Angiotensin-III is Increased in Alzheimer’s Disease in Association with Amyloid-β and Tau Pathology

Abstract

Hyperactivity of the renin-angiotensin system (RAS) is associated with the pathogenesis of Alzheimer’s disease (AD) believed to be mediated by angiotensin-II (Ang-II) activation of the angiotensin type 1 receptor (AT1R). We previously showed that angiotensin-converting enzyme-1 (ACE-1) activity, the rate-limiting enzyme in the production of Ang-II, is increased in human postmortem brain tissue in AD. Angiotensin-III (Ang-III) activates the AT1R and angiotensin type-2 receptor (AT2R), but its potential role in the pathophysiology of AD remains unexplored. We measured Ang-II and Ang-III levels by ELISA, and the levels and activities of aminopeptidase-A (AP-A) and aminopeptidase-N (AP-N) (responsible for the production and metabolism of Ang-III, respectively) in human postmortem brain tissue in the mid-frontal cortex (Brodmann area 9) in a cohort of AD (n = 90) and age-matched non-demented controls (n = 59), for which we had previous measurements of ACE-1 activity, Aβ level, and tau pathology (also in the mid-frontal cortex). We found that both Ang-II and Ang-III levels were significantly higher in AD compared to age-matched controls and that Ang-III, rather than Ang-II, was strongly associated with Aβ load and tau load. Levels of AP-A were significantly reduced in AD but AP-A enzyme activity was unchanged whereas AP-N activity was reduced in AD but AP-N protein level was unchanged. Together, these data indicate that the APA/Ang-III/APN/Ang-IV/AT4R pathway is dysregulated and that elevated Ang-III could contribute to the pathogenesis of AD.

Keywords

Alzheimer’s disease aminopeptidase-A aminopeptidase-P angiotensin-II angiotensin-III renin-angiotensin system

INTRODUCTION

It is now well established that there is a local-acting renin-angiotensin-system (RAS) within the brain that functions independently from the systemic RAS [1 –4] and that hyperactivity of brain RAS is associated with the pathogenesis of Alzheimer’s disease (AD) [5]. Most of the deleterious effects of the brain RAS in AD are believed to be associated with elevated Ang-II levels and its subsequent over-activation of angiotensin receptor type-1 (AT1R), commonly referred to as the classical axis of RAS (reviewed in [5]). Infusion of Ang-II increased both amyloid-β (Aβ) (via increased amyloidogenic processing of AβPP) and tau pathology, and reduced cognitive performance in aging Sprague Dawley rats [6, 7]. We have previously reported that angiotensin-converting enzyme-1 (ACE-1), the rate-limiting enzyme in the production of Ang-II, is increased in AD in human brain tissue [8, 9]. Epidemiological studies suggest that commonly prescribed anti-hypertensives that target brain RAS and thus reduce the activity of classical RAS (ACE-1 inhibitors and AT1R blockers) protect against cognitive decline and that re-purposing these drugs may have therapeutic potential in AD [10, 11].

Angiotensin-III (Ang-III) is a heptapeptide produced following aminopeptidase-A (AP-A) (EC 3.4.11.7) mediated cleavage of the Asp residue at the N-terminal of Ang-II [12 –15]. Ang-III is a potent activator of AT1R and AT2R [16, 17]. Evidence from experimental animal models suggest that Ang-III shares many of the biological effects that are attributed to Ang-II and that Ang-III-mediated activation of AT1R is implicated in a host of responses including increased blood pressure [18 –21], dysregulated drinking and salt intake [20], increased pituitary hormone release [22, 23], and increased vasopressin release [24]. Orally administered AP-A inhibitors, resulting in Ang-III reduction, are cardioprotective [25 –28] suggesting that Ang-III, rather than Ang-II, may be the central effector protein in RAS (reviewed in [3]). Ang-III is metabolized by aminopeptidase-N (AP-N) (EC 3.4.11.2) to generate Ang IV [29], a peptide that via inhibition of insulin-regulated aminopeptidase (IRAP), is heavily implicated in learning and memory consolidation [30, 31]. This APA/Ang-III/APN/Ang-IV/AT4R pathway remains poorly characterized within the brain and its potential role in AD is poorly understood. Recent studies indicate that serum and plasma AP-A and AP-N activities are reduced in early AD [32, 33].

In the present study, we have measured the levels of Ang-II and Ang-III, and the levels and activities of AP-A and AP-N (responsible for the metabolism ofAng-II and Ang-III, respectively) in human post-mortem brain tissue in relation to Aβ and tau pathology in AD.

MATERIALS AND METHODS

Case selection

Brain tissue was obtained from the South West Dementia Brain Bank, University of Bristol, with local research ethics committee approval. All brains had been subjected to detailed neuropathological assessment according to the NIA-AA guidelines [34] and AD pathology was a sufficient explanation for the dementia in these cases. We investigated 90 cases of AD and 59 age-matched controls whose brains were from people who had no history of dementia and had undergone extensive neuropathological assessment and had few or absent neuritic plaques, a Braak tangle stage of III or less, and no other neuropathological abnormalities. All cases had previous measurements of ACE-1 activity [8, 35], total soluble (NP-40-extracted) and insoluble (Guanidine Hydrochloride (GuHcl)-extracted) Aβ levels [36], and tau load (area fraction of cerebral cortex immunopositive for ptau) [37, 38] within the mid-frontal cortex (Brodmann area 9). The cohorts were matched closely for age-at-death and postmortem delay. The demographic data are presented in Table 1 and the MRC UK Brain Bank Network database identifiers are shown in Supplementary Table 1.

