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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, nu-merous 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 character-istic 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 precur-sor 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 hy-perphosphorylated 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 syn-thase 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

Integrative Physiology

© 2016 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.116.309686

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. (Circ Res. 2016;119:1128-1134. DOI: 10.1161/CIRCRESAHA.116.309686.)

Key Words: Alzheimer’s disease ■ Cdk5 ■ cerebrovascular disease ■ endothelial nitric oxide synthase ■ tau

Original received August 1, 2016; revision received August 26, 2016; accepted September 6, 2016. In August 2016, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.98 days.

From the Departments of Anesthesiology and Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, MN.The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.

116.309686/-/DC1.Correspondence to Zvonimir S. Katusic, MD, PhD, Department of Anesthesiology, Mayo Clinic, 200 1st St SW, Rochester, MN 55905. E-mail Katusic.

zvonimir@mayo.edu

Loss of Endothelial Nitric Oxide Synthase Promotes p25 Generation and Tau Phosphorylation in a Murine

Model of Alzheimer’s Disease

Susan A. Austin, Zvonimir S. Katusic

Short Communication

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Austin and Katusic Endothelial NO Loss Promotes Tau Phosphorylation 1129

MethodsAnimalsNos3tm1Unc/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/poly-merase chain reaction, tissue collection, confocal microscopy, Western blotting, ELISA for beta amyloid (Aβ), and statistical analysis, which are as previously described.6,7

ResultsRatio of p25/p35 Is Significantly Higher in the Brains of eNOS−/− MiceWe sought to determine whether chronic loss of endothe-lial 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 com-pared 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 phosphoryla-tion, 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 TissueTo determine whether the increased p25/p35 ratio led to in-creased 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−/− MiceThere 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 com-pared 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 TissueWe examined protein expression of p35, p25, and Cdk5 by Western blot. p25 protein levels and p25/p35 ratio were signifi-cantly 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, ex-pression of p25/p35 (Figure 3) and Cdk5 (Online Figure II) was mainly observed in neuronal tissue as demonstrated by the colo-calization 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 com-pared with wild-type and APP/PS1 mice. We did perform immu-nohistological 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 phos-phorylated GSK3β/GSK3β tended to be higher in the brains of APP/PS1/eNOS−/− mice, they did not reach statistical signifi-cance (Online Figure III; P>0.05).

Nonstandard Abbreviations and Acronyms

Aβ beta amyloid

AD Alzheimer’s disease

APP amyloid precursor protein

Cdk5 cyclin-dependent kinase 5

eNOS endothelial nitric oxide synthase

eNOS−/− eNOS knockout/deficient

GSK3β glycogen synthase kinase 3β

iNOS inducible nitric oxide synthase

nNOS neuronal nitric oxide synthase

NO nitric oxide

pTau phosphorylated tau

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Expression and Amyloidogenic Processing of APP in APP/PS1/eNOS−/− MiceWe 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 in-crease 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 be-tween APP or β-site APP cleaving enzyme 1 protein lev-els 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 ActivationTo determine whether there were compensatory changes in other important endothelial pathways, we examined protein levels of inducible NOS (iNOS), neuronal NOS (nNOS), cy-clooxygenase-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 ani-mals 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).

Figure 1. p25/35 ratio is significantly higher in the brain tissue of eNOS−/− mice as compared with wild-type, although tau phosphorylation is unchanged. A, Brain tissue from 4-month-old wild-type and eNOS−/− mice was analyzed by Western blot. A representative image is shown. Densitometric analysis for (B) p35, (C) p25, (D) p25/p35 ratio, and (E) Cdk is shown. Data are presented as relative mean optical density (OD)±SEM (n=8 animals per background, *P<0.05 from wild-type). F, Brain tissue from 4-month-old wild-type and eNOS−/− mice was analyzed by Western blot. A representative image is shown. Densitometric analysis of (G) pTau, (H) tau, and (I) pTau/tau ratio is shown. Data are presented as relative mean OD±SEM (n=8 animals per background). Cdk5 indicates cyclin-dependent kinase 5; eNOS, endothelial nitric oxide synthase; eNOS−/−, eNOS deficient; and pTau, phosphorylated tau.

