Final Report Summary - ASTROAGE (Astrocytes in aging brain exhibit altered glutamate homeostasis: Implications for age related cognitive decline?)
The effect of aging on brain function poses a serious threat to the quality of life in otherwise healthy individuals. The knowledge of the multitude of important functions in brain carried out by astrocytes is steadily increasing, however, their role in aging is still poorly investigated. A few studies on aging astrocytes have revealed age dependent reductions in glutamatergic ionotropic signaling, increased levels of GFAP and a more inflammatory state. However, there is a considerable lack of knowledge on how aging affects astrocyte energy metabolism. In humans, aging has been shown to alter TCA-cycle metabolism in neurons and astrocytes. Interestingly, the same study shows a reduction in glutamate-glutamine cycling, indicating a dysregulation of glutamate metabolism. Considered that astrocytes are of utmost importance for glutamate homeostasis in brain and that any disturbance in this homeostasis has the potential to cause neurodegeneration, more knowledge on the consequences of aging on astrocytes is needed.
Aging also affects glucose utilization in brain and ATP production is shown to be reduced in aging human tissue. Investigations of the hippocampal proteome have revealed altered expression of enzymes in the glycolytic pathway and in the TCA-cycle. Therefore, it is important to note that glutamate can be utilized as energy substrate through oxidative degradation in the TCA-cycle. Glutamate can be converted to α-ketoglutarate by transaminases or glutamate dehydrogenase (GDH), where the latter is the preferred pathway in mice astrocytes. The amount of ATP generated by oxidation of glutamate is equivalent to the amount generated by glucose, indicating that glutamate can be a valuable source of energy under low glucose conditions. Taken together, numerous findings point towards a profound role of astrocytes and glutamate dysregulation in aging. However, astrocyte (patho)physiology in aging brain is still poorly characterized and yet to be understood. The general aim of this project is, therefore, to elucidate age related effects on glutamate homeostasis and energy metabolism in astrocytes.
The specific aims of the projects were:
i) To quantify age-related changes in glutamate homeostasis in the hippocampus of mice.
ii) To characterize these changes on cellular, tissue and behavioral level.
Special focus have been put on if glutamate homeostasis is affected in brain of aging mice and if increased expression of GDH augments glutamate metabolism in the TCA cycle and counteracts age related cognitive decline.
During the course of the project we have investigated the metabolic functions of astrocytes in the hippocampus of 4 months old mice compared to 22 months old mice with regard to changes in glutamate and glucose metabolism by metabolic mapping using 13C-labelled glutamate, glucose and acetate followed by analysis of labeled isotopomers by gas chromatography coupled to mass spectrometry. To be able to carry out precise incubations of the hippocampal slices, we developed and validated an incubation chamber allowing for incubation of nine separate samples at a tightly regulated temperature, humidity and gascomposition.
To evaluate the role of GDH in astrocyte metabolism we used astrocytes cultured from animals devoid of GDH expression in the brain. On these cells we performed metabolic mapping, carbon dioxide production assays to investigate the capacity of complete glutamate oxidation.
The main finding in this project is that astrocytes in the aging hippocampus use a larger proportion of its glutamate for energy producing purposes. This can be seen from the incubations of hippocampus slices with U13-C glutamate, where the old slices show significantly higher label incorporation in all metabolites compared to slices from young adult mice. In parallel with these findings, we found an increased activity of malic enzyme and a heightened pyruvate recycling, processes required for a complete oxidation of glutamate. Take together these findings indicate that the metabolic properties of the old astrocytes are changed toward a more efficient use of glutamate for energy production. Whether this is due to a shortage of glucose, or to a dysregulation of glutamate usage in the old brain, remains to be elucidated.
Glycogen storage is affected in the old brain. We have in this project shown that glycogen accumulates in the hippocampus and cerebellum of aged compared to adult mice, while the level of glycogen in the cortex remains unchanged. Although glycogen storage is primarily an astrocytic process, it is unclear to this point whether the increase is due to increased production/decreased breakdown in the astrocytes or if it is caused by glycogen accumulation in the neurons.