Table 1

Summary of demographics of control and AD cases

	Age-at-death	Gender	Postmortem		Braak tangle stage
	(years±SD)	(F:M)	delay (h±SD)
				0–II	III-IV	V-VI
Controls (n = 59)	78.5±10.0	22:37	43.8±36	41	15	0
AD (n = 90)	78.5±9.7	55:35	45.0±25	0	13	77

Braak Tangle stage score unavailable for three control cases.

Brain tissue

The right cerebral cortex had been fixed in 10% formalin for a minimum of three weeks before the tissue was processed and paraffin blocks were taken for pathological assessment. The left cerebral hemisphere had been sliced and frozen at – 80°C until used for biochemical assessment. For each case we dissected 200 mg brain tissue from the mid-frontal cortex (Brodmann area 9) that was homogenized in a Precellys homogenizer (Stretton Scientific, Derbyshire, UK) as previously described [8, 9]. Brain homogenates were prepared in either 1% SDS lysis buffer (100 μM NaCl, 10 mM Tris pH 6, 1μM phenylmethylsulfonyl fluoride (PMSF), 1μg/ml aprotinin (Sigma Aldrich, Dorset, UK) and 1% SDS in distilled water) or 0.5% triton-X100 lysis buffer (20 mM Tris pH 7.4, 10% sucrose, 1 μM PMSF, 1μg/ml aprotinin (Sigma Aldrich) and 0.5% triton X-100 in distilled water). The samples were centrifuged at 13,000 rpm and the clarified supernatants were aliquoted and stored at – 80°C until required. Total protein was measured using the Total Protein kit (Sigma Aldrich) following manufacturer’s guidelines. Most brain tissue was obtained within 72 h of death.

Angiotensin-II sandwich ELISA

Ang-II level was measured in brain tissue homogenates (in 1% SDS lysis buffer) using a commercially available sandwich ELISA (Abcam, UK) following manufacturer’s guidelines. In brief, recombinant human Ang-II or brain tissue supernatants (50μl + 50μl PBS) were added in duplicate to wells that had been pre-coated with an anti-human Ang-II specific antibody and incubated for 2 h at room temperature. After a wash step, the plate was incubated for 2 h with a biotinylated anti-Ang-II detection antibody at room temperature. The plate was again washed followed by 30 min incubation with streptavidin:horseradish peroxidase (strep:HRP). After a final wash, TMB substrate was added for 20 min in the dark and the absorbance was read at 450 nm using a FLUOstar OPTIMA plate reader (BMG labtech, Aylesbury, Buckinghamshire, UK). The concentration of Ang-II was interpolated from a serial dilution of recombinant Ang-II (1,000-62.5 pg/ml) and measured in duplicate for each case. Ang-II level is expressed as pg/mg total protein for eachcase.

Angiotensin-III direct ELISA

Ang-III level was measured in human brain tissue homogenates (in 1% SDS lysis buffer) using a direct ELISA. Recombinant human Ang-III (Sigma Aldrich) or human brain tissue homogenates (diluted 1 in 10 in PBS) were incubated for 2 h in a clear high-binding capacity NUNC maxisorp plate (ThermoFisher Scientific, Waltham, MA, USA) at 26°C with shaking. The wells were washed five-times in PBS with 0.05% tween-20 and blocked for 1 h in 1 % PBS: BSA (Sigma Aldrich). After another five washes, the wells were incubated with biotinylated anti-human Ang-III (diluted to 2 μg/ml in PBS) (Cloud-Clone, Wuhan, China) for 2 h at 26°C with shaking, followed by a further wash step. The plate was incubated with strep:HRP (1:200) in PBS:0.01% Tween-20 at room temperature for 20 min in the dark. TMB substrate (R&D systems) was added after a further wash and left to develop in the dark for 20 minutes. Absorbance at 450 nm was read following the addition of 2 N sulphuric acid (‘stop’ solution) using a FLUOstar OPTIMA plate reader (BMG labtech). Ang-III concentrations were interpolated from a standard curve generated by serially diluting recombinant human Ang-III (20,000-312.5 pg/ml) and were measured in duplicate. Ang-III level is expressed as ng/mg total protein. The assay showed minimal cross reactivity with a number of closely-related peptides including Ang I, Ang-II, andAng 1–7.

Aminopeptidase-A sandwich ELISA

AP-A level was measured in brain tissue homogenates (in 1% SDS lysis buffer) using a commercially available competitive sandwich ELISA (FIRELISA; Human GluAP Cat. No. ELISAHu004197). In brief, brain tissue homogenates (diluted 1 in 25 in PBS) or recombinant human AP-A was added together with biotinylated antigen for 1 h at 37°C. After 5 washes with the proprietary wash buffer, streptavidin-HRP was added to each well for 1 h at 37°C. After a further five washes, TMB chromogen was added to each well and incubated for 20 min in the dark. Absorbance at 450 nm was read after the reaction was stopped using 2 N sulphuric acid using a FLUOstar OPTIMA plate reader (BMG labtech). AP-A levels were measured in duplicate for each case and were interpolated from a serial dilution of recombinant human AP-A (32-2 ng/ml). AP-A level is expressed as ng/mg total protein.

