Renin-Angiotensin System Alterations in the Human Alzheimer’s Disease Brain

Abstract

Background:

Understanding Alzheimer’s disease (AD) in terms of its various pathophysiological pathways is essential to unravel the complex nature of the disease process and identify potential therapeutic targets. The renin-angiotensin system (RAS) has been implicated in several brain diseases, including traumatic brain injury, ischemic stroke, and AD.

Objective:

This study was designed to evaluate the protein expression levels of RAS components in postmortem cortical and hippocampal brain samples obtained from AD versus non-AD individuals.

Methods:

We analyzed RAS components in the cortex and hippocampus of postmortem human brain samples by western blotting and immunohistochemical techniques in comparison with age-matched non-demented controls.

Results:

The expression of AT₁R increased in the hippocampus, whereas AT₂R expression remained almost unchanged in the cortical and hippocampal regions of AD compared to non-AD brains. The Mas receptor was downregulated in the hippocampus. We also detected slight reductions in ACE-1 protein levels in both the cortex and hippocampus of AD brains, with minor elevations in ACE-2 in the cortex. We did not find remarkable differences in the protein levels of angiotensinogen and Ang II in either the cortex or hippocampus of AD brains, whereas we observed a considerable increase in the expression of brain-derived neurotrophic factor in the hippocampus.

Conclusion:

The current findings support the significant contribution of RAS components in AD pathogenesis, further suggesting that strategies focusing on the AT₁R and AT₂R pathways may lead to novel therapies for the management of AD.

Keywords

Alzheimer’s disease angiotensin-converting enzyme angiotensin type 1 receptor angiotensin type 2 receptor renin-angiotensin system

INTRODUCTION

Alzheimer’s disease (AD) is an age-related neurodegenerative disease that affects over 5.8 million people in the United States. It is currently a leading cause of death with an incidence that is still on the rise [1, 2]. The main pathological hallmarks of AD include extracellular accumulation of amyloid-β plaques (Aβ) and intracellular phosphorylated tau (neurofibrillary tangles) in the brain [3]. None of the medications currently marketed for AD cure, prevent, or reverse its progression. The currently available drugs are simply palliative, and their efficacy decreases over time. There remains an urgent need to identify novel disease-limiting therapeutic targets that will preserve or improve cognition and address the source of AD pathology. Although the pathophysiologic mechanisms of AD are not fully understood, abnormalities in the brain renin-angiotensin system (RAS) have been reported, and several recent studies have reinforced the notion that RAS modulation may be beneficial for the treatment of several brain diseases, including AD [4 –8]. Therefore, understanding the precise contribution of RAS in AD pathology can lead to the development of novel therapeutic strategies for better management of AD.

The RAS includes the precursor angiotensinogen (AGT) that is cleaved by renin to produce angiotensin I (Ang I), which is subsequently cleaved by the angiotensin converting enzyme (ACE) to produce angiotensin II (Ang II), III (Ang III), and IV (Ang IV). The action of Ang II is mediated by two major receptor subtypes, angiotensin receptor type 1 (AT₁R) and angiotensin receptor type 2 (AT₂R). AT₁R mediates the pro-inflammatory and vasoconstrictor actions of Ang II. The localization of AT₁R and AT₂R in the human central nervous system has long been established [9]. Numerous clinical studies have implicated the RAS system in many physiological functions of the brain through the activation of the AT₁R and AT₂R pathways [9, 10]. In AD, activation of AT₁R in various cell types promotes inflammation, generates reactive oxygen species, increases activation of pro-inflammatory microglia, and reduces cerebral blood flow, known to potentially damage brain parenchyma [4, 7]. In contrast, AT₂R opposes the deleterious effects of AT₁R by promoting vasodilation, improving cerebral blood flow, and mitigating inflammation [4]. Moreover, clinical trial evidence shows that indirect AT₂R activation with angiotensin receptor blockers (ARBs) used to treat hypertension prevents cognitive decline in older adults and lowers the risk of dementia and AD [11, 12]. In addition, a degradation product of Ang II, Ang 1–7 mediates neuroprotection through activation of the Mas receptor [13]. Hence, better understanding of pathological contribution individual RAS components in AD brain is necessary for development of novel therapeutic approaches. The aim of our study was to determine the protein expression levels of RAS components in cortical and hippocampal samples obtained from age matched AD and non-AD patients’ postmortem samples, using immunoblotting and immunohistochemical techniques. We evaluated the activation of RAS components in two brain regions at cellular and molecular level along with AD pathology markers. Our findings demonstrated that AT₁R expression increased and colocalized with Aβ and p-tau in human AD brains, providing empirical evidence for the contribution of RAS-associated alteration to AD pathology.

