Sexually Dimorphic Effects of a Western Diet on Brain Mitochondrial Bioenergetics and Neurocognitive Function

A Western diet (WD), high in sugars and saturated fats, impairs learning and memory function and contributes to weight gain. Mitochondria in the brain provide energy for neurocognitive function and may play a role in body weight regulation. We sought to determine whether a WD alters behavior and metabolic outcomes in male and female rodents through impacting hippocampal and hypothalamic mitochondrial bioenergetics. Results revealed a sexually dimorphic macronutrient preference, where males on the WD consumed a greater percentage of calories from fat/protein and females consumed a greater percentage of calories from a sugar-sweetened beverage. Both males and females on a WD gained body fat and showed impaired glucose tolerance when compared to same-sex controls. Males on a WD demonstrated impaired hippocampal functioning and an elevated tendency toward a high membrane potential in hippocampal mitochondria. Comprehensive bioenergetics analysis of WD effects in the hypothalamus revealed a tissue-specific adaption, where males on the WD oxidized more fat, and females oxidized more fat and carbohydrates at peak energy demand compared to same-sex controls. These results suggest that adult male rats show a susceptibility toward hippocampal dysfunction on a WD, and that hypothalamic mitochondrial bioenergetics are altered by WD in a sex-specific manner.

Biogenetic and morphofunctional heterogeneity of mitochondria: the case of synaptic mitochondria

The mitochondria of different cells are different in their morphological and biochemical properties. These organelles generate free radicals during activity, leading inevitably to mitochondrial DNA damage. It is not clear how this problem is addressed in long-lived cells, such as neurons. We propose the hypothesis that mitochondria within the same cell also differ in lifespan and ability to divide. According to our suggestion, cells have a pool of ‘stem’ mitochondria with low metabolic activity and a pool of ‘differentiated’ mitochondria with significantly shorter lifespans and high metabolic activity. We consider synaptic mitochondria as a possible example of ‘differentiated’ mitochondria. They are significantly smaller than mitochondria from the cell body, and they are different in key enzyme activity levels, proteome, and lipidome. Synaptic mitochondria are more sensitive to different damaging factors. It has been established that neurons have a sorting mechanism that sends mitochondria with high membrane potential to presynaptic endings. This review describes the properties of synaptic mitochondria and their role in the regulation of synaptic transmission.

Abstract 15584: Mitotimer Protein Reveals Remarkable Macro and Micro Heterogeneity in the Heart; A Novel Tool for Determining Mitochondrial Turnover in the Heart

In order to study mitochondrial turnover at the level of a single mitochondrion, our laboratory has developed the MitoTimer protein. Timer is a mutant of DsRed fluorescent protein developed by Terskikh et al. The Timer protein transitions from green fluorescence to a more stable red conformation as it matures over a span of 48 h. Furthermore, the protein is very stable under physiological conditions, insensitive to variations in ionic strength, and changes in pH between 7.0 and 8.0. Notably, Timer maturation from green to red is significantly slowed in deoxygenated buffer, suggesting that molecular oxygen plays a part in fluorophore maturation. We created a construct that fused the Timer protein cDNA with the inner mitochondrial membrane signal sequence and placed it under the control of the cardiac-restricted α-myosin heavy chain promoter. This construct was used to create the α-MHC MitoTimer mice. Surprisingly, initial analysis of the hearts from these mice demonstrated a high degree of heterogeneity in the ratio of red-to-green fluorescence of MitoTimer in cardiac tissue, revealing regions of high and low oxygen tension. Further, individual mitochondria within cardiomyocytes display a higher red-to-green fluorescence relative to fluorescence of the other mitochondria in the cell, implying a block in import of newly synthesized MitoTimer caused by the lack of a high membrane potential, indicative of older, dysfunctional mitochondria. These mitochondria can be isolated and sorted from the heart by flow cytometry for further analysis. Initial studies suggest that these mice represent an elegant tool for the investigation of mitochondrial turnover in the heart.

Abstract 89: MitoTimer Mouse: a Novel Tool for the Study of Mitochondrial Turnover in vivo

In order to study mitochondrial turnover at the level of a single mitochondrion, our laboratory has developed the MitoTimer protein. Timer is a mutant of DsRed fluorescent protein developed by Terskikh et al. The Timer protein transitions from green fluorescence to a more stable red conformation as it matures over a span of 48 h. Furthermore, the protein is very stable under physiological conditions, insensitive to variations in ionic strength, and changes in pH between 7.0 and 8.0. Notably, Timer maturation from green to red is significantly slowed in deoxygenated buffer, suggesting that molecular oxygen plays a part in fluorophore maturation. We fused the Timer protein with the mitochondrial signal sequence from the cytochrome c oxidase subunit VIII (COX8) to target the protein to the inner membrane of the mitochondria, and further cloned the protein into a construct with a cardiac-restricted α-myosin heavy chain promoter. This construct was used to create the α-MHC MitoTimer mice. Surprisingly, initial analysis of the hearts from these mice reveals a remarkable degree of heterogeneity in the ratio of red-to- green fluorescence of MitoTimer in cardiac tissue. Furthermore, individual mitochondria within cardiomyocytes display a higher red-to-green fluorescence, implying a block in import of newly synthesized MitoTimer that would be caused by the lack of a high membrane potential, indicative of older, dysfunctional mitochondria. Initial studies suggest that these mice represent an elegant tool for the investigation of mitochondrial turnover in the heart.