Aminopeptidase-A enzyme activity assay

AP-A enzyme activity was measured in brain tissue homogenates (in 0.5% triton X-100 lysis buffer) using a fluorogenic peptide substrate, H-Glu-AMC-OH (AMC = 7-amido-4-methylcoumarin) (200μM) (Bachem, Bubendorf, Switzerland). The assay was performed in Nunc F16 black maxisorp 96-well plates (ThermoFisher Scientific). A series of preliminary experiments were performed to test a range of substrate concentrations (400-25 μM) using a serial dilution of recombinant human AP-A (400–3.2 ng/ml) over an extended reaction time, i.e., fluorescence was read every 5 min up to 30 min and then again at 1 and 2 h. H-Glu-AMC-OH at 200μM produced the highest fluorescent signal that became saturated at 1 h, i.e., total cleavage of substrate was observed. All subsequent experiments were performed using H-Glu-AMC-OH at 200 μM and the reaction was allowed to proceed for 1 h at 37°C. Brain tissue homogenates (triton X-100) (100μg total protein), or recombinant AP-A (serially diluted to produce a standard curve (400–3.2 ng/ml)) were diluted in assay buffer (25 mM Tris pH 8.0, 50 mM CaCl2, 200 μM NaCl) and incubated in the presence of amastatin-HCl a general aminopeptidase inhibitor [39] (10μM) (Enzo Life Sciences, Exeter, UK), or assay buffer alone, for 10 min at 37°C prior to the addition of H-Glu-AMC-OH (200 μM diluted in assay buffer). The reaction was incubated at 37°C for 1 h in the dark and fluorescence was read at an excitation/emission of 390/450 nm using a using a FLUOstar OPTIMA plate reader (BMG labtech). Enzyme activities are expressed as mMol of H-Glu-AMC-OH hydrolyzed per hour per mg of total protein (using AMC (Sigma Aldrich) as a reference).

Aminopeptidase-N sandwich ELISA

AP-N level was measured in brain tissue homogenates (in 1% SDS lysis buffer) using a commercially available ELISA kit (AP-N duoset, Cat no. DY3815) (R&D systems, Cambridge, UK). High-binding capacity NUNC Maxisorp 96-well plates (ThermoFisher Scientific) were coated with a human anti-AP-N capture antibody diluted in PBS (0.8μg/ml) overnight at room temperature. The wells were washed five time in PBS/0.05% tween-20 and blocked with 1% PBS: BSA for 1 h at room temperature. The wells were washed again and recombinant human AP-N (20-0.3125 ng/ml), or brain tissue (diluted 1 in 20 in PBS), was incubated for 2 h at room temperature. After a further five washes, the wells were incubated with streptavidin:HRP (diluted 1:200) (R&D systems) for 20 min in the dark. The wells were washed again and TMB substrate (100 μl) (R&D systems) was added and left in the dark for 20 min. Upon addition of 2 N sulphuric acid (50μl ‘stop’ solution) absorbance was read at 450 nm in a FLUOstar OPTIMA plate reader (BMG labtech). The concentration of AP-N was interpolated from a serial dilution of recombinant human AP-N and AP-N levels were measured in duplicate for each case and expressed as ng/mg totalprotein.

Aminopeptidase-N enzyme activity

AP-N enzyme activity was measured in brain tissue homogenates (in 0.5% triton X-100 lysis buffer) using an immunocapture-based activity assay using the fluorogenic peptide substrate, H-ALA-AMC (200 μM) (Bachem, Bubendorf, Switzerland). A black high binding-capacity Nunc maxisorp 96-well plate (ThermoFisher Scientific) was coated overnight at room temperature with capture antibody from the AP-N duoset ELISA kit (0.8μg/ml) (R&D systems). After five washes in 0.05% PBS:Tween-20 each well was blocked in 1% PBS:BSA for 1 h at room temperature. After another wash step, a serial dilution of recombinant human AP-N (200–1.56 ng/ml) (R&D systems) or brain tissue homogenates (125 μg total protein) were incubated for 2 h at room temperature. After another 5 washes, wells were either incubated with betstatin-HCl (20 μM) (Sigma Aldrich) diluted in assay buffer (50 mM Tris, pH 7.0), or assay buffer alone, at 37°C for 10 min prior to the addition of H-ALA-AMC (200 μM). A series of preliminary experiments were performed to test a range of H-ALA-AMC concentrations (400-25 μM) using a serial dilution of recombinant human AP-N (200–12.5 ng/ml) over an extended reaction time, i.e., fluorescence was read every 5 min up to 30 min and then again at 1 and 2 h. H-ALA-AMC at 200 μM produced the highest fluorescent signal that became saturated at 1 h, i.e., total cleavage of substrate was observed. All subsequent experiments were performed using H-ALA-AMC (200μM), the reaction was allowed to proceed for 1 h at 37°C, and fluorescence was read at excitation/emission at 390/450 nm using a FLUOstar OPTIMA plate reader (BMG labtech). AP-N enzyme activity is expressed as mMol of H-ALA-AMC hydrolyzed per hour per mg of total protein (using AMC as a reference).

AP-A and AP-N immunohistochemistry

Paraffin sections (7 μm) were dewaxed in Clearene, dehydrated in 100% ethanol and immersed in 3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity. The tissue was washed in running tap water and sections were boiled in 9 mM tri-sodium citrate, pH 6, for 5 min, left to stand for 5 min, boiled again for 5 min, and finally left to stand for 15 min at room temperature. The sections were incubated with Vectastain blocking serum (horse) (ABC kit, Vector labs, Vector Labs, Peterborough, UK) then incubated overnight with primary antibody at room temperature: Rabbit polyclonal anti-ENPEP (Aminopeptidase A) (Cat no. HPA005128, Sigma Aldrich) was diluted 1 in 75 and mouse monoclonal CD13 (AP-N) (cat no. ab7417, Abcam) was diluted 1 in 400. All sections were then incubated with biotinylated Universal Antibody followed by VectaElite ABC complex and with 3,3’-diaminobenzidine (DAB) for 7 min. After incubation with DAB, sections were washed in water and immersed in copper sulphate DAB enhancer for 4 min, counterstained with hematoxylin Gill II for 15 s, dehydrated, cleared and mounted in Clearium.