MATERIALS AND METHODS

Ethical approval

This study was approved by the UTHSC Institutional Review Board (IRB #17-05716-NHSR; Exempt Application 665258), Memphis, TN, USA, and was conducted under standard ethical procedures. All the appropriate personal protective safety procedures were followed for handling of the human brain samples.

Human brain specimens

Table 1 details the characteristics of AD brain samples used in this study. 36 brain samples from non-AD and AD patients were obtained from the Human Brain and Spinal Fluid Resource Center, which is sponsored by NIHDS/NIMH, the National Multiple Sclerosis Society, and the Department of Veterans Affairs. Samples were derived from short postmortem interval (PMI) autopsies from the University of Kentucky AD Center (UK-ADC) cohort [14]. Pre-mortem neuropathological assessments were implemented utilizing standard neuropathological procedures [15, 16]. The frontal cortical and hippocampus sections used for immunoblotting analyses were snap-frozen during the autopsy in liquid nitrogen and stored at –80°C until the time of immunoblotting. The inclusion criteria were as follows: PMI < 4 h; no clinical evidence of frontotemporal dementia or pathologic evidence of frontotemporal lobar degeneration; no cancer in the brain parenchyma; no large infarctions any brain areas or micro-infarcts within 3 cm of the brain tissue samples used for the analysis.

Table 1

A) Patient Demographics (for cortical samples)

Immunoblotting
Gender	Age (y) Mean±SEM	Diagnosis	Postmortem interval (PMI)
4 males, 3 females	81±9.18	Non-AD (n = 7)	< 4 h
3 males, 4 females	83±7.19	AD (n = 7)	< 4 h
Immunohistochemistry
2 males, 2 females	88±2.98	Non-AD (n = 4)	3–8 h
2 males, 2 females	83±2.06	AD (n = 4)	3–8 h

Table 1B

B) Patient Demographics (for hippocampal samples)

Gender	Age (y) Mean±SEM	Diagnosis	PMI
4 male, 2 females	68±8.42	Non-AD (n = 5–7)	< 4 h
5 male, 3 females	79±3.6	AD (n = 6–8)	< 4 h
Immunohistochemistry
2 male, 2 females	88±2.98	Non-AD (n = 4)	3–8 h
2 male, 2 females	83±2.06	AD (n = 4)	3–8 h

Postmortem human brains that fit the neuropathological criteria were collected from individuals who were diagnosed with AD and suffered from dementia for 5 to 12 years. These were obtained from the University of Iowa Deeded Body Program, Iowa City, IA, USA for immunofluorescent staining. Control (non-AD) brains within the same age group were obtained at routine autopsy, with no indicated history or signs of neurological or psychiatric illness. All brain specimens were isolated within 3 to 8 h of death. The temporal lobe blocks were dissected and immersion-fixed in 4% paraformaldehyde solution. Blocks were cryoprotected by freezing in the presence of 30% sucrose. Frozen sections were cut at 40μm on a sliding microtome, collected in phosphate buffered saline (PBS) and stored in cryo-storage solution (30% glycerol, 30% ethylene glycol in PBS) for immunofluorescence staining.