High membrane potential promotes alkenal-induced mitochondrial uncoupling and influences adenine nucleotide translocase conformation

Mitochondria generate reactive oxygen species, whose downstream lipid peroxidation products, such as 4-hydroxynonenal, induce uncoupling of oxidative phosphorylation by increasing proton leak through mitochondrial inner membrane proteins such as the uncoupling proteins and adenine nucleotide translocase. Using mitochondria from rat liver, which lack uncoupling proteins, in the present study we show that energization (specifically, high membrane potential) is required for 4-hydroxynonenal to activate proton conductance mediated by adenine nucleotide translocase. Prolonging the time at high membrane potential promotes greater uncoupling. 4-Hydroxynonenal-induced uncoupling via adenine nucleotide translocase is prevented but not readily reversed by addition of carboxyatractylate, suggesting a permanent change (such as adduct formation) that renders the translocase leaky to protons. In contrast with the irreversibility of proton conductance, carboxyatractylate added after 4-hydroxynonenal still inhibits nucleotide translocation, implying that the proton conductance and nucleotide translocation pathways are different. We propose a model to relate adenine nucleotide translocase conformation to proton conductance in the presence or absence of 4-hydroxynonenal and/or carboxyatractylate.

Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential

Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of ROS (reactive oxygen species). This proton leak can be induced by the lipid peroxidation products of ROS, such as HNE (4-hydroxynonenal). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and ANT (adenine nucleotide translocase), thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We have found that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent on incubation with respiratory substrate. In the presence of HNE, mitochondria energized with succinate became progressively more leaky to protons over time compared with mitochondria in the absence of either HNE or succinate. Energized mitochondria must attain a high membrane potential to allow HNE to activate uncoupling: a drop of 10–20 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11%), ANT (64%) and other pathways (25%). Our findings have shown that exogenous HNE only activates uncoupling at high membrane potential. These results suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production.

The Function of Mitochondria in Presynaptic Development at the Neuromuscular Junction

Mitochondria with high membrane potential (ΔΨm) are enriched in the presynaptic nerve terminal at vertebrate neuromuscular junctions, but the exact function of these localized synaptic mitochondria remains unclear. Here, we investigated the correlation between mitochondrial ΔΨm and the development of synaptic specializations. Using mitochondrial ΔΨm-sensitive probe JC-1, we found that ΔΨm in Xenopus spinal neurons could be reversibly elevated by creatine and suppressed by FCCP. Along naïve neurites, preexisting synaptic vesicle (SV) clusters were positively correlated with mitochondrial ΔΨm, suggesting a potential regulatory role of mitochondrial activity in synaptogenesis. Indicating a specific role of mitochondrial activity in presynaptic development, mitochondrial ATP synthase inhibitor oligomycin, but not mitochondrial Na+/Ca2+ exchanger inhibitor CGP-37157, inhibited the clustering of SVs induced by growth factor–coated beads. Local F-actin assembly induced along spinal neurites by beads was suppressed by FCCP or oligomycin. Our results suggest that a key role of presynaptic mitochondria is to provide ATP for the assembly of actin cytoskeleton involved in the assembly of the presynaptic specialization including the clustering of SVs and mitochondria themselves.

Bacterial adhesion to and viability on positively charged polymer surfaces

Secondary and tertiary amino groups were introduced into polymer chains grafted onto a polyethylene flat-sheet membrane to evaluate the effects of surface properties on the adhesion and viability of a strain of the Gram-negative bacterium Escherichia coli and a strain of the Gram-positive bacterium Bacillus subtilis. The characterization of the surfaces containing amino groups, i.e. ethylamino (EA) and diethylamino (DEA) groups, revealed that the membrane potentials are proportional to amino-group densities and contact angle hysteresis. A high bacterial adhesion rate constant k was observed at high membrane potential, which indicates that membrane potential could be used as an indicator for estimating bacterial adhesion to the EA and DEA sheets, especially in B. subtilis. The bacterial adhesion rate constant of E. coli markedly increased at a membrane potential higher than −7.8 mV, whereas that of B. subtilis increased at a membrane potential higher than −8.3 mV, at which the dominant effect on bacterial adhesion is expected to change. The viability experiments revealed that approximately 80 % of E. coli cells adhering to the sheets with high membrane potential were inactivated after a contact time of 8 h, whereas 60 % of B. subtilis cells were inactivated. Furthermore, E. coli viability significantly decreased at a membrane potential higher than −8 mV, whereas B. subtilis viability decreased as membrane potential increased, which reflects differences in cell wall structure between E. coli and B. subtilis.

Ergodicity of Spike Trains: When Does Trial Averaging Make Sense?

Neuronal information processing is often studied on the basis of spiking patterns. The relevant statistics such as firing rates calculated with the peri-stimulus time histogram are obtained by averaging spiking patterns over many experimental runs. However, animals should respond to one experimental stimulation in real situations, and what is available to the brain is not the trial statistics but the population statistics. Consequently, physiological ergodicity, namely, the consistency between trial averaging and population averaging, is implicitly assumed in the data analyses, although it does not trivially hold true. In this letter, we investigate how characteristics of noisy neural network models, such as single neuron properties, external stimuli, and synaptic inputs, affect the statistics of firing patterns. In particular, we show that how high membrane potential sensitivity to input fluctuations, inability of neurons to remember past inputs, external stimuli with large variability and temporally separated peaks, and relatively few contributions of synaptic inputs result in spike trains that are reproducible over many trials. The reproducibility of spike trains and synchronous firing are contrasted and related to the ergodicity issue. Several numerical calculations with neural network examples are carried out to support the theoretical results.