological experiments using ryanodine receptors agonist (4‐chloro‐m‐cresol) and the endoplasmic reticulum ATPase inhibitor (thapsigargin). Confocal microscopy demonstrates a high co‐localization of immunofluorescent labeled Na⁺/Ca²⁺exchanger and RyRs. Low (< 50 μmol/L) or high (> 500 μmol/L) concentrations ofl‐glutamate (l‐Glu) orl‐aspartate causes a rise inwhich is completely blocked by the Na⁺/Ca²⁺exchange inhibitors KB‐R7943 and SEA0400. The most important novel finding presented in this work is thatl‐Glu activates the reverse mode of the Na⁺/Ca²⁺exchange by inducing Na⁺entry through the electrogenic Na⁺–Glu‐co‐transporter and not through the ionophoricl‐Glu receptors, as confirmed by pharmacological experiments with specific blockers of the ionophoricl‐Glu receptors and the electrogenic Glu transporter.

There is no effective treatment for the prevalent chronic form of Chagas' disease in Latin America. Its causative agent, the protozoan parasite Trypanosoma cruzi, has an essential requirement for ergosterol, and ergosterol biosynthesis inhibitors, such as the antifungal drug posaconazole, have potent trypanocidal activity. The antiarrhythmic compound amiodarone, frequently prescribed for the symptomatic treatment of Chagas' disease patients, has also recently been shown to have antifungal activity. We now show here for the first time that amiodarone has direct activity against T. cruzi, both in vitro and in vivo, and that it acts synergistically with posaconazole. We found that amiodarone, in addition to disrupting the parasites' Ca²⁺ homeostasis, also blocks ergosterol biosynthesis, and that posaconazole also affects Ca²⁺ homeostasis. These results provide logical explanations for the synergistic activity of amiodarone with azoles against T. cruzi and open up the possibility of novel, combination therapy approaches to the treatment of Chagas' disease using currently approved drugs.

Ca2+ plays an important role in the regulation of several important activities in different trypanosomatids. These parasites possess a Ca2+ transport system in the endoplasmic reticulum (ER) involved in Ca2+ homeostasis, which has been reported to be insensitive to thapsigargin, a classical inhibitor of the sarcoplasmic-ER Ca2+ adenosine triphosphatase (ATPase) (SERCA) in most eukaryotic cells. However, currently there is a controversy regarding the existence of a thapsigargin-sensitive ER Ca2+ store in these parasites. Therefore, we decided to explore the effect of this inhibitor using different methodological approaches. First, we selected Trypanosoma evansi as a parasite model to warrant the homogeneity of the population because this parasite has only a single life cycle, i.e., bloodstream-form trypomastigotes. Second, we compared the thapsigargin effect on Ca2+ homeostasis by spectrophotometrical Ca2+ measurements using 3 different approaches: whole-cell populations, cells that have been permeabilized by treatment with digitonin, and intact single cells. Our results demonstrate that a low concentration of thapsigargin induces Ca2+ release from intracellular Ca2+ stores in this parasite, which can be observed independently of the method used. Furthermore, the addition of thapsigargin before or after nigericin did not abolish its effect, showing that thapsigargin acts specifically on the ER. In conclusion, our results indicate the presence of a nonmitochondrial thapsigargin-sensitive Ca2+ store in T. evansi.