Table 2

Primary antibodies used for immunoblotting and immunofluorescence staining

	Catalogue number	Dilution	Brand
ACE-1	SC 23908	1:500	Santa Cruz Biotechnology
ACE-2	SC-73668	1:500	Santa Cruz Biotechnology
Angiotensinogen	SC 37411	1:500	Santa Cruz Biotechnology
Ang II/III	NB100-62446	1:1000	Novus Biologicals
AT₁R	SC-513884	1:500	Santa Cruz Biotechnology
AT₂R	NBP1-77368	1:1000	Novus Biologicals
Mas receptor	AAR-013	1:2500	Alomone Labs
BDNF	ANT-010	1:2000	Alomone Labs
PPARγ	2435T	1:1000	Cell Signaling Technology
Phospho-eNOS	9571S	1:1000	Cell Signaling Technology
eNOS	32027S	1:1000	Cell Signaling Technology

Immunoblotting

Brain samples were homogenized and prepared for immunoblotting as previously described [17]. Protein concentration was calculated using BCA assay. Thirty micrograms (μg) of total protein were loaded in each well, separated by SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for non-specific binding with 5% skimmed milk and probed at 4°C overnight with primary antibodies specific for ACE-1 (1:500; SC 23908; Santa Cruz Biotechnology), ACE-2 (1:500; SC-73668; Santa Cruz Biotechnology), Angiotensinogen (1:500; SC 37411; Santa Cruz Biotechnology), Ang II (1:1000; NB100-62346; Novus Biologicals), AGTR₂ (1:1000; NBP1-77368; Novus Biologicals), AT₁R (1:500; SC-513884; Santa Cruz Biotechnology), Mas receptor (1:2500; AAR-013; Alomone Labs), brain-derived neurotrophic factor (BDNF, 1:2000; ANT-010; Alomone Labs), peroxisome proliferator- activated receptor gamma (PPARγ, 1:1000; 2435T; Cell Signaling Technology), P-eNOS (1:1000; 9571S; Cell Signaling Technology), or eNOS (1:1000; 32027S; Cell Signaling Technology). The membranes were washed with 1X TBS-T incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000, Sigma) at room temperature for 1 h. The bands were visualized using an enhanced chemiluminescent substrate system (Thermo Fisher Scientific). Their intensities were analyzed using Image J software, normalized to loading control (β-actin), and expressed as fold change.

Immunofluorescence staining

Sections of the temporal cortex were permeabilized using 0.1% PBS-TritonX-100 for 20 min. Antigen retrieval was performed for most of the antigens, using sodium citrate buffer containing 0.1% Triton-X. All tissue sections were blocked using 5% BSA for 1 h at room temperature (25°C) and incubated at 4°C with primary antibodies, including mouse anti-AT₁R (1:500), rabbit anti-NeuN (1:500), rabbit anti-GFAP (1:500), and rabbit anti-Iba-1 (1:500). The sections were washed three times with PBS and incubated for 1 h at room temperature with the respective secondary antibodies (1:200), which were tagged with DyLight™-488 (goat anti-mouse IgG 9H+L; KPL 042-03-18-06) and Alexa Fluor-594 (goat anti-rabbit IgG (H + L), ThermoFisher Scientific, A-11012). The DyLight-488-conjugated secondary antibodies were used to detect AT₁R, while the Alexa Fluor-594-conjugated secondary antibodies were used to detect NeuN, GFAP, and Iba-1. Tissue sections prepared without a primary antibody served as negative controls. The sections were washed and mounted with ProLong™ Diamond Antifade Mountant containing DAPI (4’,6-diamidino-2-phenylindole; Invitrogen P36962) to stain cellular DNA for visualization of nuclei and examined using a Zeiss 710 confocal laser scanning microscope.

Statistical analysis

All statistical analyses were performed using the student’s t-test for two groups (AD and non-AD) included in the GraphPad Prism Instat 7.0 software package. All data were described by mean values±SEM (standard error of the mean). *p < 0.05 and **p < 0.01 were considered statistically significant.