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Neuronal and astrocyte markers, NeuN, and glial fibrillary acid-ic 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 ar-ray that examined 40 cytokines (data not shown).

DiscussionOur 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 neu-ronal 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 spe-cific 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 lev-els 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 tis-sue. 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 neu-rons, and we were not able to detect tau protein in cerebral mi-crovessels or in cultured brain microvascular endothelial cells

Figure 2. p25 and p25/35 ratio are significantly increased in the brain tissue of APP/PS1/eNOS−/− mice as compared with both APP/PS1 and wild-type mice. A, Brain tissue from 4- to 5-month-old wild type, APP/PS1, and APP/PS1/eNOS−/− mice was analyzed by Western blot using p35/p25, cyclin-dependent kinase 5 (Cdk5), and actin (loading control) antibodies. A representative image is shown. Densitometric analysis for (B) p35, (C) p25, (D) p25/p35 ratio, and (E) Cdk is shown. Data are presented as relative mean optical density (OD)±SEM (n=12–14 animals per background; *P<0.05 from wild-type and &P<0.05 from APP/PS1). F, Cdk5 was isolated from brain tissue of 4-month-old wild-type, APP/PS1, and APP/PS1/eNOS−/− mice. Cdk5 enzyme activity was then measured using the ADP-Glo kinase activity assay from Promega. Data are presented as mean % of wild-type activity±SEM (n=4 animals per background; ***P<0.001 from wild-type and $P<0.001 from APP/PS1). APP indicates, amyloid precursor protein; APP/PS1, APPswe,PSEN1dE9+/−; eNOS, endothelial nitric oxide synthase; and eNOS−/−, eNOS deficient.

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(unpublished observation). In addition, our immunohistochemi-cal analysis demonstrated colocalization of pTau with the neu-ronal 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 inhibi-tory 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 in-creased 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 docu-ment 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 monophos-phate, 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 puri-fied calpain protein inhibited its activity. Consistent with our find-ings, Annamalai et al22 demonstrated that treatment of APP/PS1 mice with S-nitrosoglutathione led to decreased calpain-mediat-ed cleavage of p35 and decreased Cdk5 activity. Taken together, these data suggest that loss of endothelial NO may alter the activ-ity 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 activa-tion 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 pathol-ogy. 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 estab-lished mechanism responsible for elevated Cdk5 activity,13,14

Figure 3. p25/p35 immunoreactivity was increased in the cortex of APP/PS1/eNOS−/− mice and colocalized with the neuronal marker, NeuN. Fixed tissue sections from the brains of wild-type, APP/PS1, and APP/PS1/eNOS−/− animals were immunolabeled with anti-p25/p35 (with an anti-rabbit IgG fluorescein isothiocyanate secondary) and anti-NeuN Alexa Fluor 647 conjugated primary 4′,6′-diamiddino-2-phenylindole dilactate (DAPI) to visualize nuclei. Representative images of the cortex are shown. Magnification 40×; bar is representative of 20 μm. APP indicates, amyloid precursor protein; APP/PS1, APPswe,PSEN1dE9+/−; eNOS, endothelial nitric oxide synthase; and eNOS−/−, eNOS deficient.

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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 evi-dence supporting the role of endothelial NO in the preser-vation of brain health. These findings also have significant

Figure 4. Phosphorylated tau (pTau) and pTau/Tau ratio are significantly increased in the brain tissue of APP/PS1/eNOS−/− mice as compared with both APP/PS1 and wild-type mice, and pTau is localized to neuronal tissue. A, Brain tissue from 4- to 5-month-old wild-type, APP/PS1, and APP/PS1/eNOS−/− mice was analyzed by Western blot. A representative image is shown. Densitometric analysis for (B) pTau, (C) tau, and (D) pTau/tau ratio is shown. Data are presented as relative mean optical density (OD)±SEM (n=4–6 animals per background; **P<0.01, ***P<0.001 from wild-type and &P<0.05 from APP/PS1). E, Fixed tissue sections from the brains of wild-type, APP/PS1, and APP/PS1/eNOS−/− animals were immunolabeled with anti-pTau (with an anti-rabbit IgG fluorescein isothiocyanate secondary) and anti-NeuN Alexa Fluor 647 conjugated primary 4′,6′-diamiddino-2-phenylindole dilactate (DAPI) to visualize nuclei. Representative images of the cortex are shown. Magnification 40×; bar is representative of 20 μm. APP indicates, amyloid precursor protein; APP/PS1, APPswe,PSEN1dE9+/−; eNOS, endothelial nitric oxide synthase; and eNOS−/−, eNOS deficient.

