Betaine-The dark knight of the brain

The role of betaine in the liver and kidney has been well documented, even from the cellular and molecular point of view. Despite literature reporting positive effects of betaine supplementation in Alzheimer's, Parkinson's and schizophrenia, the role and function of betaine in the brain are little studied and reviewed. Beneficial effects of betaine in neurodegeneration, excitatory and inhibitory imbalance and against oxidative stress in the central nervous system (CNS) have been collected and analysed to understand the main role of betaine in the brain. There are many 'dark' aspects needed to complete the picture. The understanding of how this osmolyte is transported across neuron and glial cells is also controversial, as the expression levels and functioning of the known protein capable to transport betaine expressed in the brain, betaine-GABA transporter 1 (BGT-1), is itself not well clarified. The reported actions of betaine beyond BGT-1 related to neuronal degeneration and memory impairment are the focus of this work. With this review, we underline the scarcity of detailed molecular and cellular information about betaine action. Consequently, the requirement of detailed focus on and study of the interaction of this molecule with CNS components to sustain the therapeutic use of betaine

Bile acid interactions with neurotransmitter transporters

Synthesized in the liver from cholesterol, the bile acids (BAs) primary role is emulsifying fats to facilitate their absorption. BAs can cross the blood-brain barrier (BBB) and be synthesized in the brain. Recent evidence suggests a role for BAs in the gut-brain signaling by modulating the activity of various neuronal receptors and transporters, including the dopamine transporter (DAT). In this study, we investigated the effects of BAs and their relationship with substrates in three transporters of the solute carrier 6 family. The exposure to obeticholic acid (OCA), a semi-synthetic BA, elicits an inward current (I-BA) in the DAT, the GABA transporter 1 (GAT1), and the glycine transporter 1 (GlyT1b); this current is proportional to the current generated by the substrate, respective to the transporter. Interestingly, a second consecutive OCA application to the transporter fails to elicit a response. The full displacement of BAs from the transporter occurs only after exposure to a saturating concentration of a substrate. In DAT, perfusion of secondary substrates norepinephrine (NE) and serotonin (5-HT) results in a second OCA current, decreased in amplitude and proportional to their affinity. Moreover, co-application of 5-HT or NE with OCA in DAT, and GABA with OCA in GAT1, did not alter the apparent affinity or the I-max, similar to what was previously reported in DAT in the presence of DA and OCA. The findings support the previous molecular model that suggested the ability of BAs to lock the transporter in an occluded conformation. The physiological significance is that it could possibly avoid the accumulation of small depolarizations in the cells expressing the neurotransmitter transporter. This achieves better transport efficiency in the presence of a saturating concentration of the neurotransmitter and enhances the action of the neurotransmitter on their receptors when they are present at reduced concentrations due to decreased availability of transporters

Bile Acids Gate Dopamine Transporter Mediated Currents

Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Sterols such as cholesterol interact with monoamine transporters, including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward. This study explores the interactions of the BA, obeticholic acid (OCA), with DAT and characterizes the regulation of DAT activity via both electrophysiology and molecular modeling. We expressed murine DAT (mDAT) in Xenopus laevis oocytes and confirmed its functionality. Next, we showed that OCA promotes a DAT-mediated inward current that is Na+-dependent and not regulated by intracellular calcium. The current induced by OCA was transient in nature, returning to baseline in the continued presence of the BA. OCA also transiently blocked the DAT-mediated Li+-leak current, a feature that parallels DA action and indicates direct binding to the transporter in the absence of Na+. Interestingly, OCA did not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, regarding DA. Docking simulations performed for investigating the molecular mechanism of OCA action on DAT activity revealed two potential binding sites. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., J. Biol. Chem., 2014, 289, 25764\u201325773; Cheng and Bahar, Structure, 2015, 23, 2171\u20132181). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is coordinated by the DAT residues R445 and D436. Binding to that site may stabilize the inward-facing (IF) open state by preventing the re-formation of the IF-gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors

Lifestyles and socio-cultural factors among children aged 6-8 years from five Italian towns: The MAPEC-LIFE study cohort