RESULTS

Altered levels of Ang II type 1, type 2, and Mas receptors in both cortical and hippocampal regions of brains from AD patients

To elucidate the association between angiotensin receptor such as AT₁R, AT₂R, and Mas receptor with AD pathology, immunoblotting was performed on cortical and hippocampal tissues from AD and control (non-AD) brain. Our results showed that AT₁R protein levels were slightly increased in both the cortex and hippocampus of AD compared to non-AD brains but that this increase was only statistically significant in the hippocampus (p = 0.02) (Fig. 1A, D). Immunofluorescence/confocal microscopy was used to show that AT₁R localized in the immediate vicinity of key AD pathology markers, amyloid beta and phospho-tau (ser262) (Fig. 1G). We observed no statistically significant changes in AT₂R protein levels in the hippocampus and cortex of AD and non-AD brains (Fig. 1B, E). Additionally, expression of the Mas receptor remained the same in the cortex, however, the expression of Mas receptor decreased in hippocampus (Fig. 1C, F). To examine the cell-specificity of AT₁R accumulation in AD brains, we used immunofluorescence/confocal microscopy to show apparent co-localization of AT₁R with cell specific markers NeuN (neurons), GFAP (astrocytes), and Iba-1 (microglia and macrophages) (Fig. 1H).

Fig. 1

Alterations in AT₁R, AT₂R, and Mas receptor protein levels in AD compared to non-AD brains. A-F, Immunoblot analysis with antibodies specific for AT₁R (A, D), AT₂R (B, E), and Mas receptors (C, F) in the hippocampus (A, B, C) or cortex (D, E, F) of AD and non-AD brains, with beta-actin used as a loading control; G) confocal immunofluorescence microscopy in AD and non-AD brains with antibodies specific for AT₁R and phospho-tau-ser 262 (upper panels) or AT₁R and amyloid beta (lower panels). A) AD brains showed significantly higher levels of AT₁R in the hippocampus compared to non-AD brains (p = 0.02). B and E the levels of cortical (B) and hippocampal (E) AT₂R were not significantly different in AD and non-AD brains. The change in levels of AT₁R in cortical AD samples did not reach statistical significance. G) The localization of AT₁R coincided with that of Aβ and phospho-tau (serine262) proteins in the cortical AD samples. H) AT₁R also colocalized with cell-specific markers NeuN (neurons), GFAP (astrocytes), and Iba-1 (microglia/macrophages). Representative images are shown. Scale bar represents 50μm. The values were expressed as mean ± SEM; *p < 0.05 was considered statistically significant. AT₁R, Ang II receptor type 1; AT₂R, Ang II receptor type 2; Aβ, amyloid-β; phospho-tau (ser262), phosphorylated (ser 262) tau protein; GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium binding adaptor molecule 1; NeuN, neuronal nuclear antigen.

Expression of ACE-1, angiotensinogen, and angiotensin II in AD brains

The RAS includes the precursor AGT that is cleaved by renin to produce angiotensin I, which is subsequently cleaved by ACE-1 to produce angiotensin II, III, and IV. Previous studies have shown an increase in ACE-1 levels in many parts of the brain, including the cortex [18, 19]. A high level of ACE expression has been detected in the basal ganglia and cerebrospinal fluid of patients with psychiatric disorders [20, 21]. ACE plays a critical role in the degradation of Aβ and low ACE activity may result in neuronal damage and plaque deposits in AD [22]. In addition, the endopeptides generated by ACE activity, including Ang II, bradykinin, and substance P, play well characterized roles in several neurodegenerative disorders [23].

The relative levels of proteins ACE-1, ACE-2, AGT, and Ang II in the cortex and hippocampus of brains from AD patients were analyzed to determine the association between AD and alterations in these RAS components. Our immunoblot analysis showed no statistically significant changes in levels of ACE-1 (Fig. 2A, C), ACE-2 (Fig. 2B, D), or AGT (Fig. 3A, C) in the hippocampus or cortex of the AD brain compared to the non-AD brain. Interestingly, we observed a statistically significant increase in the level of Ang II proteins in the hippocampus (p = 0.02) but not in the cortex of AD brains (Fig. 3B, D).

Fig. 2

Lack of statistically significant changes in ACE-1 and ACE-2 levels in the brains of AD patients. A-D, Immunoblot analyses for ACE-1 (A, C) and ACE-2 (B, D) in the hippocampus (A, B) or cortex (C, D) of AD and non-AD brains, with beta-actin used as a loading control. A and C, ACE-1 expression was not significantly different in hippocampal (A) and cortical (C) samples from AD compared to non-AD brains. B and D, The apparent change in the relative levels of ACE-2 proteins did not reach statistical significance in the hippocampus (B) or the cortex (D) of AD brains. The values were expressed as mean±SEM; *p < 0.05 was considered statistically significant. ACE-1, angiotensin converting enzyme 1; ACE-2, angiotensin converting enzyme 2.