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implications for the development of therapies designed to treat and prevent mild cognitive impairment and AD.

Sources of FundingThis 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.

DisclosuresNone.

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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 dys-function, 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 dis-play increased levels of p25, an aberrant activator of cyclin-dependent kinase 5, which is one of the primary kinases responsible for tau hy-perphosphorylation, 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-de-pendent kinase 5 enzyme activity, and hyperphosphorylated tau.

Cardiovascular risk factors are associated with a higher inci-dence 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 phos-phorylation, 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 sup-port the concept that preservation of the endothelial NO pathway is a therapeutic target in the prevention and treatment of AD.

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Susan A. Austin and Zvonimir S. KatusicPhosphorylation in a Murine Model of Alzheimer's Disease

Loss of Endothelial Nitric Oxide Synthase Promotes p25 Generation and Tau

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2016 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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Supplemental Material

Methods Animals Nos3tm1Unc/J (eNOS-/-), stock #002684, and C57BL/6 (wild-type) mice, stock #000664, were purchased from Jackson Laboratory (Bar Harbor, ME). APPswe,PSEN1dE9+/- (APP/PS1) mice on the C57BL/6 background were originally purchased from Jackson laboratory as stock # 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 #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 2-3 litters with viable pups and these resultant litters average 4-5 pups. We wish to point out that APP/PS1/eNOS-/- mice are found in very small numbers in the resulting litters, approaching 1 mouse in every 22-25 mice born. Male mice were sacrificed at 4-5 months of age by a lethal dose of pentobarbital. Genotyping Genotyping was performed using APP, PS1, and eNOS primers according to Jackson Laboratories. Briefly, DNA was isolated from a 2-3 mm piece of mouse tail using the PureLink Genomic DNA mini kit (Invitrogen) following manufacturer’s instructions. APP transgene primer sequences were 5’- AGG ACT GAC CAC TCG ACC AG -3’ and 5’ CGG GGG TCT AGT TCT GCA T -3’. The positive control gene used was TCR alpha with the sequences 5’- CAA ATG TTG CTT GTC TGG TG -3’ and 5’- GTC AGT CGA GTG CAC AGT TT -3’. PS1 transgene primer sequences were 5’- AAT AGA GAA CGG CAG GAG CA -3’ and 5’- GCC ATG AGG GCA CTA ATC AT -3’. I12 was used as the positive control gene and the sequences were 5’- CTA GGC CAC AGA ATT GAA AGA TCT -3’ and 5’- GTA GGT GGA AAT TCT AGC ATC ATC C -3’. The sequence for mutant (the knock out) eNOS was 5’- AAT TCG CCA ATG ACA AGA CG -3’. The wild type eNOS sequence was 5’- AGG GGA ACA AGC CCA GTA GT -3’. And the common reverse primer for eNOS was 5’- CTT GTC CCC TAG GCA CCT CT -3’. Tissue collection Brains were carefully removed and immediately placed in ice cold modified Krebs-Ringer bicarbonate solution containing 118.6 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 25.1 mmol/L NaHCO3, 0.026 mmol/L EDTA, 10.1 mmol/L glucose plus protease inhibitors as previously described[1]. Large cerebral arteries, including basilar and cerebral arteries were carefully removed prior to homogenization. Glucose, Cholesterol, and Triglyceride measurements Blood was collected via right ventricular puncture. Glucose was measured in whole blood using Accu Check (Roche Diagnostics, Indianapolis, IN). Blood was centrifuged (2,000 rpm, 10 mins, 4°C) and stored at -80° until all samples were collected. Total triglyceride and cholesterol levels were measured using the Hitachi 912 chemistry analyzer (Roche Diagnostics).

