Monocarboxylate Transporters In Tumor Cells And Mitochondrial DNA Toxicity Mice

Monocarboxylate transporters are a family of transmembrane proteins which can transport monocarboxylate through the cytolemma. Most common monocarboxylate in the mammalian are lactate, pyruvate and ketone bodies. Monocarboxylate transporters are a big family and fourteen MCTs have been identified. But only MCT1, MCT2 and MCT3 are shown to catalyze proton coupled transport of lactate in human. Lactate is the end product of glycolysis and quantitatively the most important monocarboxylate. In glycolysis, glucose is enzymatically broken down to pyruvate, which in turn either enters the mitochondria for oxidation in the Krebs cycle or is reversibly converted to lactate by the enzyme lactate dehydrogenase. Export of lactate from the cell is important to maintain high rates of glycolysis in excess of what can be oxidized via the Krebs cycle. This needs the normal function of monocarboxylate tranporters. Uncontrolled proliferation is a main characteristic of cancer. The fast proliferation results in an increased demand for nutrients. These nutrients include glucose, amino acids, and fatty acids. The proliferating cancer cells have few mitochondria in cytoplasm and use cytosolic glycolysis to yield ATP even with adequate oxygen. This is called'Warburg Effect'. High rate of the glycolysis must have the accumulation of lactate and pyruvate in the cytoplasm. Transport monocarboxylate out the cell and keep the balance of production and consumption are also attributed to monocarboxylate transporters.Mitochondria are crucial for the viability of multicellular organisms. Mitochondrial dynamics are particularly important in neurons, where the energy demand of synaptic regions of axons is intense. It is estimated that the human brain accounts for 20% of the body's resting metabolism. Defects in mitochondrial distribution can therefore cause localized energy and Ca2+ defects in affected neurons, resulting in synaptic dysfunction. The importance of mitochondrial dynamics in proper neuronal function is illustrated by several neurodegenerative diseases with such defects, including Parkinson's disease and Alzheimer's disease. Mitochondria are strictly dependent on the nuclear gene products; yet they also contain their own genome, which provides components for the respiratory electron transport chain (ETC). We have previously generated transgenic mice that conditionally express a mutated DNA-repair enzyme (mutUNG1) in forebrain neurons using a Tet-on inducible system that drives human mutUNG1 expression under the contro1 of the Ca2+/calmodulin-dependent kinaseⅡα(CαMKⅡα)-promoter. This creates mtDNA damage in the form of apyrimidinic-(AP) sites. The resulting mice are characterized by mtDNA toxicity, excessive apoptosis, neurodegeneration, and behavioral impairments. We show here that the hippocampus of mutUNG1-expressing mice have severely altered mitochondrial dynamics and reaches a state of oxidative stress, reflected in strong upregulation of endogenous antioxidant enzymes and increased oxidative DNA damage—features comparable to the early symptoms of neurodegenerative pathologies like Alzheimer's disease. So the mutUNG1 model can be used for the study of neurodegenerative diseases mechanism.For this research, we firstly analyzed the different MCTs expression on membranes of human T-47D breast cancer cells and human glioblastoma T98G cells by normoxic or cycle hypoxic cultivation. The data from electronic photographs by post-embedding immunogold electron microscopy showed the different regulations of MCTs on tumor cell membranes in various oxygen conditions. The differential expression and regulation of MCTs in hypoxic and normoxic tumour cells opens possibilities for the innovation of tumour therapy through the selective targeting of monocarboxylate metabolism.The next experiment for the analysis of mtDNA damage in forebrain neurons in the mutUNG1-expressing mice model reveals physiological features that mirror aspects of widespread neurological pathologies, like Alzheimer's disease. Ultimately, understanding the cellular mechanisms underlying neurological pathologies and their progression, where mitochondrial dysfunction may often play a prominent role, is important for proper treatment. To date, mouse models have provided many answers that would be difficult to obtain using other biological systems, including the demonstration that well functioning mitochondria with intact mtDNA are necessary for neuronal viability.Finally by the experiments with metabolic associated proteins in hippocampus and cerebellum of mutUNG1-expressing and wild type mice, we got a better understanding of affects on synaptic information transfer, glucose transport and lactate metabolism by mitochondrial DNA toxicity. For the first time we portrayed the distribution and expression of MCT1 and MCT2 in hippocampus stratum radiatum and cerebellum by transmission electron microscopy. It's a morphology basis for brain energy metabolism especially for lactate and glucose in normal and pathological situations. Part I:Alterations of monocarboxylate transporter densities during hypoxia in brain and breast tumour cellsObjective:To analyze the monocarboxylate transporter densities in breast cancer T-47D and glioblastoma T98G cell membranes during cycle hypoxic and nomoxic cultures.Methods:1. Human breast cancer T-47D and glioblastoma T98G cell were cultured separately under 4% cycle hypoxia or 