Fig. 3

The relative expression levels of AGT and Ang II proteins were altered in the cortex and hippocampus of brains from AD patients. A-D, Immunoblot analysis with antibodies specific for Ang (A, C) and Ang II (B, D) in the hippocampus (A, B) or cortex (C, D) of AD and non-AD brains, with beta-actin used as a loading control. A and C, There was no statistically significant difference in levels of Ang detected by immunoblot in the hippocampus (A) and cortex (C) of AD and non-AD brains. B and D, A statistically significance increase in the levels of Ang II was observed in the hippocampus (p = 0.02) (B) but not in the cortex (D) of AD brains, compared to non-AD brains. Values were expressed as mean ± SEM; *p < 0.05 was considered statistically significant. AGT, angiotensinogen; Ang II, angiotensin II.

Modification in protein expression of BDNF, PPARγ, and eNOS in postmortem brains of AD patients

To assess pathways associated with neuroprotection in AD, we analyzed the expression of the anti-inflammatory markers BDNF, PPARγ, and phospho-endothelial nitric oxide synthase (eNOS) in the cortical and hippocampal tissue of the AD brains. We did not detect a statistically significant difference in levels of PPARγ or phosphor-eNOS in the hippocampus (Fig. 4A, C) or cortex of AD brains (Fig. 4D, F). BDNF levels exhibited a statistically significant increase in hippocampal tissue (p = 0.03) of AD brains, while there was no significant change in the cortex (Fig. 4B, D). Moreover, there was no remarkable difference in the ratio of these markers that would correlate with neurogenesis and anti-inflammatory response [24 –26]. This observation illustrates that the levels of anti-inflammatory markers were not higher in the AD brain than the non-AD brain.

Fig. 4

BDNF levels were elevated in the hippocampus of brains from AD patients. A-D, Immunoblot analysis with antibodies specific for PPARγ (A, D), BDNF (B, E), and p-eNOS (C, F) in the hippocampus (A, B, C) or cortex D, E, F) of AD and non-AD brains, with beta-actin used as a loading control. A and D, PPARγ levels did not exhibit a statistically significant difference in hippocampus and cortex of AD brains compared to non-AD brains. B) A marked increase in BDNF was detected in the hippocampus of AD brains (p = 0.03). E, no statistically significant difference was detected for BDNF levels in the cortex of AD brains. C and F, The ratio of Phospho-eNOS/eNOS expression remained unchanged in the hippocampus and cortex of AD brains. Values were expressed as mean ± SEM; *p < 0.05 was considered statistically significant. BDNF, brain-derived neurotrophic factor; e-NOS, endothelial nitric oxide synthase; phospho-eNOS, phosphorylated eNOS; PPARγ, peroxisome proliferator-activated receptor gamma.

DISCUSSION

Although the contribution of the RAS system in hemodynamic homeostasis has been extensively studied and long been established, the existence of a local RAS system in the brain that regulates learning, memory, and emotional stress has gained recent attention [27]. An association between RAS and AD pathology has become a target area for development of therapeutic interventions designed to mitigate the cognitive impairment resulting from AD [7]. In the present study, we demonstrated that hyperactivation of the RAS system contributes to AD pathology in the postmortem human brain. We found that the levels of AT₁R protein were significantly increased in the hippocampus and specifically in astrocytes, neurons, and microglia of AD brains. AT₁R co-localized with the key pathological marker of AD, Aβ and phospho-tau. These results are consistent with previous reports [9, 28] of elevated levels of AT₁R in AD that are involved in cognitive impairment in mouse models of AD [29]. Further, treatment with many ARBs such as losartan and valsartan improved cognitive function in hypertensive subjects [28 , 30–32]. The beneficial effect of ARB drugs is mediated by direct activation of AT₂R and a similar activation of AT₂R by compound 21 ameliorated classical AD biomarkers in a mouse model of AD [33]. Interestingly, we found that levels of AT₂R remained unchanged in the cortex and hippocampus of AD versus non-AD brains. Studies have demonstrated that deletion of AT₂R in mice is associated with poor cognitive function [34]. AT₂R stimulation counteracts several harmful effects of AT₁R and is correlated with neuro-regeneration [35], anti-inflammation, memory improvement [36], and oxidative stress mitigation [37]. Further, the expression of Mas receptor was marginally decreased in the hippocampus of AD brains, but not in the cortex. Activation of Mas receptor with Ang (1–7) counteract the detrimental effect of Ang II and exerts neuroprotective effects [13, 38]. Consistently, exogenous administration of Ang-(1–7) attenuated cognitive impairment in mouse model of AD [39].