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Blood pressure Mice were trained for blood pressure measurements. Systolic blood pressure was measured in non-anesthetized mice using the tail cuff method as previously described[2] (Harvard Apparatus Ltd, Kent, England). Confocal Microscopy Mice were killed by an overdose of pentobarbital and perfused with PBS followed by 4% paraformaldehyde. Brains were dissected and fixed in a 4% paraformaldehyde solution. Tissue was embedded in paraffin and longitudinal sections (5 µm) were cut. Tissue was deparaffinized and rehydrated prior to antigen retrieval using sodium citrate heat-induced epitope retrieval. Briefly, sections were heated at 98°C for 20 minutes in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0). Tissue was permeabilized using 0.1% Triton X-100 in 2% fetal bovine serum. Sections were incubated with anti-pTau (Santa Cruz, Dallas, TX), anti-p35/p25 (Cell Signaling, Danvers, MA), or anti-Cdk5 (Millipore, Temecula, CA) antibodies and anti-NeuN (neuronal marker) Alexa Fluor 647 conjugated antibody. Sections were incubated with FITC conjugated secondary antibodies for visualization of pTau, p25/p35, and Cdk antibodies. To examine for neurofibrillary tangles, sections were incubated with anti-neurofibrillary tangle antibodies (Chemicon, and Millipore, Temecula, CA). 4’,6’-diamiddino-2-phenylindole dilactate (DAPI) was used to visualize nuclei. Sections were visualized using a Zeiss LSM 780 laser scanning confocal microscope. Western blotting To perform Western blot analyses, tissue homogenates were lysed in ice cold Triton lysis buffer (10 mmol/L Hepes, 50 mmol/L NaF, 50 mmol/L NaCl, 5 mmol/L EDTA, 5 mmol/L EGTA, 100 μmol/L Na3VO4, 50 mmol/L Na pyrophosphate and 1% Triton X-100) as previously described[1]. Equal protein amounts were resolved by SDS-PAGE and transferred to nitrocellulose membranes for Western blotting. Blots were probed with primary antibodies. BACE1, Cdk5, p35/p25, nNOS, pTau (Ser 396), GSK3β and pGSK3β (Ser 9) were purchased from Cell Signaling (Danvers, MA). Iba-1 was obtained from Abcam, (Cambridge, MA) and CD68 and MHC II were purchased from Santa Cruz Biotechnology (Dallas, TX). APP was obtained from Upstate Cell Signaling (Temecula, CA) and COX-1 was purchased from Invitrogen (Camarillo, CA). COX-2, eNOS, and iNOS were purchased from BD Transduction Laboratories (San Jose, CA). The GFAP antibody was purchased from StemCell Technologies (Vancouver, BC, Canada). Mn SOD, EC SOD, and CuZn SOD were purchased from Enso Life Sciences (Farmingdale, NY). The NeuN antibody was purchased from Millipore (Billerica, MA) and the PGI2S antibody was obtained from Cayman Chemical (Ann Arbor, MI). Catalase and Actin (loading control) were purchased from Sigma-Aldrich (St. Louis, MO). Intracellular superoxide anion Brain tissue, cut into small pieces, was incubated in Kreb’s-Hepes with 50 µmol/L dihydroethidium (Molecular Probes, Eugene, OR) at 37° for 15 minutes. Brain tissue was homogenized in methanol and intracellular superoxide anions were quantified using HPLC-based fluorescence and normalized using mg protein[3].