Statistical analysis

Parametric statistical tests were used for comparisons between groups and ANOVA with Bonferroni post-hoc test was used for multiple-group comparisons. Pearson analysis was used to assess the correlation between pairs of variables. Statistical tests were performed using SPSS version 23. p-values <0.05 were considered statistically significant.

RESULTS

Study cohort

The control (mean age-at-death±SD = 78.5±10 years and postmortem delay±SD = 43.8±36 h) and AD cohorts (mean age-at-death±SD = 78.5 years±9.7 years and postmortem delay±SD = 45.0 h±25 h) were matched closely for age-at-death and postmortem delay (Table 1). Ang-II and Ang-III levels (Supplementary Figure 1), and AP-A and AP-N level and enzyme activity (Supplementary Figure 2) did not vary according to age-at-death or postmortem delay.

The control and AD cohorts varied with regard to the ratio of male: female with a higher ratio of males in the control (F 22: M 37) and females in the AD group (F 55: M 35) (Table 1). Ang-II and Ang-III level did not differ between genders although a non-significant trend of higher Ang-II was observed in females compared to males in both the control and AD group (Supplementary Figure 3). Ang-III level, and AP-A and AP-N enzyme activity and protein levels were not influenced by gender (Supplementary Figure 3).

Angiotensin II level is increased in AD and does not correlate with parenchymal Aβ and tau load

Ang-II level was significantly elevated in AD (mean±SEM = 58.30±2.33 pg/mg total protein) compared with controls (mean±SEM = 39.49±3.34 pg/mg total protein) (p < 0.0001) (Fig. 1A). When the cases were stratified according to Braak tangle stage, an indicator of disease severity, Ang-II level was significantly increased in Braak stage V-VI (mean±SEM = 56.42±2.62 pg/mg total protein) compared to Braak stage 0–II (mean±SEM = 38.63±3.40 pg/mg total protein) (p < 0.001) (Fig. 1B). Ang-II level was increased in Braak stage III-IV (mean±SEM = 49.96±4.45 pg/mg total protein) but was not significantly different to Braak stage 0–II. Ang-II level did not correlate with insoluble Aβ level (r = 0.12; p = 0.20) (Fig. 1C) or parenchymal tau load (r = 0.16; p = 0.06) (Fig. 1D).

Fig.1

Angiotensin-II (Ang-II) level is increased in the mid-frontal cortex in Alzheimer’s disease (AD). A) Bar chart showing significantly elevated Ang-II levels in AD compared to age-matched controls (p < 0.0001 using an unpaired t-test). B) Bar chart showing increased Ang-II level in relation to disease severity, indicated by Braak tangle stage. Angiotensin-II level was significantly increased in Braak stage V-VI compared to 0–II (p < 0.001 using a one-way ANOVA with Bonferroni post-hoc analysis). In A and B, the bars represent the mean and SEM. C, D) Scatterplots showing a trend towards positive correlations between Ang-II level and (C) guanidine-HCl-extracted insoluble amyloid-β (Aβ) level and (D) parenchymal tau load. The best-fit linear regression lines (inner line) and 95% confidence intervals are superimposed. Each dot represents a single case. The green circles indicate controls and the red circles indicate AD cases.

Angiotensin III level is increased in AD and correlates strongly with parenchymal Aβ and tau load

Ang-III level was significantly elevated in AD (mean±SEM = 154.79±2.90 ng/mg total protein) compared with controls (mean±SEM = 127.01±3.27 ng/mg total protein) (p < 0.0001) (Fig. 2A). When the cases were stratified according to Braak tangle stage, an indicator of disease severity, the level of Ang-III was significantly increased in Braak stage V-VI (mean±SEM = 157.49±3.18 ng/mg total protein) compared to Braak stage 0–II (mean±SEM = 127.66±3.99 ng/mg total protein) (p < 0.0001) (Fig. 2B). Ang-III level was unchanged between Braak stage 0–II and III-IV (mean±SEM = 134.19±4.28 ng/mg total protein). A strongly significant positive correlation was observed between the level of Ang-III and the concentration of Aβ (r = 0.49, p < 0.0001) (Fig. 2C) and between Ang-III and parenchymal tau load (0.28, p < 0.01) (Fig. 2D). Multiple regression analysis (Ang-III as the dependent variable and Aβ and ptau load as the predictors) indicates that Aβ alone is a strong independent predictor of Ang-III (p < 0.0001) whereas ptau is not (p = 0.072).

Fig.2

Angiotensin-III (Ang-III) level is increased in the mid-frontal cortex in Alzheimer’s disease (AD). A) Bar chart showing significantly elevated Ang-III levels in AD compared to age-matched controls (p < 0.0001 using unpaired t-test). B) Bar chart showing increased angiotensin-III level in relation to disease severity, indicated by Braak tangle stage. Ang-III level was significantly increased in Braak stage V-VI compare to 0–II (p < 0.0001 using one-way ANOVA with Bonferroni post-hoc analysis). In A and B, the bars represent the mean and SEM. C, D) Scatterplots showing highly significant positive correlations between Ang-III level and (C) insoluble Aβ level and (D) parenchymal tau load. The best-fit linear regression lines (inner line) and 95% confidence intervals are superimposed. Each dot represents a single case. The green circles indicate controls and the red circles indicate AD cases.