Fig. 5

A schematic illustration of the association between RAS activation and AD-pathology. Ang II accelerates inflammation, oxidative stress, vascular damage, and cell death through the AT₁R in AD. AT₂R activation neutralizes the detrimental effects of AT₁R stimulation thereby enhancing Aβ plaque clearance and preserving cognitive function. Activation of Mas receptor counteracts the detrimental effects of Ang II by producing BDNF, eNOS, and PPARγ, all of which contribute to neuroprotection and neurogenesis, hence decreasing the risk of AD.

We detected a statistically significant elevation in Ang II protein levels in the hippocampus but not the cortex of AD brains. These results are consistent with the observation that elevated levels of Ang II and subsequent stimulation of AT₁R are associated with hyperactivation of the RAS system in AD [39, 40]. Administration of Ang II to older rats is associated with elevated levels of Aβ and tau in the brain, which correlates with cognitive impairment [41, 42]. Interestingly, we did not detect a statistically significant change in levels of the ACE-1 or ACE-2 proteins. ACE-1 is the rate-limiting component of the RAS system that converts Ang I to Ang II and is elevated in AD [9 , 43–45]. Recent in vitro studies suggest that ACE-1 may contribute to the metabolism of Aβ [46 –48]. Overexpression of ACE-1 is linked to an increased degradation of Aβ whereas suppression of ACE can result in accumulation of Aβ [46], although paradoxically, increased ACE-1 activity is observed in AD brains that have demonstrated Aβ accumulation [9, 44]. Hyperactivation of ACE-1 also increases levels of Ang II, which causes vasoconstriction, reduced blood flow, and impaired clearance of Aβ, thereby neutralizing the favorable effects of ACE-1 in Aβ cleavage [49]. Immunohistochemical data collected from human brain samples exhibited low levels of ACE-1 in the cortex and hippocampus and high levels of ACE-1 in the substantia nigra, basal ganglia, and choroid plexus [9]. Elevated Ang-II increases AT₁R and AT₂R expression in brain regions in AD [9, 10] and AT₂R signaling promotes the production of BDNF, which is essential for neurogenesis [25]. Although we did not observe a difference in ACE-1 levels in AD versus non-AD brains a hypothetical increase in ACE-1 activity would be consistent with our observed increases in Ang II and AT₁R in the AD hippocampus.

Another RAS component, ACE-2, converts Ang-II to Ang (1–7) and is involved in neuroprotection [50, 51]. ACE-2 activity opposes that of ACE-1 and is diminished in regions of the brain with Aβ and tau accumulation [52]. We detected no statistically significant changes in the levels of ACE-2 expression in the AD hippocampus or cortex, possibly due to a smaller sample size, which is not consistent with the recent clinical findings of an upregulation of ACE-2 in the AD brain [53]. ACE-2, which converts Ang. II to Ang (1–7), resulting in decreased blood pressure [54], has been implicated in neurogenesis and the anti-inflammatory response [52, 55].

Lower expression of ACE-2 leads to accumulation of Ang II and reduction in Ang (1–7) [7]. Ang (1–7) counteracts the destructive effects of ACE-1, Ang II, and AT₁R [56, 57], and improves memory performance [58, 59]. Our data showed a trend towards an increase in Ang II and a decrease in ACE-2 expression in the cortex of the AD compared to non-AD brains, although neither change was statistically significant. It is likely that the activity of ACE-2 may not be affected by the increased accumulation of Ang II in the hippocampus of AD patients. Ang II is implicated in oxidative stress, neuroinflammation, cognitive impairment, and Aβ accumulation through AT₁R activation [60 –64].

