Altered Gene Expression Within the Renin-Angiotensin System in Normal Aging and Dementia

Abstract

The renin-angiotensin system (RAS) is dysregulated in Alzheimer's disease (AD). In this study, we have explored the hypothesis that an -age--related imbalance in brain RAS is a trigger for RAS dysregulation in AD. We characterized RAS gene expression in the frontal cortex from (i) a cohort of normal aging (n = 99, age range = 19-96 years) and (ii) a case-control cohort (n = 209) including AD (n = 66), mixed dementia (VaD + AD; n = 50), pure vascular dementia (VaD; n = 42), and age-matched controls (n = 51). The AD, mixed dementia, and age-matched controls were further stratified by Braak tangle stage (BS): BS0-II (n = 48), BSIII-IV (n = 44), and BSV-VI (n = 85). Gene expression was calculated by quantitative PCR (qPCR) for ACE1, AGTR1, AGTR2, ACE2, LNPEP, and MAS1 using the 2-∆∆Cq method, after adjustment for reference genes (RPL13 and UBE2D2) and cell-specific calibrator genes (NEUN, GFAP, PECAM). ACE1 and AGTR1, markers of classical RAS signaling, and AGTR2 gene expression were elevated in normal aging and gene expression in markers of protective downstream regulatory RAS signaling, including ACE2, MAS1, and LNPEP, were unchanged. In AD and mixed dementia, AGTR1 and AGTR2 gene expression were elevated in BSIII-IV and BSV-VI, respectively. MAS1 gene expression was reduced at BSV-VI and was inversely related to parenchymal Aβ and tau load. LNPEP gene expression was specifically elevated in VaD. These data provide novel insights into RAS signaling in normal aging and dementia.

Keywords: Angiotensin-II type 1 receptor (AGT1R); Angiotensin-II type 2 receptor (AGT2R); Angiotensin-converting enzyme (ACE).

Figure 1.

RAS gene expression in the frontal cortex in normal aging. ACE1, AGTR1, and AGTR2 gene expression was measured by qPCR in a normal aging cohort (n = 99) split into quartiles: <52 years (n = 25), 55–71 (n = 25), 72–85 (n = 25), and 86 years and older (n = 24). Genes were calibrated to either reference genes or cell-specific markers (where appropriate) and expressed using the 2^−∆∆Cq method. Each dot represents an individual case measured in triplicate. The geometric mean and 95% confidence interval are shown. *p < .05, **p < .01, ***p < .001, ****p < .0001. RAS = renin–angiotensin system.

Figure 2.

Laser-scanning confocal imaging of the cell-specific expression of ACE-1 (ACE) and ACE-2 proteins, and AGTR1, AGTR2, and MAS1 gene expression in the frontal cortex. (A) Representative images of immunofluorescence labeling of ACE-1 (red), ACE-2 (green), and GFAP (blue) in frontal cortex showing vascular and perivascular expression of ACE-1 and ACE-2 (left), ACE-2 expression in astrocytes (center), and likely neuronal expression of ACE-2 in GFAP-negative cells with neuronal morphology (right). (B–D) Representative RNAscope images of cell-specific expression of (B) AGTR1, (C) AGTR2, (D) MAS1 transcripts (green) in the frontal cortex, co-labeled with PECAM1 (left), GFAP (center), and NeuN (right) probes (red) and DAPI (blue). Scale bars = 10 µm. ACE = angiotensin-converting enzyme.

Figure 3.

Altered RAS receptor gene expression in the frontal cortex in relation to Braak tangle stage in dementia. AGTR1 and AGTR2 gene expression is elevated, whereas LNPEP and MAS1 gene expression is reduced, in relation to Braak tangle stage groups: 0–II (n = 48), III–IV (n = 44), and V–VI (n = 85) in a cohort comprising Alzheimer’s disease, mixed (AD/VAD), and age-matched controls. Gene expression was measured by qPCR and calibrated to reference genes and cell-specific markers where appropriate. Relative gene levels are expressed using the 2^−∆∆Cq method. Each dot represents an individual case measured in triplicate. The geometric mean and 95% confidence interval are shown. *p < .05, **p < .01, ***p < .001, ****p < .0001. RAS = renin–angiotensin system.

Figure 4.

ACE1, ACE2, AGTR1, and AGTR2 gene expression is unaltered but LNPEP and MAS1 gene is dysregulated in the frontal cortex in dementia. Gene expression was measured by qPCR in a cohort (n = 209) divided into diagnoses group: age-matched controls (n = 51), Alzheimer’s disease (AD; n = 66), AD and VaD (mixed; n = 50), and vascular dementia (VaD; n = 42). Genes were calibrated to reference genes and expressed as using the 2^−∆∆Cq method. Each dot represents an individual case measured in triplicate. The geometric mean and 95% confidence interval are shown. *p < .05, **p < .01, ***p < .001, ****p < .0001.