Aminopeptidase-A level is reduced in AD

AP-A is the rate-limiting enzyme in the production of Ang-III. AP-A level was significantly reduced in AD (mean±SEM = 1285.45±47.35 ng/mg total protein) compared to controls (mean±SEM = 1696±66.35 ng/mg total protein) (p < 0.0001) (Fig. 3A) and was significantly lower in Braak stage V-VI (mean±SEM = 1255.67±47.96 ng/mg total protein) compared to Braak stage 0–II (mean±SEM = 1732.5±86.96 ng/mg total protein) (p < 0.0001). No difference was observed between Braak stage III-IV (mean±SEM = 1632.52±97.73 ng/mg total protein) and 0–II. Despite the reduction in AP-A level, a trend toward higher AP-A activity was observed in AD (mean±SEM = 2.11±0.23 mMol/h/mg protein) compared to controls (mean±SEM = 1.73±0.18 mMol/h/mg protein) (Fig. 3C) and in relation to Braak tangle stage (BS) (mean±SEM = 1.75±0.20 mMol/h/mg protein in BS 0–II; 1.92±0.43 mMol/h/mg protein in BS III-IV and 2.08±0.24 mMol/h/mg protein in BS V-VI) (Fig. 3D) although neither reached statistical significance.

Fig.3

Aminopeptidase-A (AP-A) protein levels are significantly reduced in Alzheimer’s disease (AD) but enzyme activity is unaltered. A) Bar charts showing significantly reduced AP-A level in AD (p < 0.0001 using an unpaired t-test). B) AP-A level was significantly reduced in Braak stage V-VI compared to Braak stage 0–II (p < 0.0001 using one-way ANOVA with Bonferroni post-hoc analysis). C, D) Bar charts show a trend toward higher AP-A enzyme activity in AD in relation to disease severity (despite reduced protein level), indicated by highest activity in Braak tangle stage V-VI, although the difference was not statistically significant. Bars represent the mean and SEM.

Aminopeptidase-N level is unchanged but enzyme activity is reduced in AD

AP-N level was unchanged in AD (mean±SEM 55.85±1.91 ng/mg total protein) compared to controls (mean±SEM 53.16±2.05 ng/mg total protein) (Fig. 4A) and did not vary according to Braak tangle stage (mean±SEM 52.71±2.40 ng/mg total protein in BS 0–II; 53.2±3.18 ng/mg total protein in BS III-IV and 56.27±2.10 ng/mg total protein in BS V-VI) (Fig. 4B). AP-N activity was significantly lower in AD (mean±SEM 2.24±0.16 mMol/h/mg total protein) compared with age-matched controls (mean±SEM 2.86±0.22 mMol/h/mg protein) (p < 0.05) (Fig. 3C). A trend toward reduced AP-N activity in relation to increasing Braak stage severity was observed (mean±SEM 2.89±0.25 mMol/h/mg protein in BS 0–II; 2.39±0.35 mMol/h/mg protein and 2.24±0.17 mMol/h/mg protein in BS V-VI), however, there were no significant differences between Braak tangle stage groups.

Fig.4

Aminopeptidase-N (AP-N) enzyme activity is reduced in Alzheimer’s disease (AD) but protein levels are unaltered. A, B) Bar charts showing unchanged AP-N levels in AD compared to age-matched controls in relation to disease stage severity. C, D) Bar charts showing significantly lower AP-N activity in AD compared to age-matched controls (p < 0.05 using an unpaired t-test) and in relation to disease severity, indicated by lower activity in Braak tangle stage V-VI, although this difference was not statistically significant. Bars represent the mean and SEM.

Aminopeptidase-A localization in human brain tissue

AP-A immunolabeling of the vasculature was observed within the temporal neocortex and subiculum in both AD and controls (Fig. 5). Perivascular AP-A labeling was also present in areas of focal white matter ischemic damage (Fig. 5). AP-A labeling of macrophages and microglia was observed, particularly in association with Aβ plaques and occasional neuronal labeling was present in both AD and controls. A similar staining pattern was observed in the frontal and parietal neocortex (not shown).

Fig.5

Aminopeptidase-A (AP-A) localization in human brain tissue. AP-A distribution in the temporal neocortex (a and b, scale bars = 50 μm) and subiculum (c, scale bar = 100 μm) of an Alzheimer’s disease (AD) patient. AP-A also labelled cells in the temporal neocortex (d and f, scale bars = 50 μm) and an area of focal white matter ischemic damage (e, scale bar = 100 μm) in controls. Prominent vascular labeling (g, scale bar = 50 μm) and labeling was also present in the subiculum (h, scale bar = 100 μm) of controls. AP-A immunolabeling was observed in macrophages and microglia in both AD cases and controls. Staining was also seen in these cell types in association with Aβ plaques. Occasional neuronal immunolabeling was seen in both AD cases and controls. High levels of labeling were seen in an area of focal white matter ischemic damage (d and f) while prominent vascular labeling was present in some subjects.

Aminopeptidase-N localization in human brain tissue

AP-N neuronal labeling was observed within the frontal neocortex, CA2 region of the hippocampus, temporal neocortex, and parietal neocortex in AD (Fig. 6a-d). A similar distribution of AP-N was observed in controls, albeit with much lower intensity (Fig. 6e-h). AP-N immunolabeling was not observed in the vasculature.

Fig.6

Aminopeptidase-N (AP-N) localization in human brain tissue. AP-N distribution in the frontal neocortex (a), CA2 region of the hippocampus (b), temporal neocortex (c) and parietal neocortex (d) of an Alzheimer’s disease (AD) patient. AP-N also labelled some neurons, albeit with less intensity, in the frontal neocortex (e), CA2 region of the hippocampus (f), temporal neocortex (g) and parietal neocortex (h) of control subjects. No vessel staining was observed in any of the cases examined. Scale bar = 50 μm for all sections.

