High Concentration of Ketone Body ß-Hydroxybutyrate Modifies Synaptic Vesicle Cycle and Depolarizes Plasma Membrane of Rat Brain Synaptosomes.

Ketoacidosis is a dangerous complication of diabetes mellitus in which plasma levels of ketone bodies can reach 20-25 mM. This condition is life-threatening. In contrast, a ketogenic diet, achieving plasma levels of ketone bodies of about 4-5 mM, can be used for treating different brain diseases. However, the factors leading to the conversion of the neuroprotective ketone bodies' action to the neurotoxic action during ketoacidosis are still unknown. We investigated the influence of high concentration (25 mM) of the main ketone body, ß-hydroxybutyrate (BHB), on intrasynaptosomal pH (pHi), synaptic vesicle cycle, plasma membrane, and mitochondrial potentials. Using the fluorescent dye BCECF-AM, it was shown that BHB at concentrations of 8 and 25 mM did not influence pHi in synaptosomes. By means of the fluorescent dye acridine orange, it was demonstrated that 25 mM of BHB had no effect on exocytosis but inhibited compensatory endocytosis by 5-fold. Increasing buffer capacity with 25 mM HEPES did not affect endocytosis. Glucose abolished BHB-induced endocytosis inhibition. Using the fluorescent dye DiSC3(5), it was shown that 25 mM of BHB induced a significant plasma membrane depolarization. This effect was not impacted by glucose. Using the fluorescent dye rhodamine-123, it was shown that BHB alone (25 mМ) did not alter the potential of intrasynaptosomal mitochondria.Importantly, the high concentration of BHB (25 mМ) causes the depolarization of the plasma membrane and stronger inhibition of endocytosis compared with the intermediate concentration (8 mM).

Calcium release from intracellular stores is involved in mitochondria depolarization after lowering extracellular pH in rat brain synaptosomes.

In the brain, pH can be lowered in both healthy and disease states. Previously, we showed that moderate extracellular acidification (down to pHo 7.0), but not intracellular acidification, leads to mitochondrial depolarization in synaptosomes. This indicates that the plasma membranes of neuronal presynaptic endings have proton receptors that can induce mitochondrial dysfunction when activated. In the present paper we attempt to identify this hypothetical receptor. First, we have demonstrated that lowering pHo to 7.0 does not induce sodium influx as monitored by the fluorescent dye Sodium Green. This fact, in conjunction with the absence of calcium influx in the same conditions - demonstrated previously, excludes ion channels as possible receptors. However, we showed that acidification-induced mitochondrial depolarization is sensitive to thapsigargin - an inhibitor of calcium release from intracellular stores, U73122 - an inhibitor of phospholipase C, as well as Cu2+ and Zn2+, which can block the metabotropic proton receptor ovarian cancer G protein-coupled receptor 1 (OGR1). Furthermore, using fluorescent dye Fluo-3 we have demonstrated that moderate extracellular acidification induces a cytosolic calcium increase. Excess calcium was scavenged by mitochondria (monitored by fluorescent dye Rhod-2). Our results suggest that the metabotropic OGR1 is a hypothetical presynaptic receptor for low pH. Its activation leads to phospholipase C activation and calcium release from the endoplasmic reticulum followed by accumulation in mitochondria, which likely causes a decrease in mitochondrial membrane potential.

ß-Hydroxybutyrate supports synaptic vesicle cycling but reduces endocytosis and exocytosis in rat brain synaptosomes.

The ketogenic diet is used as a prophylactic treatment for different types of brain diseases, such as epilepsy or Alzheimer's disease. In such a diet, carbohydrates are replaced by fats in everyday food, resulting in an elevation of blood-borne ketone bodies levels. Despite clinical applications of this treatment, the molecular mechanisms by which the ketogenic diet exerts its beneficial effects are still uncertain. In this study, we investigated the effect of replacing glucose by the ketone body ß-hydroxybutyrate as the main energy substrate on synaptic vesicle recycling in rat brain synaptosomes. First, we observed that exposing presynaptic terminals to nonglycolytic energy substrates instead of glucose did not alter the plasma membrane potential. Next, we found that synaptosomes were able to maintain the synaptic vesicle cycle monitored with the fluorescent dye acridine orange when glucose was replaced by ß-hydroxybutyrate. However, in presence of ß-hydroxybutyrate, synaptic vesicle recycling was modified with reduced endocytosis. Replacing glucose by pyruvate also led to a reduced endocytosis. Addition of ß-hydroxybutyrate to glucose-containing incubation medium was without effect. Reduced endocytosis in presence of ß-hydroxybutyrate as sole energy substrate was confirmed using the fluorescent dye FM2-10. Also we found that replacement of glucose by ketone bodies leads to inhibition of exocytosis, monitored by FM2-10. However this reduction was smaller than the effect on endocytosis under the same conditions. Using both acridine orange in synaptosomes and the genetically encoded sensor synaptopHluorin in cortical neurons, we observed that replacing glucose by ß-hydroxybutyrate did not modify the pH gradient of synaptic vesicles. In conclusion, the nonglycolytic energy substrates ß-hydroxybutyrate and pyruvate are able to support synaptic vesicle recycling. However, they both reduce endocytosis. Reduction of both endocytosis and exocytosis together with misbalance between endocytosis and exocytosis could be involved in the anticonvulsant activity of the ketogenic diet.

Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes.

Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K(+)-induced Ca(2+)-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound's fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.

Role of iron, zinc and reduced glutathione in oxidative stress induction by low pH in rat brain synaptosomes.

Brain ischemia leads to a decrease in pHo. We have shown previously in synaptosomes that the extracellular acidification induces depolarization of mitochondria followed by synthesis of superoxide anions and oxidative stress. Here, we investigated the effects of lowered pHo on oxidative stress and membrane potentials in synaptosomes treated by the iron chelator deferoxamine and zinc chelator TPEN. We demonstrated that chelating of metals has no impact on superoxide anion synthesis and intrasynaptosomal mitochondria depolarization. Meanwhile, deferoxamine was able to inhibit oxidative stress induced by low pHo and hydrogen peroxide application. Compared to deferoxamine, TPEN was less effective but it decreased the DCF fluorescence induced by pHo 6.0 which had no effects in other oxidative stress models. We found that the chelators were able to inhibit slightly plasma membrane depolarization. Synaptosomes preincubation at low pHo caused no effects on the reduced glutathione level. Depletion of glutathione by CDNB produced no additional increase in the DCF fluorescence induced by pHo 7.0. Our results suggest that free iron is crucial for the development of oxidative stress elicited by acidification in synaptosomes. Chelating of this metal seems to be a promising strategy for protecting the neuronal presynaptic terminals against oxidative stress developed at stroke.