Loss of Endothelial Nitric Oxide Synthase Promotes p25 Generation and Tau Phosphorylation in a Murine Model of Alzheimer’s DiseaseNovelty and Significance
Rationale: Alzheimer’s disease has an unknown pathogenesis; however, cardiovascular risk factors are associated with a higher incidence of Alzheimer’s disease. A defining feature of endothelial dysfunction induced by cardiovascular risk factors is reduced bioavailable endothelial nitric oxide (NO). We previously demonstrated that endothelial NO acts as an important signaling molecule in neuronal tissue.
Objective: We sought to determine the relationship between the loss of endothelial NO synthase (eNOS) and tau phosphorylation in neuronal tissue.
Methods and Results: We used eNOS knockout (−/−) mice as well as an Alzheimer’s disease mouse model, amyloid precursor protein (APP)/PSEN1dE9+/− (PS1) that lacked eNOS (APP/PS1/eNOS−/−) to examine expression of tau kinases and tau phosphorylation. Brain tissue from eNOS−/− mice had statistically higher ratios of p25/p35, indicative of increased cyclin-dependent kinase 5 activity as compared with wild-type (n=8, P<0.05). However, tau phosphorylation was unchanged in eNOS−/− mice (P>0.05). Next, we determined the role of NO in tau pathology in APP/PS1/eNOS−/−. These mice had significantly higher levels of p25, a higher p25/p35 ratio (n=12–14; P<0.05), and significantly higher cyclin-dependent kinase 5 activity (n=4; P<0.001). Importantly, APP/PS1/eNOS−/− mice also had significantly increased tau phosphorylation (n=4–6; P<0.05). No other changes in amyloid pathology, antioxidant pathways, or neuroinflammation were observed in APP/PS1/eNOS−/− mice as compared with APP/PS1 mice.
Conclusions: Our data suggests that loss of endothelial NO plays an important role in the generation of p25 and resulting tau phosphorylation in neuronal tissue. These findings provide important new insights into the molecular mechanisms linking endothelial dysfunction with the pathogenesis of Alzheimer’s disease.
Alzheimer’s disease (AD) is a huge social and economic burden affecting over 5 million people in the United States.1 Although a definitive cause of AD is unknown, numerous cardiovascular risk factors are associated with higher incidence of AD.2,3 A common feature of these cardiovascular risk factors is endothelial dysfunction. A defining characteristic of endothelial dysfunction is reduced bioavailability of endothelial nitric oxide (NO).4,5 We previously demonstrated that endothelial NO acted as an important signaling molecule in neuronal tissue.6,7 Loss of endothelial NO led to several AD-related pathologies, including increased amyloid precursor protein (APP) expression and amyloidogenic processing6,7 and cognitive impairment in aged eNOS+/− and eNOS−/− mice.7,8 Based on these observations, we hypothesized that endothelial NO might also affect the phosphorylation of tau. Tau pathology is a hallmark of AD, where it becomes hyperphosphorylated and dissociates from the microtubules to form the primary component of neurofibrillary tangles. Several kinases are implicated in the phosphorylation of tau, primarily cyclin-dependent kinase (Cdk) 5 and glycogen synthase kinase (GSK) 3β.9,10 Therefore, we sought to determine the role of endothelial NO in modulating kinase expression and tau phosphorylation in brain tissue of endothelial nitric oxide synthase (eNOS) knockout mice (eNOS−/−) as well as in an AD mouse model that also lacked eNOS.
Editorial, see p 1052
In This Issue, see p 1039
Nos3tm1Unc/J (eNOS−/−), stock no 002684, and C57BL/6 (wild-type) mice, stock no 000664, were purchased from Jackson Laboratory (Bar Harbor, ME). APPswe,PSEN1dE9+/− (APP/PS1) mice on the C57BL/6 genetic background were originally purchased from Jackson laboratory as stock no 005864. Subsequently, APP/PS1 mice were transferred from Jackson Laboratory to Mutant Mouse Resource & Research Centers (MMRRC), and mice were purchased from MMRRC as stock no 034832-JAX. eNOS−/− mice were bred with C57BL/6 mice to generate eNOS+/− mice. To generate wild-type, APP/PS1, and APP/PS1/eNOS−/− mice used in experiments, eNOS+/− (female) and APP/PS1/eNOS+/− (male) mice were bred in house. The average breeding pair has 2 to 3 litters with viable pups, and these resultant litters average 4 to 5 pups. We wish to point out that APP/PS1/eNOS−/− mice are found in small numbers in the resulting litters, approaching 1 mouse in every 22 to 25 mice born. Male mice were euthanized at 4 to 5 months of age by a lethal dose of pentobarbital.