DISCUSSION

In the present study, we measured Ang-II and Ang-III levels in relation to pathological markers of AD. Our data indicate that Ang-II and Ang-III levels are both increased in AD but that Ang-III levels correlate strongly with parenchymal Aβ and tau load whereas Ang-II levels did not. We also found dysregulation of the APA/Ang-III/APN/Ang-IV/AT4R pathway in AD. AP-A (responsible for the conversion of Ang-II to Ang-III) levels were significantly reduced in AD but enzyme activity was unaltered. AP-N (responsible for the conversion of Ang-III to Ang-IV) activity was reduced in AD despite AP-N protein levels remaining unchanged. Together, these data suggest dysregulation of the APA/Ang-III/APN/Ang-IV/AT4R pathway might contribute to elevated Ang-III in AD due to increased conversion of Ang-II to Ang-III and reduced metabolism of Ang-III to Ang-IV. Ang-III level, and the enzymes involved in its processing, may contribute to the pathogenesis of AD.

Hyperactivity of the classical axis of brain RAS (ACE-1/Ang-II/AT1R) via Ang-II-mediated signaling through the AT1R receptor is implicated in the pathogenesis of AD (reviewed in [5]). We have previously reported that ACE-1 activity(rate-limiting in Ang-II production) is increased in human postmortem brain tissue in association with higher parenchymal Aβ load [8]. In the present study, we measured Ang-II and Ang-III levels in human postmortem brain tissue in AD. Ang-II was elevated in AD but did not correlate with Aβ and tau levels. In contrast, Ang-III was significantly increased in AD and correlated strongly with both Aβ level and parenchymal tau load. Ang-III has been shown to bind with equipotency to AT1r and AT2r and share many of the physiological functions of Ang-II and studies have shown that orally administered AP-A inhibitors, which presumably reduce the level of Ang-III, are cardio-protective [25 –28]. It is conceivable that Ang-III, rather than Ang-II, may be the central effector in the classical axis of brain RAS.

We have also explored the pathways responsible for Ang-III metabolism, commonly referred to as the APA/Ang-III/APN/Ang-IV/AT4R pathway, which involves AP-A-mediated conversion of Ang-II to Ang-III and further downstream processing of Ang-III by AP-N to Ang-IV. Our data suggest that increased Ang-III is associated with a non-significant trend toward increased AP-A activity, i.e., increased conversion of Ang-II to Ang-III, in combination with significantly reduced AP-N activity, i.e. reduced conversion of Ang-III to Ang-IV.

It is currently unclear why there is a discrepancy between AP-A level (significantly reduced) and activity (non-significantly elevated) but this could indicate that there is post-translational modification in AD that enhances that activity of AP-A relative to reduced AP-A protein level. These findings in brain tissue are in contrast to previous studies showing a gender-specific reduction (lower in males) in plasma and serum AP-A activity in AD [32, 33]; whether this is due to differences between the brain and periphery (as we have also observed for ACE-1 activity in brain tissue [8 , 35]) remains unknown. Our immunohistochemical studies revealed that AP-A was mostly localized within microglia surrounding Aβ plaques in AD suggesting that there may be upregulation of a specific pool of AP-A in conjunction with an immune response associated with AD pathology. Alternatively, the trend toward increased AP-A activity in AD may simply reflect a compensatory response of AP-A in the presence of increasing Ang-II level. AP-A generates highly amyloidogenic and neurotoxic N-terminal truncated and pyroglutamated (AβpE3) Aβ₄₂ species that could also contribute directly to AD pathogenesis [40, 41].

AP-N activity was significantly reduced in AD in contrast to protein levels that either remained unchanged (determined by ELISA) or appeared to be elevated (immunohistochemical analysis).A reduction in AP-N activity could however potentially mediate reduced conversion of Ang-III to Ang-IV resulting in elevated Ang-III level, which would not only impact on disease pathology but could also influence downstream signaling pathways involved in cognition - Ang-IV mediated inhibition of its receptor IRAP is heavily implicated in learning and memory processes [30, 31]. Serum and plasma AP-N activities are also reduced in AD [32, 33]. Together, our data indicate that Ang-III may be increased in AD as a result of dysregulation of the APA/Ang-III/APN/Ang-IV/AT4R pathway and could contribute not only to disease pathology but also to memory impairment.

Emerging data has identified a number of regulatory pathways within brain RAS that are involved in the metabolism of Ang-II and counter-regulate the activity of the ‘classical’ RAS axis (reviewed in [4]). ACE-2 converts Ang-II to Ang 1-7, which via the MAS receptor counter-regulates the actions of the classical axis, known as the ACE2/Ang(1–7)/MASR pathway (reviewed in [42, 43]). We recently showed that ACE-2 activity is highly significantly reduced in AD and is inversely correlated with both Aβ level and tau parenchymal load [44]. Ang 1–7 (via the MAS receptor) is also implicated in long-term potentiation [45] and therefore loss of the activity within the ACE2/Ang(1–7)/MASR pathway may not only be associated with disease pathology, but as for Ang-IV, may adversely impact on learning and memory processes.

These data indicate that elevated Ang-III level in AD is strongly linked with disease pathology and is possibly associated with dysregulation of the APA/APN/Ang-IV/AT4R pathway within brain RAS. Longitudinal studies, such as those described by Gard et al. [32] who measured changes in serum aminopeptidases activity in healthy and mild-moderate AD at various stages of disease, will be useful in determining whether changes within these RAS pathways contribute to the early pathological changes in AD.

