Visualization of Spatiotemporal Energy Dynamics of Hippocampal Neurons by Mass Spectrometry during a Kainate-Induced Seizure

We report the use of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry combined with capillary electrophoresis (CE) mass spectrometry to visualize energy metabolism in the mouse hippocampus by imaging energy-related metabolites. We show the distribution patterns of ATP, ADP, and AMP in the hippocampus as well as changes in their amounts and distribution patterns in a murine model of limbic, kainate-induced seizure. As an acute response to kainate administration, we found massive and moderate reductions in ATP and ADP levels, respectively, but no significant changes in AMP levels—especially in cells of the CA3 layer. The results suggest the existence of CA3 neuron-selective energy metabolism at the anhydride bonds of ATP and ADP in the hippocampal neurons during seizure. In addition, metabolome analysis of energy synthesis pathways indicates accelerated glycolysis and possibly TCA cycle activity during seizure, presumably due to the depletion of ATP. Consistent with this result, the observed energy depletion significantly recovered up to 180 min after kainate administration. However, the recovery rate was remarkably low in part of the data-pixel population in the CA3 cell layer region, which likely reflects acute and CA3-selective neural death. Taken together, the present approach successfully revealed the spatiotemporal energy metabolism of the mouse hippocampus at a cellular resolution—both quantitatively and qualitatively. We aim to further elucidate various metabolic processes in the neural system

Induction of Erythroid Differentiation in Human Erythroleukemia Cells by Depletion of Malic Enzyme 2

Malic enzyme 2 (ME2) is a mitochondrial enzyme that catalyzes the conversion of malate to pyruvate and CO2 and uses NAD as a cofactor. Higher expression of this enzyme correlates with the degree of cell de-differentiation. We found that ME2 is expressed in K562 erythroleukemia cells, in which a number of agents have been found to induce differentiation either along the erythroid or the myeloid lineage. We found that knockdown of ME2 led to diminished proliferation of tumor cells and increased apoptosis in vitro. These findings were accompanied by differentiation of K562 cells along the erythroid lineage, as confirmed by staining for glycophorin A and hemoglobin production. ME2 knockdown also totally abolished growth of K562 cells in nude mice. Increased ROS levels, likely reflecting increased mitochondrial production, and a decreased NADPH/NADP+ ratio were noted but use of a free radical scavenger to decrease inhibition of ROS levels did not reverse the differentiation or apoptotic phenotype, suggesting that ROS production is not causally involved in the resultant phenotype. As might be expected, depletion of ME2 induced an increase in the NAD+/NADH ratio and ATP levels fell significantly. Inhibition of the malate-aspartate shuttle was insufficient to induce K562 differentiation. We also examined several intracellular signaling pathways and expression of transcription factors and intermediate filament proteins whose expression is known to be modulated during erythroid differentiation in K562 cells. We found that silencing of ME2 leads to phospho-ERK1/2 inhibition, phospho-AKT activation, increased GATA-1 expression and diminished vimentin expression. Metabolomic analysis, conducted to gain insight into intermediary metabolic pathways that ME2 knockdown might affect, showed that ME2 depletion resulted in high orotate levels, suggesting potential impairment of pyrimidine metabolism. Collectively our data point to ME2 as a potentially novel metabolic target for leukemia therapy

Altered Expression of Human Mitochondrial Branched Chain Aminotransferase in Dementia with Lewy Bodies and Vascular Dementia

© 2016, The Author(s). Cytosolic and mitochondrial human branched chain aminotransferase (hBCATc and hBCATm, respectively) play an integral role in brain glutamate metabolism. Regional increased levels of hBCATc in the CA1 and CA4 region of Alzheimer’s disease (AD) brain together with increased levels of hBCATm in frontal and temporal cortex of AD brains, suggest a role for these proteins in glutamate excitotoxicity. Glutamate toxicity is a key pathogenic feature of several neurological disorders including epilepsy associated dementia, AD, vascular dementia (VaD) and dementia with Lewy bodies (DLB). To further understand if these increases are specific to AD, the expression profiles of hBCATc and hBCATm were examined in other forms of dementia including DLB and VaD. Similar to AD, levels of hBCATm were significantly increased in the frontal and temporal cortex of VaD cases and in frontal cortex of DLB cases compared to controls, however there were no observed differences in hBCATc between groups in these areas. Moreover, multiple forms of hBCATm were observed that were particular to the disease state relative to matched controls. Real-time PCR revealed similar expression of hBCATm mRNA in frontal and temporal cortex for all cohort comparisons, whereas hBCATc mRNA expression was significantly increased in VaD cases compared to controls. Collectively our results suggest that hBCATm protein expression is significantly increased in the brains of DLB and VaD cases, similar to those reported in AD brain. These findings indicate a more global response to altered glutamate metabolism and suggest common metabolic responses that might reflect shared neurodegenerative mechanisms across several forms of dementia

H55N polymorphism as a likely cause of variation in citrate synthase activity of mouse skeletal muscle

The aim of this study was to investigate the mechanisms underlying low activity of citrate synthase (CS) in A/J mice compared with other inbred strains of mice. Enzyme activity, protein content, and mRNA levels of CS were studied in the quadriceps muscles of A/J, BALB/cByJ, C57BL/6J, C3H/HeJ, DBA/2J, and PWD/PhJ strains of mice. Cytochrome c protein content was also measured. The results of the study indicate that A/J mice have a 50–65% reduction in CS activity compared with other strains despite similar levels of Cs mRNA and lack of differences in CS and cytochrome c protein content. CS from A/J mice also showed lower Michaelis constant (Km) for both acetyl CoA and oxaloacetate compared with the other strains of mice