A detailed methods section is provided in the Online Data Supplement and includes detailed methods regarding genotyping/polymerase chain reaction, tissue collection, confocal microscopy, Western blotting, ELISA for beta amyloid (Aβ), and statistical analysis, which are as previously described.6,7
Ratio of p25/p35 Is Significantly Higher in the Brains of eNOS−/− Mice
We sought to determine whether chronic loss of endothelial NO affected protein kinases known to be involved in the phosphorylation of tau. First, we examined the levels of Cdk5 and its activators p35 and p25. Although there was no difference between protein levels of Cdk5 or p35, p25 tended to be increased in the brain tissue from eNOS−/− mice as compared with wild-type mice (Figure 1A–1E). Importantly, the increased ratio of p25/p35, an established index of increased Cdk5 activity, was significantly higher in the eNOS−/− brain tissue as compared with wild-type (Figure 1D; P<0.05).
We also examined the levels of GSK3β and Akt, 2 other kinases involved in aberrant tau phosphorylation. There were no differences observed in their expression or phosphorylation, suggesting that their expression and phosphorylation in the brain are unaffected by loss of eNOS (Online Figure I).
No Alteration in Tau Phosphorylation in eNOS−/− Brain Tissue
To determine whether the increased p25/p35 ratio led to increased tau phosphorylation, we measured protein levels of tau and phosphorylated tau (pTau) in the brains of wild-type and eNOS−/− mice. No differences were observed in tau or pTau levels in brain tissue (Figure 1F–1I; P>0.05).
Characterization of APP/PS1/eNOS−/− Mice
There was no difference in body weight between wild-type, APP/PS1, or APP/PS1/eNOS−/− mice (Online Table I). Systolic blood pressure was significantly elevated in APP/PS1/eNOS−/− mice as compared with both wild-type and APP/PS1 mice (Online Table I; P<0.001 from wild-type and P<0.01 from APP/PS1). Next, we examined circulating levels of glucose, total cholesterol, high-density lipoprotein cholesterol, and triglycerides (Online Table I). Of these, only triglycerides were significantly different between the groups. Triglycerides were significantly higher in APP/PS1/eNOS−/− mice as compared with both wild-type and APP/PS1 mice (Online Table I; P<0.01 from wild-type and P<0.05 from APP/PS1).
p25 and Tau Phosphorylation in APP/PS1/eNOS−/− Brain Tissue
We examined protein expression of p35, p25, and Cdk5 by Western blot. p25 protein levels and p25/p35 ratio were significantly higher in the brains of APP/PS1/eNOS−/− mice as compared with both wild-type and APP/PS1 mice (Figure 2A–2D; *P<0.05 from wild-type and &P<0.05 from APP/PS1). Importantly, p25 and the p25/p35 ratio were not significantly different in the APP/PS1 brain tissue as compared to wild-type (Figure 2). Notably, Cdk5 enzyme activity was significantly higher in Cdk5 isolated from APP/PS1/eNOS−/− brain tissue as compared with Cdk5 from wild-type and APP/PS1 tissue (Figure 2F; P<0.001 from wild-type and APP/PS1). Immunohistochemical analysis of p25/p35 showed increased immunoreactivity in both the cortex (Figure 3) and hippocampus (data not shown) of APP/PS1/eNOS−/− mice as compared with wild-type and APP/PS1 mice. Furthermore, expression of p25/p35 (Figure 3) and Cdk5 (Online Figure II) was mainly observed in neuronal tissue as demonstrated by the colocalization with NeuN, a neuronal marker.
