L-arginine metabolism in animal models of Alzheimer’s disease

Alzheimer’s disease (AD) is a frequent neurodegenerative condition among the elderly, it is the most common form of dementia and currently has no cure. Amyloid beta (Aβ) proteins are found as plaque accumulations in the AD brain and are thought to be central to the disease process and as such are used to model AD. In the non-transgenic model of AD, Aβ proteins are injected into the rat brain to model rapid induction of the disease. Transgenic (Tg) mice have mutations that result in age-dependent Aβ accumulation in the brain, behavioural impairments and are used to model progressive disease conditions. L-arginine is a semi-essential amino acid with a number of bioactive metabolites. Nitric oxide (NO) is critically involved in neurotransmission, learning and memory and has an important role in maintaining cerebral blood flow. However excessive NO concentrations lead to neurotoxicity and neurodegeneration. Polyamines (putrescine, spermidine and spermine) are essential in maintaining normal cellular function and also like NO, have dose-dependent effects. The third L-arginine metabolic pathway results in agmatine (decarboxylated arginine), a novel putative neurotransmitter that directly participates in learning and memory processes. It stands at the crossroads of arginine metabolism regulating the production of NO and polyamines, and exogenous treatment has memory enhancing, anti-inflammatory and neuroprotective properties. Recent research has demonstrated altered metabolism of L-arginine in human AD brains and also in the rat brain following the injection of Aβ25-35 (the toxic domain of full-length Aβ), suggesting the involvement of altered L-arginine metabolism in Aβ mediated neurodegenerative processes warrants further investigation. The present thesis aimed to build on these novel reports by i) investigating how Aβ affects arginine metabolism in the brain and/or blood using two Aβ-centric models of AD; ii) evaluating the safety of oral agmatine delivery; and iii) exploring the therapeutic potential of agmatine for AD. Experiment 1 investigated time-course changes in L-arginine metabolism in the rat prefrontal cortex (PFC) and sub-regions of the hippocampus (HPC) following a single intracerebroventricular (icv.) infusion of pre-aggregated Aβ25-35. Aβ25-35 (30 nmol/rat) altered arginine metabolism in the brain 24 and 72 h (Experiment 1.1), and 42 and 97 days (Experiment 1.2) post-infusion in a time-, region- and neurochemical-dependent manner. These findings demonstrate that icv. infusion of Aβ25-35 results in both acute and prolonged changes in arginine metabolism in the brain, that might contribute to pathology and altered behavioural performance. Experiment 2 characterised time-course changes in behaviour and L-arginine metabolism in six brain regions (including the PFC and HPC), and the plasma of APPswe/PS1ΔE9 transgenic (Tg) and wild-type (WT) mice at 7 and 13 months of age. Tg mice displayed significant spatial learning and memory deficits in the Morris water maze and Barnes circular maze, and altered arginine metabolic profiles in the brain (including reduced agmatine levels in the HPC) at 13 months of age. Interestingly, the plasma arginine metabolic profile was markedly altered in Tg mice at 7, but not 13, months of age. These results demonstrate that age-related behavioural deficits in Tg mice parallel arginine metabolic profile changes in the brain and an altered plasma arginine metabolic profile precedes these changes. Experiment 3 assessed the safety of oral delivery of agmatine (via gavage) in WT and Tg mice. In Experiment 3.1, agmatine administered to WT mice at 300, 600 and 900 mg/kg for 7 days did not affect animals’ body weight gain, but resulted in reduced locomotor activity in the open field when tested 30 min after dosing, indicating these doses may have been too high. Dose-dependent increases in agmatine levels in the hippocampus and plasma were found 30 min following dosing, suggesting that agmatine (via gavage) can get into the blood and brain rapidly. In Experiment 3.2, both WT and Tg mice were treated with agmatine at 0 and 300 mg/kg via gavage once daily for 90 days over a 105 day period, from 9 months of age. Sub-chronic agmatine treatment had no effects on animals’ food or water intake, general behaviour in the elevated plus maze and open field, body and organ weights and organ histology. Agmatine treatment significantly increased levels of agmatine in all six brain regions of both WT and Tg mice when examined 24 h after the final dosing, and higher levels were found in Tg mice when compared to WT mice in three of these regions. Plasma agmatine concentrations were not different between groups. These findings suggest that agmatine (at 300 mg/kg) via gavage over 3 months was well tolerated and non-toxic in experimental mice, and that agmatine accumulates in the brain under conditions of chronic delivery. Furthermore, Tg mice at 13 months of age may have increased permeability for agmatine to cross the blood brain barrier and a greater tendency for agmatine to accumulate in the brain, which may be beneficial. Experiment 4 aimed to explore the therapeutic potential of agmatine for AD using APPswe/PS1ΔE9 mice at lower doses than used in the toxicity study. Tg mice displayed mild impairments in the radial arm water maze relative to WT mice at 13 months of age. In both genotypes, there was a trend of behavioural improvement following agmatine treatment at 50 and 200 mg/kg doses (via gavage). Agmatine treatment significantly increased levels of agmatine in the brain when examined 30 min after the final dosing in both WT and Tg mice, and again higher levels were found in Tg mice. Moreover, agmatine treatment seemed to normalise altered L-arginine neurochemistry in the Tg brains to the WT levels. In summary, the present thesis, for the first time, demonstrated that arginine metabolism in the brain was affected by Aβ in two models of AD, following icv. infusion of pre-aggregated Aβ25-35 and during chronic age-dependent Aβ accumulation in the APPswe/PS1ΔE9 Tg mouse. While the Aβ cascade hypothesis has dominated the AD field for over 2 decades, Aβ has recently been considered as a consequence, rather than the cause, of AD. It has been suggested that cerebrovascular endothelial dysfunction (hence reduced cerebral blood flow) during advanced aging triggers the neurodegenerative process in AD. Hence the interactions between Aβ and arginine metabolism need to be further investigated. The present findings demonstrated that agmatine oral delivery (via gavage) was safe and well tolerated by both WT and Tg mice. Agmatine was able to reach the brain rapidly after dosing and to accumulate in the brain following sub-chronic gavage treatment. Moreover, agmatine treatment appeared to have beneficial effects in both WT and Tg mice, which merits future research to further explore the therapeutic potential of agmatine for AD using different disease models