Abstract
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and neuropathological hallmarks of amyloid-β (Aβ ) plaques and neurofibrillary tangles, with aging as a major risk factor. The amyloid cascade hypothesis proposes a causative role of Aβ in the disease development and has dominated the AD field for over 20 years. Recently this hypothesis has been increasingly challenged: Is Aβ the primary cause for the 95% of sporadic late-onset AD cases, or is it secondary to some other process? It has been proposed that cerebrovascular endothelial dysfunction during advanced aging, together with other risk factors, triggers the neurodegenerative processes in AD. L-arginine is a semi-essential amino acid with a number of bioactive metabolites. The gaseous signalling molecule nitric oxide (NO) derived from endothelial NO synthase (eNOS) plays a critical role in maintaining normal cerebral blood flow (CBF). In AD brains, there are dramatically reduced eNOS protein expression and altered arginine metabolic profile, and the plaques and tangles are associated with reduced capillary expression of eNOS. Interestingly, mice with complete eNOS deficiency display amyloidogenic processing of amyloid precursor protein, increased Aβ levels and tau phosphorylation in the brain, and memory deficits. This thesis aimed to further explore the implication of eNOS dysfunction for AD by investigating the effects of complete and partial eNOS deficiency on CBF, neurovascular coupling (NVC), L-arginine metabolism and its inter-related urea cycle.
Experiment 1 systematically investigated how behavioural function, basal CBF, NVC and brain and plasma arginine metabolic profiles changed in male and female mice with complete eNOS deficiency (eNOS-/-) at 14 months of age. Both male and female eNOS-/- mice displayed altered behaviour in the Y-maze and open field tests. A real-time microcirculation imager revealed a significant sex difference in the basal CBF and significantly increased perfusion response to whisker stimulations in the Barrel cortex (NVC) in eNOS-/- mice relative to wildtype (WT) controls regardless of sex. Treatment with 7-nitroindazole (a selective neuronal NO synthase (nNOS) inhibitor) blocked the increased perfusion response to whisker stimulations in eNOS-/- mice, indicating the role of nNOS in NVC. Neurochemically, the most intriguing changes in the brain were markedly reduced glutamine levels in the fontal cortex, hippocampus, parahippocampal region and cerebellum of eNOS-/- mice at both sexes. In the plasma, there were increased L-arginine, but decreased L-citrulline levels in eNOS-/- mice relative to their sex-matched WT controls. These findings demonstrate altered behavioural function, NVC, and brain (glutamine in particular) and plasma arginine metabolic profiles in 14-month eNOS-/- mice.
In order to rule out the contribution of age to the abovementioned changes, Experiment 2 systematically determined how basal CBF, NVC and brain and plasma L-arginine metabolic profiles changed in younger eNOS-/- mice at 4 months of age. While there were no changes in baseline CBF, 4-month old eNOS-/- mice displayed significantly increased perfusion response to whisker stimulations, reduced glutamine levels in all four brain regions examined, and increased plasma L-arginine and L-citrulline levels. In conjunction with Experiment 1, these findings suggest that complete eNOS deficiency results in early and long-lasting changes in NVC, global reduction in brain glutamine and increases in plasma L-arginine.
Glutamine is the principal mechanism for detoxifying ammonia in the brain, whereas the urea cycle is the key pathway to dispose of ammonia in the liver. Experiment 3 quantified the brain (hippocampus) and plasma urea and/or ammonia levels in eNOS-/- mice at 4 and 14 months of age, and investigated how the enzymes/proteins involved in the glutamate-glutamine and urea cycles and the formation, metabolism and transport of ammonia changed in the hippocampus of male eNOS-/- mice at 14 months of age. Male and female eNOS-/- mice at both age points displayed reduced plasma ammonia levels, but increased plasma and hippocampal urea levels. Reverse transcription quantitative PCR (RT-qPCR) revealed increased mRNA expression of glutamine synthetase (forming glutamine from glutamate and ammonia), glutaminase (converting glutamine to glutamate and ammonia), glutamate dehydrogenase (a reversible enzyme that can utilize ammonia in hyperammonemic conditions) and aquaporin-4 (an ammonia channel) in the hippocampus of eNOS-/- mice, with no changes in the urea cycle enzymes and urea transporter-B. These results demonstrate that complete eNOS deficiency leads to an early and long-lasting build-up of urea in the hippocampus and plasma, along with altered glutamine and ammonia formation and metabolism in the brain. The inverse relationship between ammonia and urea levels in the plasma perhaps indicate increased conversion of ammonia to urea in the liver.
Experiment 4 investigated the effects of partial eNOS deficiency on basal CBF, NVC and brain and plasma arginine metabolic profiles and urea levels using male and female eNOS+/- mice at 4 and 14 months of age. As in Experiment 1, female mice showed an increase in basal CBF regardless of age and genotype. At the 4-month age point, there were no genotype-related changes in NVC and brain arginine metabolism. At 14-month age point, however, male (but not female) eNOS+/- mice displayed increased perfusion response to whisker stimulations, and eNOS+/- mice at both sexes had significantly decreased levels of L-arginine and its 5 downstream amino acid metabolites in the temporal cortex (an auditory cortex) along with some changes in the hippocampus. There were no genotype-related changes in plasma arginine metabolic profiles and brain and plasma urea at both age points. These findings demonstrate that altered NVC and brain arginine metabolism in eNOS+/- mice are sex-, age- and brain region-specific. A 50% decrease in eNOS protein does not cause a global reduction of glutamine in the brain and a build-up of urea in the brain and plasma seen in eNOS-/- mice.
The amyloid cascade hypothesis of AD has been challenged off late giving rise to a need for exploring other mechanisms. Earlier research has shown increased Aβ levels and tau phosphorylation in the brain and memory deficits in mice with complete eNOS deficiency. The present thesis, for the first time, demonstrates that a loss of eNOS-derived NO leads to early and long-lasting alterations in NVC and metabolic profile changes in the brain and plasma (glutamine and urea in particular). A 50% reduction in eNOS protein, especially in combination with ageing, preferentially affects arginine metabolism in the auditory cortex and in the hippocampus to a lesser extent. Taken together, eNOS deficiency may act as an early-onset trigger for the neurodegenerative processes in AD by causing metabolic, as well as cerebrovascular dysfunction.