AbstractHyperactivity of the classical renin angiotensin system (cRAS) (ACE-1/Ang-II/AT1R) within the brain exerts damaging effects and contributes to the pathogenesis of Alzheimer’s disease (AD). Alternative ‘downstream’ regulatory RAS pathways: the non-classical RAS axis (ACE-2/Ang (1- 7)/MasR) and the alternative regulatory RAS axis (rRAS) (APA/Ang-III/APN/Ang-IV/IRAP) have recently been discovered that counter-regulate the damaging effects of cRAS signalling, whilst also regulating synaptic function and boosting learning and memory. Despite a strong association of an imbalance in brain RAS pathways in relation to AD pathology and cognitive decline, the role of ACE-1 and the involvement of the other rRAS pathways in relation to AD pathogenesis remains unclear. This thesis describes a series of investigations of both cRAS and rRAS, and non-classical RAS components, in AD in post-mortem brain tissue aimed to test the general hypothesis that alterations of both the classical and regulatory axes of brain RAS can influence AD pathogenesis and are related to vascular dysfunction.
For this thesis I have studied mid-frontal cortex (Brodmann area 8/9) from post-mortem confirmed AD cases (n= 70) and age-matched controls (n= 48) that were matched closely for age-at-death and post-mortem delay (PMD). I developed specific ACE-1 N-domain and C-domain activity assays using immunocapture-based fluorogenic substrates. I measured the level of angiotensin peptides (Ang-I and Ang (1-7)) and characterised the expression, enzyme activity and distribution of rRAS receptors including MasR, Ang-IV, and IRAP by ELISA and immunohistochemistry. I used previously measured markers of disease pathology (parenchymal Aβ/Tau and insoluble Aβ40 and 42) and measured markers of cerebrovascular dysfunction including brain ischaemia (VEGF) and tissue oxygenation (MAG:PLP1 ratio) and examined the relationship between alterations of brain RAS components with these markers. I also explored the novel non-AT1R and non-AT2R receptor (Neurolysin) and its substrate (Neurotensin) using an ELISA.
Divergent ACE-1 C-domain (increased) and N-domain (reduced) enzyme activity was observed in AD that potentially favours Ang-II production and limits Aβ clearance and provides further insight into the complex role of ACE-1 in AD. These data potentially indicate that C-domain ACEIs may have greater therapeutic benefit for AD. Ang-I level was reduced and the ratio of Ang-II:Ang-I was increased in AD - as would be expected with overactivation of ACE-1. Changes were observed in mid-frontal cortex in the rRAS pathways indicative of dysregulation in AD: the Ang-II:Ang (1-7) ratio, a proxy marker of ACE-2 activity, was reduced in AD; IRAP activity was also reduced in AD, however, other components of the rRAS pathways including Ang (1-7), and MasR and Ang-IV and IRAP protein level remained unchanged in AD (although they were related to ischaemic damage). Interestingly, the non-AT1R and non-AT2R binding protein (neurolysin), previously not studied in AD, was reduced in relation to disease severity and ischaemia in AD.
In conclusion, these data support recent findings within the field that indicate that regulatory RAS pathways are dysfunctional in AD associated with disease pathology and vascular function. This thesis also offers insights into novel RAS components, such as neurolysin and neurotensin and their involvement in AD, that will require further investigation. The RAS remains a pivotal and complex system that underpins disease pathogenesis in AD and may offer new therapeutic targets in the future.
|Date of Award||12 May 2020|
|Sponsors||Imam Abdulrahman Bin Faisal University|
|Supervisor||J S Miners (Supervisor) & Patrick Gavin Kehoe (Supervisor)|