In addition to changes in glucose/glycogen metabolism, we also discovered changes in content of the TCA cycle intermediates fumarate and citrate in the aged animals. In the aged animals, the level of fumarate was increased in cortex but remained unaffected in hippocampus and cerebellum. Citrate was, on the other hand, reduced in the old cortex and unaffected in hippocampus. The conclusion from these data is that not only are metabolic properties affected differently by aging in different brain areas brain areas, but the intermediates in the TCA-cycle are also differentially regulated. This could in turn indicate an imbalance in the reactions in the TCA-cycle or, as fumarate is increased, an adaptation to a higher flux through the left side of the cycle caused for instance by a higher input of carbon from glutamate in to the cycle.
The experiments using 15N-labelled glutamate yielded in one important result regarding the activity of GDH in the slices. There has been a discussion in the field regarding whether the high activity of GDH in cultured astrocytes is a culture artifact or not. The evidence provided by our incubation data show that GDH is highly active in acute hippocampus preparations as well.
Abrogation of GDH activity affects not only glutamate metabolism, but also glucose utilization in astrocytes. Carbon dioxide production from glutamate is severely hampered in astrocytes lacking GDH and the cells are unable to respond to glucose deprivation by increasing their glutamate oxidation, seen in a carbon dioxide production assay using 14C labeled glutamate as substrate. Surprisingly, they also have a significantly reduced capacity for glucose oxidation when using 14C-glucose as the labeled substrate. This indicates an intimate coupling between glutamate input to the TCA cycle via GDH and glucose metabolism. The cause for this connection is not clear, but it can be speculated that the astrocytes need the supply of carbon coming from glutamate to be able to maintain their pool of TCA cycle intermediates at an optimum level.
Basic research is recognized by the European Union [COM(2004)353] as having an important impact on economic and social progress. The “ASTRO AGE” project addresses questions fundamental for our understanding of astrocytes contribution to brain aging processes. A thorough understanding of these processes is crucial for increasing our chances to in the future be able to counteract the cognitive decline caused by aging. The results obtained during the project period have shed new light on the important metabolic function of astrocytes during aging. It has been shown that glutamate plays a more diverse role in brain than previously thought, by acting as a valuable energy substrate. It has been known that aging impairs glucose utilization in brain, a fact thought to be largely explained by a reduced glucose uptake. However, results from this project show that glycogen dysregulation and accumulation can be a part of the puzzle. Taken together, the results obtained in the “ASTRO AGE” project lays the foundation for new and more in depth studies of the role of astrocyte metabolism in brain aging.
Contact details: Professor Helle Waagepetersen
Dept. of Drug Design and Pharmacology
Faculty of Health and Medical Sciences
School of Pharmaceutical Sciences
Universitetsparken 2
2100 Copenhagen Ø
Phone nr. +45 35336470
helle.waagepetersen@sund.ku.dk
www.neuromet.dk
Aging also affects glucose utilization in brain and ATP production is shown to be reduced in aging human tissue. Investigations of the hippocampal proteome have revealed altered expression of enzymes in the glycolytic pathway and in the TCA-cycle. Therefore, it is important to note that glutamate can be utilized as energy substrate through oxidative degradation in the TCA-cycle. Glutamate can be converted to α-ketoglutarate by transaminases or glutamate dehydrogenase (GDH), where the latter is the preferred pathway in mice astrocytes. The amount of ATP generated by oxidation of glutamate is equivalent to the amount generated by glucose, indicating that glutamate can be a valuable source of energy under low glucose conditions. Taken together, numerous findings point towards a profound role of astrocytes and glutamate dysregulation in aging. However, astrocyte (patho)physiology in aging brain is still poorly characterized and yet to be understood. The general aim of this project is, therefore, to elucidate age related effects on glutamate homeostasis and energy metabolism in astrocytes.
The specific aims of the projects were:
i) To quantify age-related changes in glutamate homeostasis in the hippocampus of mice.
ii) To characterize these changes on cellular, tissue and behavioral level.
Special focus have been put on if glutamate homeostasis is affected in brain of aging mice and if increased expression of GDH augments glutamate metabolism in the TCA cycle and counteracts age related cognitive decline.
During the course of the project we have investigated the metabolic functions of astrocytes in the hippocampus of 4 months old mice compared to 22 months old mice with regard to changes in glutamate and glucose metabolism by metabolic mapping using 13C-labelled glutamate, glucose and acetate followed by analysis of labeled isotopomers by gas chromatography coupled to mass spectrometry. To be able to carry out precise incubations of the hippocampal slices, we developed and validated an incubation chamber allowing for incubation of nine separate samples at a tightly regulated temperature, humidity and gascomposition.