Footnotes

ACKNOWLEDGMENTS

This work was supported by Alzheimer’s Research UK (ARUK-PG2015-11). The South West Dementia Brain Bank is part of the Brains for Dementia Research program, jointly funded by Alzheimer’s Research UK and Alzheimer’s Society, and is supported by BRACE (Bristol Research into Alzheimer’s and Care of the Elderly) and the Medical Research Council.

Authors’ disclosures available online ().

Appendix

References

Phillips

, de Oliveira

(2008) Brain renin angiotensin in disease. J Mol Med (Berl) 86, 715–722.

Wright

, Harding

(2011) Brain renin-angiotensin–a new look at an old system. Prog Neurobiol 95, 49–67.

Wright

, Harding

(2013) The brain renin-angiotensin system: A diversity of functions and implications for CNS diseases. Pflugers Arch 465, 133–151.

Wright

, Kawas

, Harding

(2013) A Role for the Brain RAS in Alzheimer’s and Parkinson’s Diseases. Front Endocrinol (Lausanne) 4, 158.

Kehoe

, Miners

, Love

(2009) Angiotensins in Alzheimer’s disease - friend or foe? Trends Neurosci 32, 619–628.

Tian

, Zhu

, Xie

, Shi

(2012) Central angiotensin II-induced Alzheimer-like tau phosphorylation in normal rat brains. FEBS Lett 586, 3737–3745.

Zhu

, Shi

, Zhang

, Wang

, Liu

, Chen

, Tong

(2011) Central angiotensin II stimulation promotes beta amyloid production in Sprague Dawley rats. PLoS One 6, e16037.

Miners

, Ashby

, Van Helmond

, Chalmers

, Palmer

, Love

, Kehoe

(2008) Angiotensin-converting enzyme (ACE) levels and activity in Alzheimer’s disease, and relationship of perivascular ACE-1 to cerebral amyloid angiopathy. Neuropathol Appl Neurobiol 34, 181–193.

Miners

, Ashby

, Baig

, Harrison

, Tayler

, Speedy

, Prince

, Love

, Kehoe

(2009) Angiotensin-converting enzyme levels and activity in Alzheimer’s disease: Differences in brain and CSF ACE and association with ACE1 genotypes. Am J Transl Res 1, 163–177.

10.

Ashby

, Kehoe

(2013) Current status of renin-aldosterone angiotensin system-targeting anti-hypertensive drugs as therapeutic options for Alzheimer’s disease. Expert Opin Investig Drugs 22, 1229–1242.

11.

Corbett

, Pickett

, Burns

, Corcoran

, Dunnett

, Edison

, Hagan

, Holmes

, Jones

, Katona

, Kearns

, Kehoe

, Mudher

, Passmore

, Shepherd

, Walsh

, Ballard

(2012) Drug repositioning for Alzheimer’s disease. Nat Rev Drug Discov 11, 833–846.

12.

Chauvel

, Llorens-Cortes

, Coric

, Wilk

, Roques

, Fournie-Zaluski

(1994) Differential inhibition of aminopeptidase A and aminopeptidase N by new beta-amino thiols. J Med Chem 37, 2950–2957.

13.

Healy

, Wilk

(1993) Localization of immunoreactive glutamyl aminopeptidase in rat brain. II. Distribution and correlation with angiotensin II. Brain Res 606, 295–303.

14.

Ramirez

, Arechaga

, Garcia

, Sanchez

, Lardelli

, de Gandarias

(1990) Mn2(+)-activated aspartate aminopeptidase activity, subcellular localization in young and adult rat brain. Brain Res 522, 165–167.

15.

Rich

, Moon

, Harbeson

(1984) Inhibition of aminopeptidases by amastatin and bestatin derivatives. Effect of inhibitor structure on slow-binding processes. J Med Chem 27, 417–422.

16.

Mukoyama

, Nakajima

, Horiuchi

, Sasamura

, Pratt

, Dzau

(1993) Expression cloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane receptors. J Biol Chem 268, 24539–24542.

17.

Murphy

, Alexander

, Griendling

, Runge

, Bernstein

(1991) Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature 351, 233–236.

18.

Abhold

, Sullivan

, Wright

, Harding

(1987) Binding, degradation and pressor activity of angiotensins II and III after aminopeptidase inhibition with amastatin and bestatin. J Pharmacol Exp Ther 242, 957–962.

19.

Wright

, Jensen

, Roberts

, Sardinia

, Harding

(1989) Structure-function analyses of brain angiotensin control of pressor action in rats. Am J Physiol 257, R1551–R1557.

20.

Wright

, Morseth

, Abhold

, Harding

(1985) Pressor action and dipsogenicity induced by angiotensin II and III in rats. Am J Physiol 249, R514–R521.

21.

Wright

, Sullivan

, Petersen

, Harding

(1985) Brain angiotensin II and III binding and dipsogenicity in the rabbit. Brain Res 358, 376–379.

22.

Phillips

(1987) Functions of angiotensin in the central nervous system. Annu Rev Physiol 49, 413–435.

23.

Saavedra

(1992) Brain and pituitary angiotensin. Endocr Rev 13, 329–380.

24.

Zini

, Fournie-Zaluski

, Chauvel

, Roques

, Corvol

, Llorens-Cortes

(1996) Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: Predominant role of angiotensin III in the control of vasopressin release. Proc Natl Acad Sci U S A 93, 11968–11973.

25.