20% normoxic conditions.2. PH, lactate and glucose measurements in medium while culture.3. Primary antibodies'specificity test by Western blotting.4. Labeled MCTs on cell membranes by Post-embedding immunogold electron microscopy.5. Analyze electronic micrographs by Image J plus in Point-density.Results:1. The energy metabolism style was found different between hypoxic and normoxic cells by pH, lactate and glucose measurements.2. All five MCT antibodies have proper specificities by Western blotting test.3. When normoxic and hypoxic cells were compared, the membrane density of MCT1 was significantly higher in cells grown with 4% oxygen than in normoxic cells, both for T-47D and T98G cells (all p<0.0001, Student's t-test). The membrane density of MCT4 in hypoxic T-47D and T98G cells was significantly higher than that of normoxic tumour cells (all p<0.0001, Student's t-test). In contrast, the density of MCT2 revealed a difference between T-47D and T98G cells. In T-47D cells, the density of MCT2 on plasma membranes in hypoxic cells was significantly higher than that of normoxic cells (all p<0.0001, Student's t-test). However, in T98G cells, the density of MCT2 on plasma membranes in cells grown with 4% oxygen was significantly lower than that of normoxic cells (all p<0.0001, Student's t-test)Conclusion:1. For tumor cells the energy metabolism style can be changed by oxygen conditions..2. The differential expression and regulation of MCTs in hypoxic and normoxic tumour cells opens possibilities for the innovation of tumour therapy through the selective targeting of monocarboxylate metabolism.PartⅡ:Mitochondrial DNA toxicity compromises mitochondrial dynamicsObjective:To explore the mitochondrial DNA toxicity comprises mitochondrial dynamics in cerebellum and hippocampus.Methods:1. MutUNG1-expressing mice modeling.2. Total DNA for mitochondrial DNA copy number and RNA expression analysis by RT-PCR.3. Proteins in hippocampus analyzed and compared between mutUNG1-expressing mice and wild type mice by confocal immunoflourence.Results:1. Mitochondrial dynamics are compromised in the hippocampus of muUNG1-expressing mice..2. Mitochondrial DNA copy number and mitochondrial gene expression are greatly reduced in mutUNG1-expressing mice.3. Mitochondrial DNA-encoded ETC complexes are compromised by mutUNG1 expression.4. Mitochondrial DNA expression induces astrogliosis in the hippocampus.5. The antioxidant defense system is induced in the hippocampus of mutUNG1-expressing mice.6. Oxidative stress in the hippocampus causes DNA damage and relocalization of Apel. Conclusion:The analysis of mitochondrial DNA damage in forebrain neurons in the mutUNG1-expressing mice model reveals physiological features that mirror aspects of widespread neurological pathologies, like Alzheimer's disease. Ultimately, understanding the cellular mechanisms underlying neurological pathologies and their progression, where mitochondrial dysfunction may often play a prominent role, is important for proper treatment.PartⅢ:Alterations of energy metabolism associated proteins in mitochondrial DNA toxicity miceObjective:To analyze the distribution and regulation of energy metabolism associated proteins (AMPA receptors, NMDA receptors, glucose transporter 1, MCT1 and MCT2) in hippocampus and cerebellum by post-embedding immunogold electron microscopy within mitochondrial DNA toxicity mice and wild type mice.Methods:1. MutUNG1-expressing mice modeling.2.3 four month old mutUNG1-expressing mice and 3 same genotype wild type mice were perfumed and brains were removed for transmission electronic microscopy tissue preparation.3. Ultra-thin brain tissue were made and connected with primary antibodies (NR2A/B, GluR1, GluR2/3, GLUT1, MCT1, MCT2) and after that reacted with 10 nm gold particles conjugated secondary antibodies.4. Electronic micrographs were taken from hippocampus and cerebellum and analyzed by Image J with plug in Point-density or Synapses.Results:1. PSD length of NMDA receptors (NR2A/B) decreased in stratum radiatum and increased in hilus in mutUNG1-expressing mice compared with wild type mice. 2. mutUNG1 expression induced a loss of AMPA (GluRl, GluR2/3) receptors and anomalies in PSD length.3. mutUNG1 expression induced mitochondria clustered around the nucleus in granular cells within hippocampus.4. No difference between mutUNG1-expressing mice and wild type mice for the densities of GLUT1 on luminal and abluminal membranes of endothelial cell around micro-vessel.5. Densities of MCT1 in hippocampus were higher than that in cerebellum. The expression decreased in hippocampus within mutUNG1-expressing mice.6. Densities of MCT2 on astrocyte end-feet in hippocampus and granular layer of cerebellum were higher than that in molecular layer of cerebellum. However, in molecular layer, many MCT2 labeled synapses were found. As MCT1, in hippocampus, the expression decreased in mutUNG1-expressing mice.Conclusion:By the experiments with metabolic associated proteins in hippocampus and cerebellum of mutUNG1-expressing and wild type mice, we got a better understanding of affects on synaptic information transfer, glucose transport and lactate metabolism by mitochondrial DNA toxicity. For the first time we portrayed the distribution and expression of MCT1 and MCT2 in hippocampus stratum radiatum and cerebellum by transmission electron microscopy. It's a morphology basis for brain energy metabolism especially for lactate and glucose in normal and pathological situations.