To evaluate the role of GDH in astrocyte metabolism we used astrocytes cultured from animals devoid of GDH expression in the brain. On these cells we performed metabolic mapping, carbon dioxide production assays to investigate the capacity of complete glutamate oxidation.
The main finding in this project is that astrocytes in the aging hippocampus use a larger proportion of its glutamate for energy producing purposes. This can be seen from the incubations of hippocampus slices with U13-C glutamate, where the old slices show significantly higher label incorporation in all metabolites compared to slices from young adult mice. In parallel with these findings, we found an increased activity of malic enzyme and a heightened pyruvate recycling, processes required for a complete oxidation of glutamate. Take together these findings indicate that the metabolic properties of the old astrocytes are changed toward a more efficient use of glutamate for energy production. Whether this is due to a shortage of glucose, or to a dysregulation of glutamate usage in the old brain, remains to be elucidated.
Glycogen storage is affected in the old brain. We have in this project shown that glycogen accumulates in the hippocampus and cerebellum of aged compared to adult mice, while the level of glycogen in the cortex remains unchanged. Although glycogen storage is primarily an astrocytic process, it is unclear to this point whether the increase is due to increased production/decreased breakdown in the astrocytes or if it is caused by glycogen accumulation in the neurons.
In addition to changes in glucose/glycogen metabolism, we also discovered changes in content of the TCA cycle intermediates fumarate and citrate in the aged animals. In the aged animals, the level of fumarate was increased in cortex but remained unaffected in hippocampus and cerebellum. Citrate was, on the other hand, reduced in the old cortex and unaffected in hippocampus. The conclusion from these data is that not only are metabolic properties affected differently by aging in different brain areas brain areas, but the intermediates in the TCA-cycle are also differentially regulated. This could in turn indicate an imbalance in the reactions in the TCA-cycle or, as fumarate is increased, an adaptation to a higher flux through the left side of the cycle caused for instance by a higher input of carbon from glutamate in to the cycle.
The experiments using 15N-labelled glutamate yielded in one important result regarding the activity of GDH in the slices. There has been a discussion in the field regarding whether the high activity of GDH in cultured astrocytes is a culture artifact or not. The evidence provided by our incubation data show that GDH is highly active in acute hippocampus preparations as well.
Abrogation of GDH activity affects not only glutamate metabolism, but also glucose utilization in astrocytes. Carbon dioxide production from glutamate is severely hampered in astrocytes lacking GDH and the cells are unable to respond to glucose deprivation by increasing their glutamate oxidation, seen in a carbon dioxide production assay using 14C labeled glutamate as substrate. Surprisingly, they also have a significantly reduced capacity for glucose oxidation when using 14C-glucose as the labeled substrate. This indicates an intimate coupling between glutamate input to the TCA cycle via GDH and glucose metabolism. The cause for this connection is not clear, but it can be speculated that the astrocytes need the supply of carbon coming from glutamate to be able to maintain their pool of TCA cycle intermediates at an optimum level.
Basic research is recognized by the European Union [COM(2004)353] as having an important impact on economic and social progress. The “ASTRO AGE” project addresses questions fundamental for our understanding of astrocytes contribution to brain aging processes. A thorough understanding of these processes is crucial for increasing our chances to in the future be able to counteract the cognitive decline caused by aging. The results obtained during the project period have shed new light on the important metabolic function of astrocytes during aging. It has been shown that glutamate plays a more diverse role in brain than previously thought, by acting as a valuable energy substrate. It has been known that aging impairs glucose utilization in brain, a fact thought to be largely explained by a reduced glucose uptake. However, results from this project show that glycogen dysregulation and accumulation can be a part of the puzzle. Taken together, the results obtained in the “ASTRO AGE” project lays the foundation for new and more in depth studies of the role of astrocyte metabolism in brain aging.
Contact details: Professor Helle Waagepetersen
Dept. of Drug Design and Pharmacology
Faculty of Health and Medical Sciences
School of Pharmaceutical Sciences
Universitetsparken 2
2100 Copenhagen Ø
Phone nr. +45 35336470
helle.waagepetersen@sund.ku.dk
www.neuromet.dk