Background: Lifestyles profoundly determine the quality of an individual’s health and life since his childhood. Many diseases in adulthood are avoidable if health-risk behaviors are identified and improved at an early stage of life. The aim of the present research was to characterize a cohort of children aged 6–8 years selected in order to perform an epidemiological molecular study (the MAPEC_LIFE study), investigate lifestyles of the children that could have effect on their health status, and assess possible association between lifestyles and socio-cultural factors. Methods: A questionnaire composed of 148 questions was administered in two different seasons to parents of children attending 18 primary schools in five Italian cities (Torino, Brescia, Pisa, Perugia and Lecce) to obtain information regarding the criteria for exclusion from the study, demographic, anthropometric and health information on the children, as well as some aspects on their lifestyles and parental characteristics. The results were analyzed in order to assess the frequency of specific conditions among the different seasons and cities and the association between lifestyles and socio-economic factors. Results: The final cohort was composed of 1,164 children (50.9 boys, 95.4% born in Italy). Frequency of some factors appeared different in terms of the survey season (physical activity in the open air, the ways of cooking certain foods) and among the various cities (parents’ level of education and rate of employment, sport, traffic near the home, type of heating, exposure to passive smoking, ways of cooking certain foods). Exposure to passive smoking and cooking fumes, obesity, residence in areas with heavy traffic, frequency of outdoor play and consumption of barbecued and fried foods were higher among children living in families with low educational and/or occupational level while children doing sports and consuming toasted bread were more frequent in families with high socio-economic level. Conclusions: The socio-economic level seems to affect the lifestyles of children enrolled in the study including those that could cause health effects. Many factors are linked to the geographical area and may depend on environmental, cultural and social aspects of the city of residence

Investigation of the interaction of bile acids with the dopamine transporter DAT

Bile acids (BAs) are cholesterol derivatives primarily involved in dietary fat absorption; new evidence supports a possible additional role in regulating the reward system. Bile diversion, a bariatric surgery used to treat obesity, increases circulating BAs, and in mice reduces both food and cocaine reward-related behavior. Feeding mice addicted to cocaine with Obeticholic acid (OCA), a bile acid analogue, FDA-approved drug, and an agonist of the plasma membrane Takeda G protein-coupled receptor 5 (TGR5), induced the same effects as the bariatric surgery. The interactions of OCA with murine DAT (mDAT) heterologously expressed in X. laevis oocytes investigated by two-electrode voltage clamp showed that OCA promotes a DAT-mediated transient Na+-inward current, not dependent on the presence of hTGR5 and that OCA did not affect DA affinity and DAT kinetic parameters. Docking simulations highlighted possible binding sites for OCA on DAT suggesting that OCA could stabilize DAT in an occluded conformation (Romanazzi et al., 2021). Oocytes expressing mDAT exposed to a second OCA perfusion do not elicit OCA–inward transient current and only after DA perfusion, OCA-current was visible again. The modeling suggests that the OCA stabilizes DAT in an occluded conformation, the experiments let to hypothesize that the DA binding allows the displacement of OCA, and lets DAT complete the transport cycle. When the same experiment was repeated in the presence of other DAT substrates like norepinephrine (NE) and serotonin (5HT), agonists with lower apparent affinity, OCA elicits an inward transient current, but smaller in amplitude compared to the one recorded after DA perfusion. We hypothesize that this occurs because NE or 5HT results in a weaker displacement of the OCA, thus leaving some transporters bound to OCA, in the occluded conformation. Moreover, reducing DA concentration from 30 to 3μM (proximal to the K05), the second exposure to OCA-elicited a smaller current than the first, supporting the hypothesis that some transporters stay in occluded OCA-bind conformation, lowering the amplitude of the current elicited by the second OCA exposure. These experiments agree with the model: OCA binds and freezes DAT in an occluded conformation that can be recovered by the presence of the substrates. The lithocholic acid (LCA) a natural bile acid behaves similarly to OCA (Romanazzi et al., 2021). Exposing mDAT to LCA or OCA in sequence point out that the perfusion order changes the responses to the BA. OCA induces a current after LCA perfusion, but LCA does not after the application of OCA. These data suggest a stronger interaction of OCA with mDAT. The different apparent affinity could be caused by the R groups at positions 5 and 6 of the steroid-base ring, common to both BA. Modeling the interactions of R- groups with DAT residues may suggest possible determinants involved in the mechanism of action. These are preliminary results; the ongoing experiment will require to determining the apparent affinity and IC50 of BA in the presence of different substrates and in the presence of cocaine and amphetamine. Future work will be focused on the OCA binding site, studying DAT mutants suggested by docking experiments. Testing the BAs on other SLC6 transporters will shed a light on the specificity of the interaction

The “www” of Xenopus laevis Oocytes: The Why, When, What of Xenopus laevis Oocytes in Membrane Transporters Research

After 50 years, the heterologous expression of proteins in Xenopus laevis oocytes is still essential in many research fields. New approaches and revised protocols, but also classical methods, such as the two-electrode voltage clamp, are applied in studying membrane transporters. New and old methods for investigating the activity and the expression of Solute Carriers (SLC) are reviewed, and the kinds of experiment that are still useful to perform with this kind of cell are reported. Xenopus laevis oocytes at the full-grown stage have a highly efficient biosynthetic apparatus that correctly targets functional proteins at the defined compartment. This small protein factory can produce, fold, and localize almost any kind of wild-type or recombinant protein; some tricks are required to obtain high expression and to verify the functionality. The methodologies examined here are mainly related to research in the field of membrane transporters. This work is certainly not exhaustive; it has been carried out to be helpful to researchers who want to quickly find suggestions and detailed indications when investigating the functionality and expression of the different members of the solute carrier families