Studies on the consumption, regeneration and restoration of ATP in cultured brain astrocytes

An impaired energy metabolism in brain cells has been connected with various neurodegenerative diseases and traumatic injuries. Studies have demonstrated a sudden loss of up to 50% of adenosine triphosphate (ATP) - the primary energy currency in mammalian cells - during events such as stroke or traumatic brain injury. However, little is known on the contribution of different brain cell types to such conditions, on the metabolic processes involved in such a rapid ATP depletion, as well as on the mechanisms by which brain cells attempt to restore their ATP levels. To address these questions for brain astrocytes, this thesis has focused on the ATP consumption, regeneration and restoration in this cell type by using astrocyte-rich primary cultures as model system. Cultured astrocytes contain high specific contents of ATP (31 ± 3 nmol/mg), but much lower levels of adenosine diphosphate (ADP; 2.9 ± 2.1 nmol/mg) and adenosine monophosphate (AMP; 1.7 ± 2.1 nmol/mg). Cytosolic glycolysis allowed glucose-fed astrocytes to maintain a high ATP levels for at least 30 minutes by regenerating ATP from ADP, even in the presence of the mitochondrial complex III inhibitor antimycin A. Also, in the absence of glucose, astrocytes maintained their high ATP levels, but a treatment with the glycolysis inhibitor 2-deoxyglucose (2DG) resulted in a 50% decrease in ATP content and the presence of antimycin A depleted cellular ATP levels completely within 30 minutes. Inhibition of both ATP-generating pathways, glycolysis and oxidative phosphorylation, using 2DG and one of several inhibitors of mitochondrial oxidative phosphorylation led to a significantly accelerated, additive ATP decline. During the initial 30 minutes, the ATP loss was accompanied by a transient increase in ADP content and a gradual rise in AMP content, which was accompanied by a pronounced decline in both the adenylate energy charge and the total adenosine phosphate (ATP, ADP plus AMP) pool. Another way for the phosphorylation of ADP to generate ATP is the creatine kinase mediated reaction with creatine phosphate (CrP). In astrocytes, CrP (25.9 ± 10.8 nmol/mg) is used as immediately available energy supplier and short-term energy backup. Results of this thesis show that the cellular CrP content strongly depends on the extracellular creatine availability and decreases with ongoing culture age. Moreover, ATP-decline-studies demonstrate that astrocytes rapidly deplete their CrP pools to phosphorylate ATP during starvation. Especially during the rapid ATP decline in the presence of antimycin A, astrocytes sacrifice their CrP content to delay the loss of ATP in the first 5 minutes of incubation. For ATP restoration studies, the astrocytic ATP content was first reduced to 25% of its initial level by treating the cells for 60 minutes with the mitochondrial uncoupler BAM15 in the absence of glucose. After BAM15 removal, astrocytes were then exposed to various substrates that would potentially foster ATP restoration. Glucose as exclusive substrate restored ATP slowly and incompletely within 6 hours, whereas adenosine as exclusive substrate led to a rapid but transient ATP restoration. In contrast, the combination of glucose as an energy source and adenosine as a precursor for AMP resulted in a complete and sustained restoration of ATP within 60 minutes that was mediated by adenosine kinase. However, due to its neuroactive properties, adenosine is unsuitable for in vivo applications. As potential alternatives, combinations of sugars, purine bases and purine nucleosides were studied and guanosine and inosine were found to strongly accelerate ATP restoration in the presence of glucose as an energy source combined with adenine as source for the base needed for AMP synthesis via the purine salvage pathway. In conclusion, the results obtained for this thesis demonstrate the ability of cultured astrocytes to compensate for the impairment of one of their two main ATP-generating pathways and underlines the importance of mitochondrial ATP regeneration for astrocytes. Concerning ATP restoration in ATP-depleted astrocytes, the results obtained identify the application of combinations of an energy source such as glucose, a ribose source such as purine nucleosides and an adenine source such as adenine as the most effective strategy for restoring a normal astrocytic ATP content following severe ATP depletion.