Levels of total tau were unchanged between wild-type, APP/PS1, and APP/PS1/eNOS−/− mice (Figure 4A and 4C), whereas protein levels of pTau and the ratio of pTau/Tau were significantly increased in the brains of the APP/PS1/eNOS−/− mice as compared with the other mice (Figure 4A, 4B, and 4D; ***P<0.001, **P<0.01 from wild type and &P<0.05 from APP/PS1). However, although pTau tended to be higher in APP/PS1 mice (P>0.05), the pTau/tau ratio was not different between wild-type and APP/PS1 mice (Figure 4A–4D). Immunohistochemical analysis of pTau showed cellular localization within neurons as indicated by colocalization with the neuronal marker. Increased pTau immunoreactivity was observed in the cortex (Figure 4E) and hippocampus (data not shown) of APP/PS1/eNOS−/− as compared with wild-type and APP/PS1 mice. We did perform immunohistological examinations of brain tissue using an antibody for neurofibrillary tangles but did not observe any evidence of these tangles in the brain sections from APP/PS1 or APP/PS1/eNOS−/− mice at the 3 to 4 months of age we examined (data not shown).
Although phosphorylated GSK3β and the ratio of phosphorylated GSK3β/GSK3β tended to be higher in the brains of APP/PS1/eNOS−/− mice, they did not reach statistical significance (Online Figure III; P>0.05).
Expression and Amyloidogenic Processing of APP in APP/PS1/eNOS−/− Mice
We examined protein levels of APP and β-site APP cleaving enzyme 1 in the brain tissue of wild type, APP/PS1, and APP/PS1/eNOS−/− mice. There was a significant increase in APP protein expression in APP/PS1 and APP/PS1/eNOS−/− mice as compared with wild-type mice (Online Figure IVA and IVB; P<0.05 as compared with wild-type) and an increase in β-site APP cleaving enzyme 1 protein levels in APP/PS1/eNOS−/− as compared with wild-type mice (Online Figure IVC; *P<0.05); however, there was no difference between APP or β-site APP cleaving enzyme 1 protein levels between APP/PS1 and APP/PS1/eNOS−/− mice (Online Figure IV).
We measured circulating levels of Aβ40 and Aβ42 in these mice and found no significant differences between APP/PS1 and APP/PS1/eNOS−/− mice (data not shown, n=4–6 animals per background; P>0.05). Furthermore, when we examined brain tissue levels of soluble Aβ40 and Aβ42, we found no differences (data not shown, n=4–6 animals per background; P>0.05).
NOS Isoforms, Cyclooxygenase Pathway, Antioxidant Systems, and Microglia Activation
To determine whether there were compensatory changes in other important endothelial pathways, we examined protein levels of inducible NOS (iNOS), neuronal NOS (nNOS), cyclooxygenase-1, cyclooxygenase-2, and prostacyclin synthase. Importantly, iNOS and nNOS protein levels were unchanged by the loss of eNOS (Online Figure V). Furthermore, levels of cyclooxygenase enzymes and prostacyclin synthase were not altered in APP/PS1/eNOS−/− mice (data not shown, n=6–8 animals per background; P>0.05). Levels of the major antioxidant enzymes were unchanged between wild-type, APP/PS1, and APP/PS1/eNOS−/− mice (Online Figure VI). Furthermore, levels of 2-hydroxyethiudium, a measure of superoxide anion, were not different in APP/PS1/eNOS−/− mice (Online Figure VIF). Neuronal and astrocyte markers, NeuN, and glial fibrillary acidic protein, respectively, were unchanged (data not shown, n=4–6 animals per background, P>0.05). Importantly, microglial markers, cluster of differentiation 68, major histocompatibility complex II, and ionized calcium-binding adaptor molecule-1 were not different between the 3 groups of mice (Online Figure VII). Levels of interleukin-1α, as measured by ELISA, were not altered in the brains of APP/PS1/eNOS−/− mice (Online Figure VII). Finally, no differences were seen between wild-type, APP/PS1, or APP/PS1/eNOS−/− brain tissue in a proinflammatory array that examined 40 cytokines (data not shown).