Gao

, Marc

, Iturrioz

, Leroux

, Balavoine

, Llorens-Cortes

(2014) A new strategy for treating hypertension by blocking the activity of the brain renin-angiotensin system with aminopeptidase A inhibitors. Clin Sci (Lond) 127, 135–148.

26.

Llorens-Cortes

(2014) Orally active aminopeptidase A inhibitors reduce blood pressure: A new strategy for treating hypertension. Biol Aujourdhui 208, 217–224.

27.

Marc

, Gao

, Balavoine

, Michaud

, Roques

, Llorens-Cortes

(2012) Central antihypertensive effects of orally active aminopeptidase A inhibitors in spontaneously hypertensive rats. Hypertension 60, 411–418.

28.

Marc

, Llorens-Cortes

(2011) The role of the brain renin-angiotensin system in hypertension: Implications for new treatment. Prog Neurobiol 95, 89–103.

29.

Palmieri

, Bausback

, Ward

(1989) Metabolism of vasoactive peptides by vascular endothelium and smooth muscle aminopeptidase M. Biochem Pharmacol 38, 173–180.

30.

Albiston

, Diwakarla

, Fernando

, Mountford

, Yeatman

, Morgan

, Pham

, Holien

, Parker

, Thompson

, Chai

(2011) Identification and development of specific inhibitors for insulin-regulated aminopeptidase as a new class of cognitive enhancers. Br J Pharmacol 164, 37–47.

31.

Albiston

, Morton

, Ng

, Pham

, Yeatman

, Ye

, Fernando

, De Bundel

, Ascher

, Mendelsohn

, Parker

, Chai

(2008) Identification andcharacterization of a new cognitive enhancer based on inhibition of insulin-regulated aminopeptidase. FASEB J 22, 4209–4217.

32.

Gard

, Fidalgo

, Lotter

, Richardson

, Farina

, Rusted

, Tabet

(2017) Changes of renin-angiotensin system-related aminopeptidases in early stage Alzheimer’s disease. Exp Gerontol 89, 1–7.

33.

Puertas Mdel

, Martinez-Martos

, Cobo

, Lorite

, Sandalio

, Palomeque

, Torres

, Carrera-Gonzalez

, Mayas

, Ramirez-Exposito

(2013) Plasma renin-angiotensin system-regulating aminopeptidase activities are modified in early stage Alzheimer’s disease and show gender differences but are not related to apolipoprotein E genotype. Exp Gerontol 48, 557–564.

34.

Montine

, Phelps

, Beach

, Bigio

, Cairns

, Dickson

, Duyckaerts

, Frosch

, Masliah

, Mirra

, Nelson

, Schneider

, Thal

, Trojanowski

, Vinters

, Hyman

, National Institute on Aging, Alzheimer’s Association (2012) National Institute on Aging-Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease: A practical approach. Acta Neuropathol 123, 1–11.

35.

Miners

, van Helmond

, Raiker

, Love

, Kehoe

(2010) ACE variants and association with brain Abeta levels in Alzheimer’s disease. Am J Transl Res 3, 73–80.

36.

van Helmond

, Miners

, Kehoe

, Love

(2010) Higher soluble amyloid beta concentration in frontal cortex of young adults than in normal elderly or Alzheimer’s disease. Brain Pathol 20, 787–793.

37.

Ballard

, Chalmers

, Todd

, McKeith

, O’Brien

, Wilcock

, Love

, Perry

(2007) Cholinesterase inhibitors reduce cortical Abeta in dementia with Lewy bodies. Neurology 68, 1726–1729.

38.

Chalmers

, Wilcock

, Vinters

, Perry

, Ballard

, Love

(2009) Cholinesterase inhibitors may increase phosphorylated tau in Alzheimer’s disease. J Neurol 256, 717–720.

39.

Aoyagi

, Tobe

, Kojima

, Hamada

, Takeuchi

, Umezawa

(1978) Amastatin, an inhibitor of aminopeptidase A, produced by actinomycetes. J Antibiot (Tokyo) 31, 636–638.

40.

Antonios

, Saiepour

, Bouter

, Richard

, Paetau

, Verkkoniemi-Ahola

, Lannfelt

, Ingelsson

, Kovacs

, Pillot

, Wirths

, Bayer

(2013) N-truncated Abeta starting with position four: Early intraneuronal accumulation and rescue of toxicity using NT4X-167, a novel monoclonal antibody. Acta Neuropathol Commun 1, 56.

41.

Brannstrom

, Ohman

, Nilsson

, Pihl

, Sandblad

, Olofsson

(2014) The N-terminal region of amyloid beta controls the aggregation rate and fibril stability at low pH through a gain of function mechanism. J Am Chem Soc 136, 10956–10964.

42.

Jiang

, Gao

, Lu

, Zhang

(2013) ACE2-Ang-(1-7)-Mas axis in brain: A potential target for prevention and treatment of ischemic stroke. Curr Neuropharmacol 11, 209–217.

43.

, Sriramula

, Lazartigues

(2011) ACE2/ANG-(1-7)/Mas pathway in the brain: The axis of good. Am J Physiol Regul Integr Comp Physiol 300, R804–R817.

44.

Kehoe

, Wong

, Al Mulhim

, Palmer

, Miners

(2016) Angiotensin-converting enzyme 2 is reduced in Alzheimer’s disease in association with increasing amyloid-beta and tau pathology. Alzheimers Res Ther 8, 50.

45.

Hellner

, Walther

, Schubert

, Albrecht

(2005) Angiotensin-(1-7) enhances LTP in the hippocampus through the G-protein-coupled receptor Mas. Mol Cell Neurosci 29, 427–435.