In this study, elevated levels of Ang II and AT₁R suggested the presence of inflammation and oxidative stress in the AD compared to non-AD brains. AT₂R signaling contributes to neuroprotection via producing neurotrophins such as vascular endothelium growth factor [65], BDNF, nerve growth factor, neuotrophin-3 [25], and PPARγ [66]. Previous reports observed reduced levels of BDNF in AD brains [24, 67]. BDNF is a neurotrophin widely distributed in the CNS, and is associated with neurogenesis, neuronal repair, neural survival, and synaptic plasticity [68]. In addition, BDNF modulates cognition and memory; higher expression of BDNF ameliorated cognitive impairment [69]. We detected a minor reduction in BDNF expression in the cortex of the AD brain, in agreement with previous reports [24, 67] and significant elevation in the hippocampus which is inconsistent with previous findings [70, 71]. It has been demonstrated that high expression of BDNF in brain could slow down the cognitive impairment progression in people who have AD [72]. The elevated BDNF expression could be considered as a compensatory mechanism to increase the level of other neurotrophins to counteract the detrimental effect of RAS activation in the hippocampus. Consistently, elevated expression of BDNF has been previously demonstrated in human AD hippocampus [73].

We observed no significant changes in PPARγ protein levels in cortical and hippocampal regions of brains from AD patients. It has been reported that AT₁R [26] and ACE signaling [74] mediates the inactivation of PPARγ. AT₁R activation in the AD brain contributes to neuroinflammation by inhibiting PPARγ, which can further aggravate reactive oxygen species production. The Ang II receptor antagonist telmisartan produces its beneficial effect on cognitive impairment partly through activation of PPARγ [75]. Interestingly, we observed a slight trend toward a decrease in eNOS in the AD brain compared to non-AD brains, although it was not statistically significant. Ang II mitigates the protective effect of eNOS through AT₁R activation [76]. In the present study, eNOS reduction could be due to the stimulation of AT₁R in AD. Our results support the recent finding that eNOS is an important contributor to the pathogenesis of AD [77].

CONCLUSION

In summary, exploring the pathophysiology of AD is helpful for understanding the complexity of the disease and development of novel therapeutic targets. In this study, we demonstrated that activation of the neural RAS system in the AD brain is mediated through activation of AT₁R. Additionally, AT₁R is expressed in CNS cells, including neurons, astrocytes, and microglia, in close association with Aβ and neurofibrillary tangles, which supports the hypothesis that RAS activation, contributes to AD pathogenesis. Several pieces of evidence suggest that inhibition of AT₁R using ARBs including candesartan reduces AD associated pathology and neuro inflammation. However, ARB increases the possibility of lowering blood pressure. On the other hand, AT₂R upregulation counteracts the deleterious effect of AT₁R and triggers its neuroprotective effect. Furthermore, AT₂R upregulation using C21 might be a promising therapeutic approach for the management of AD as it does not affect blood pressure. Taken together, future investigation is needed to evaluate the neuroprotective effect of AT₂R stimulation in animal models of AD. Although the RAS system can be considered a double-edged sword since it can have both positive and negative effects on the brain, the precise mechanism by which RAS contributes to AD pathology remains to be established.

Footnotes

ACKNOWLEDGMENTS

This work was supported by startup funds from the Department of Anatomy and Neurobiology, UTHSC Memphis TN (TI), and a grant from the National Institutes of Health (R01-NS097800 to TI). We thank Dr. Peter T. Nelson (University of Kentucky Sanders-Brown Center on Aging) for providing human brain specimens from the University of Kentucky AD Center (UK-ADC) cohort. The authors are also grateful to Dr. Asgar Zaheer, Department of Neurology, University of Missouri, Columbia, MO, USA, for providing human brain sections for immunostaining.

Authors’ disclosures available online ().

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