Our results are significant because they continue to demonstrate the importance of endothelial dysfunction as a plausible factor in the development of AD pathology. We demonstrate for the first time that loss of endothelial NO leads to alterations in neuronal p25, an aberrant activator of the tau kinase, Cdk5. Indeed, in our APP/PS1/eNOS−/− mice, increased p25 is accompanied by statistically higher enzymatic activity of Cdk5 and increased levels of pTau. It is important to note that this effect seems specific to loss of endothelial NO in the AD mouse model. Although we cannot completely rule out that alterations in NO produced by iNOS and nNOS may contribute to the upregulation of Cdk activity and increased pTau levels, these seem unlikely. First, in our previous studies, we reported that the loss of endothelial NO in eNOS−/− mice resulted in decreased microvascular levels of NOx, while overall brain NOx levels were unchanged,6 which suggests that loss of eNOS did not lead to compensatory changes in nNOS- or iNOS-produced NO within the brain tissue. Importantly, loss of eNOS alone, as seen in the eNOS−/− mice, was sufficient to increase generation of p25. Second, a search of the literature did not return any results suggesting that iNOS or nNOS protein levels or enzyme activity were increased in young APP/PS1 mice. In addition, we report that expression of iNOS and nNOS is not elevated in APP/PS1/eNOS−/− mice, thereby, reinforcing our conclusion that loss of eNOS function is primarily responsible for elevated phosphorylation of pTau. Finally, we did report increased systolic blood pressure in APP/PS1/eNOS−/− mice and, therefore, cannot completely rule out hypertension as a contributing factor in the changes we report.
It is established that tau is predominantly expressed in neurons, and we were not able to detect tau protein in cerebral microvessels or in cultured brain microvascular endothelial cells (unpublished observation). In addition, our immunohistochemical analysis demonstrated colocalization of pTau with the neuronal marker, NeuN. Cdk5 and GSK3β are the most relevant kinases involved in tau phosphorylation.9,10 Increased Cdk5 activity is associated with hyperphosphorylation of tau, paired helical fragments, and neurite death.11,12 The increased pTau we observed in APP/PS1/eNOS−/− mice seems to be mediated by increased activity of Cdk5 because loss of endothelial NO did not lead to changes in other tau kinases, namely GSK3β and Akt. Although phosphorylated GSK3β tended to be higher in APP/PS1/eNOS−/− mice, this phosphorylation site is an inhibitory site that would lead to decreased GSK3β activity and, thus, is not likely to be responsible for the increased pTau we see. Indeed, we report here for the first time that loss of eNOS led to an increased p25/p35 ratio. It is reported that increased p25/p35 ratio, caused by increased generation of p25 by calpain, leads to an aberrant activation of Cdk5, promoting tau phosphorylation and neurodegeneration.13,14 Importantly, our observed increased in p25 and p25/p35 ratio was accompanied by increased Cdk5 enzymatic activity in APP/PS1/eNOS−/− mice. Notably, in the present study, increased tau phosphorylation was seen in APP/PS1/eNOS−/− mice and not in eNOS−/− mice or APP/PS1 mice as compared with age-matched wild-type mice at 4 months of age. This suggests that there may be a synergistic effect between the loss of eNOS and amyloid alterations present in the APP/PS1 mouse, resulting in early appearance of tau pathology. Indeed, prior studies established that pTau was detected in adult APP/PS1 mice, only at 7 to 10 months of age.15,16
Tau acts to stabilize microtubules within neurons. Hyperphosphorylation of tau can cause tau to dissociate from the microtubules, thereby, leading to neurofibrillary tangle formation and eventually neuronal dysfunction and death.17 We did not observe neurofibrillary tangles in the 4-month-old APP/PS1/eNOS−/− mice; however, the earlier appearance of increased pTau in APP/PS1/eNOS−/− as compared with what is reported in the literature for APP/PS1 mice suggests that the loss of endothelial NO may accelerate the pathology timeline in these mice. Future studies will need to be performed to document the time course of pathological and cognitive alterations in these mice as they age.
