Abstract
Alzheimer's disease (AD) is the most common cause of dementia in the aged. It is characterized by progressive memory loss and the presence of intracellular neurofibrillary tangles and extracellular amyloid beta (Aβ) plaques. The most commonly held view regarding the primary pathophysiological event in AD centres on the amyloid cascade hypothesis, which states that AD begins with the assembly and accumulation of Aβ peptides derived from the amyloid precursor protein (APP). However, the causality of Aβ in AD has been increasingly challenged largely due to the failure of Aβ-centred clinical trials. A critical question has therefore been raised: Is Aβ truly the primary cause of AD, particularly for the 95% of sporadic late-onset AD cases?
Recent research has postulated that cerebrovascular endothelial dysfunction during advanced ageing, together with other risk factors, triggers the neurodegenerative processes in AD. Nitric oxide (NO) is a gaseous signalling molecule produced by nitric oxide synthase (NOS) from L-arginine, and NO derived from endothelial NOS (eNOS) plays a critical role in maintaining normal cerebral blood flow (CBF). eNOS deficiency is evident in AD brains and is positively correlated with the load of plaques and tangles. Interestingly, mice with eNOS deficiency display memory deficits, increased amyloidogenic processing of APP, increased Aβ levels and tau phosphorylation in the brain. APPswe/PS1dE9 (APP/PS1) mice show age-related Aβ deposition in the brain and memory deficits, and are commonly used Aβ model for the early stage of AD. Given the role of eNOS in the pathogenesis of AD, partial eNOS deficiency was added to APP/PS1 mice to produce APP/PS1/eNOS+/− mice. This thesis characterized the APP/PS1/eNOS+/− mouse model and explored the therapeutic potential of agmatine, a known stimulator of eNOS with memory enhancing, anti-inflammatory and neuroprotective properties.
Experiment 1 crossed male complete eNOS deficient mice with female APP/PS1 mice to generate APP/PS1/eNOS+/− mice, and then assessed the effects of partial eNOS deficiency on cognitive function, CBF and neurovascular coupling (NVC), Aβ deposition in the brain and microglial immunoreactivity in APP/PS1 mice. Male APP/PS1/eNOS+/− mice at 8-months of age displayed a more severe spatial working memory deficit, however had no changes in CBF and NVC, relative to age-matched APP/PS1 mice. Immunohistochemistry and immunoblotting revealed significantly increased Aβ plaque load in the brains of APP/PS1/eNOS+/− mice, concomitant with upregulated beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1; hence increased Aβ production) and low density lipoprotein receptor-related protein 1 (LRP-1; speculatively reduced Aβ clearance), and downregulated insulin-degrading enzyme (IDE; hence reduced Aβ clearance) and increased immunoreactivity and expression of microglia. These results demonstrated that partial eNOS deficiency exacerbated behavioural dysfunction, Aβ brain deposition (due to increased Aβ production, but reduced Aβ clearance) and microglial pathology in 8-months old male APP/PS1 mice.
Using both male and female APP/PS1/eNOS+/- and APP/PS1 mice at 4-, 6- and 8-months of age, Experiment 2 further characterized AD-like pathology in the new model with a focus on the Aβ load in the brain and the enzymes involved in Aβ production and clearance. Immunohistochemistry revealed no genotype and sex differences in Aβ load in the brain at 4-months of age. At 6-months of age, more Aβ plaques were evident in the cortex of APP/PS1/eNOS+/− mice and female mice in both genotype groups. At 8-months of age, there were marked genotype and sex differences in Aβ load in both the cortex and hippocampus with more plaques in APP/PS1/eNOS+/- mice and the females regardless of genotype. Western blot revealed increased BACE-1 protein (at 8 months only), but an early and persistent reduction in IDE protein expression (at all age points) in APP/PS1/eNOS+/- mice. These results demonstrated that partial eNOS deficiency exacerbated Aβ brain deposition (from 6 months of age) and had an early and persistent effect on Aβ clearance in APP/PS1 mice. These findings further support that reduced clearance of Aβ is one of the key player linking vascular changes to AD. Moreover, sex differences in Aβ burden were evident in both APP/PS1 and APP/PS1/eNOS+/- mice at 6- and 8-months of age, with more severe plaque pathology in the females.
Recent research has implicated altered brain arginine metabolism in the pathogenesis of AD. Experiment 3 systematically investigated how the brain and plasma arginine metabolic profiles changed in 8-months old male and female APP/PS1/eNOS+/- mice, the age point with accelerated accumulation of amyloid plaques in the brain. Despite the reported age-related changes in L-arginine metabolism in the brain and/or blood in APP/PS1 mice, this experiment demonstrated a general lack of genotype effect in both APP/PS1 and APP/PS1/eNOS+/- mice at 8-months of age regardless of sex.
Agmatine (decarboxylated arginine) stimulates eNOS and has memory enhancing and neuroprotective properties. As a preliminary study, Experiment 4 assessed the effects of long-term daily agmatine sulphate supplementation to APP/PS1/eNOS+/- mice at the dose of 50 mg/kg via food chow from 4-months of age for a period of 4 months. At 8-months of age, all animals were assessed behaviourally, followed by the measurements of CBF, NVC, Aβ plague load in the brain and microglial immunoreactivity. While agmatine supplementation showed limited rescuing effects, there was reduced hyperactivity (males only), but increased eNOS expression (43% increase in males), Aβ load (both sexes) and ionized calcium binding adaptor molecule 1 (Iba-1) expression (males only) in APP/PS1/eNOS+/-AGM mice relative to APP/PS1/eNOS+/- mice.
The present study developed APP/PS1/eNOS+/− mouse model and for the first time demonstrated that APP/PS1/eNOS+/− mice display impaired spatial learning, increased Aβ plaque load and exacerbated microglial pathology in the brain relative to APP/PS1 mice. Moreover, partial eNOS deficiency augmented the amyloidogenic processing of APP and reduced cellular and system Aβ clearance. In conclusion, our findings further support the role of endothelial dysfunction in the pathogenesis of AD and also provide the scientific basis for developing preventive and/or therapeutic strategies by targeting endothelial dysfunction. The findings from current study also make a strong case for the use of APP/PS1/eNOS+/− model as a clinically more relevant model for early-stage AD that could be employed to investigate AD pathophysiology and search for viable therapeutic targets.