There are 2 mechanisms by which NO can mediate signaling changes: cyclic guanosine monophosphate and S-nitrosylation. Treatment of Tg2576 mice with sildenafil, a phosphodiesterase 5 inhibitor that increases levels of cyclic guanosine monophosphate, led to decreased Cdk5 activity, as well as GSK3β activity.18 Furthermore, it is reported that S-nitrosylation can inhibit calpain activity, an enzyme responsible for cleavage of p35 to p25.19,20 Won et al21 reported that S-nitrosoglutathione treatment of purified calpain protein inhibited its activity. Consistent with our findings, Annamalai et al22 demonstrated that treatment of APP/PS1 mice with S-nitrosoglutathione led to decreased calpain-mediated cleavage of p35 and decreased Cdk5 activity. Taken together, these data suggest that loss of endothelial NO may alter the activity of calpain, thus, leading to increased Cdk5 activity. Although it is reported that several other stimuli, such as oxidative stress, Aβ exposure, and neuroinflammation,23–25 can all lead to activation of calpain, we did not observe any changes in Aβ levels, the antioxidant enzyme system or superoxide anion production, or inflammatory markers between APP/PS1 and APP/PS1/eNOS−/− mice, making these unlikely sources of calpain activation.
The results in this study provide novel findings regarding yet another role for vascular dysfunction in AD-related pathology. We report that loss of endothelial NO, a primary feature of endothelial dysfunction, leads to increased p25 production. Importantly, increased p25, increased p25/35 ratio, is an established mechanism responsible for elevated Cdk5 activity,13,14 and indeed, Cdk5 enzyme activity was significantly higher in APP/PS1/eNOS−/− mice. Furthermore, loss of endothelial NO in APP/PS1, an AD mouse model, led to statistically higher phosphorylation of tau. Our data, thus, provide significant evidence supporting the role of endothelial NO in the preservation of brain health. These findings also have significant implications for the development of therapies designed to treat and prevent mild cognitive impairment and AD.
Sources of Funding
This work was supported by National Institutes of Health grants HL-111062 and HL-131515, the Mayo Alzheimer’s Disease Research Center (Z.S. Katusic), American Heart Association (AHA) Postdoctoral Fellowship (AHA no 12POST8550003; S.A. Austin) and AHA Scientist Development Award (AHA no 14SDG20410063; S.A. Austin), and the Mayo Foundation.
In August 2016, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.98 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.116.309686/-/DC1.
- Nonstandard Abbreviations and Acronyms
- beta amyloid
- Alzheimer’s disease
- amyloid precursor protein
- cyclin-dependent kinase 5
- endothelial nitric oxide synthase
- eNOS knockout/deficient
- glycogen synthase kinase 3β
- inducible nitric oxide synthase
- neuronal nitric oxide synthase
- nitric oxide
- phosphorylated tau
- Received August 1, 2016.
- Revision received August 26, 2016.
- Accepted September 6, 2016.
- © 2016 American Heart Association, Inc.
- Faraci FM
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Novelty and Significance
What Is Known?
Cardiovascular risk factors are associated with a higher incidence of Alzheimer’s disease (AD).
A common feature of cardiovascular risk factors is endothelial dysfunction, specifically, a loss of bioavailable endothelial nitric oxide (NO).
Hyperphosphorylated tau is the primary component of neurofibrillary tangles, one of the hallmark pathologies of AD.
What New Information Does This Article Contribute?
Endothelial nitric oxide synthase (eNOS)–deficient (eNOS−/−) mice display increased levels of p25, an aberrant activator of cyclin-dependent kinase 5, which is one of the primary kinases responsible for tau hyperphosphorylation, and a statistically higher p25/p35 ratio.
We generated a novel AD mouse model that also lacks eNOS, APP/PS1/eNOS−/− mice.
APP/PS1/eNOS−/− mice displayed increased p25, p25/p35, cyclin-dependent kinase 5 enzyme activity, and hyperphosphorylated tau.
Cardiovascular risk factors are associated with a higher incidence of AD. Loss of bioavailable endothelial NO is a common feature of these risk factors. The results of this study provide evidence that loss of endothelial NO leads to increased tau phosphorylation, a major mechanism responsible for the development of neurodegeneration in AD. Our findings identify a new role for endothelial NO in the pathogenesis of AD. Presented results support the concept that preservation of the endothelial NO pathway is a therapeutic target in the prevention and treatment of AD.