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Mouse Cytokine Array Brain tissue was collected and homogenized as described above. Brain tissue lysates were analyzed using Mouse Cytokine Array Panel A (# ARY006, R&D Systems, Minneapolis, MN) per manufacturer’s instructions. This array contained nitrocellulose membranes with 40 different cytokine antibodies. IL-1α ELISA Brain tissue levels of IL-1α were measured using a commercially available colorimetric ELISA kit following manufacturer’s instructions (R&D Systems, Minneapolis, MN). Aβ ELISA Circulating plasma levels of Aβ1-40 and Aβ1-42 were measured using a commercially available colorimetric ELISA kit following manufacturer’s instructions (Invitrogen, Camarillo, CA). Cdk5 enzyme activity Cdk5/p35 was precipitated, using anti Cdk-5 (Santa Cruz Biotechnology, Dallas, TX), from wild type, APP/PS1, and APP/PS1/eNOS-/- mouse brain tissue lysate. The Cdk5 kinase assay was then performed in kinase buffer (40 mmol/L Tris [pH 7.5], 20 mmol/L MgCl2, 0.1 mg/mL BSA) with 150 µmol/L ATP and 5 µg Histone H1 protein in a final reaction volume of 25 µL for 30 mins. at room temperature. The ADP-Glo Kinase assay was used according to manufacturer’s instructions (Promega, Madison, WI). The ADP-Glo kinase assay is a luminescent assay that measures the amount of ADP formed from the kinase reaction. Statistical analysis Data are represented as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey-Kramer post hoc comparison or the unpaired Student’s t-test for parametric data. For non-parametric data statistical analysis was performed using Kruskal-Wallis with Dunn’s multiple comparison as the post hoc comparison.

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References

1. Austin SA, Santhanam AV, Katusic ZS (2010) Endothelial nitric oxide modulates expression and processing of amyloid precursor protein. Circulation research 107: 1498-1502.

2. d'Uscio LV, Smith LA, Katusic ZS (2011) Differential effects of eNOS uncoupling on conduit and small arteries in GTP-cyclohydrolase I-deficient hph-1 mice. American journal of physiology Heart and circulatory physiology 301: H2227-2234.

3. Santhanam AV, d'Uscio LV, Smith LA, Katusic ZS (2012) Uncoupling of eNOS causes superoxide anion production and impairs NO signaling in the cerebral microvessels of hph-1 mice. Journal of neurochemistry 122: 1211-1218.

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Supplemental Tables & Figures

Wild type APP/PS1 Tg APP/PS1/eNOS-/-

Body weight (g) 29.73 ± 0.66 30.51 ± 1.00 29.28 ± 0.60 Systolic blood pressure 103.56 ± 2.73 117.95 ± 3.26 137.58 ± 7.28 ****, # Glucose (mg/dL) 218.57 ± 12.71 218.91 ± 14.02 211.38 ± 13.09 Total cholesterol (mg/dL) 67.57 ± 4.16 61.17 ± 3.90 67.86 ± 2.69 HDL cholesterol (mg/dL) 50.71 ± 3.58 46.67 ± 3.25 51.08 ± 2.42 Triglycerides (mg/dL) 74.31 ± 6.78 68.83 ± 4.22 103.62 ± 6.37 **, &

**P<0.01 from wild type; ***P<0.001 from wild type & P<0.05 from APP/PS1; # P<0.01 from APP/PS1

Online Table I. Characteristics of wild type, APP/PS1, and APP/PS1/eNOS-/- mice. Body weight, systolic blood pressure, glucose, total cholesterol, HDL cholesterol, and triglycerides were measured. Data are presented as mean ± SEM (n=6-14 animals per background, **P<0.01, ***P<0.001 from wild type and &P<0.05, #P<0.01 from APP/PS1).

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Online Figure I

Online Figure I. Protein and phosphorylation levels of GSK3β and Akt are unaltered in the brains of eNOS-/- mice. A, Brain tissue from 4 month old wild type and eNOS-/- mice was analyzed by Western blot. A representative image is shown. Densitometric analysis for B, pGSK3β, C, GSK3β, and D, pGSK3β/GSK3β ratio, is shown. E, Brain tissue from 4 month old wild type and eNOS-/- mice was analyzed by Western blot. A representative image is shown. Densitometric analysis for F, pAkt, G, Akt, and H, pAkt/Akt ratio, is shown. Data is presented as relative mean O.D. ± SEM (n=8 animals per background).

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Online Figure II

Online Figure II. Cdk5 immunoreactivity was found primarily in neuronal cells in the cortex. Fixed tissue sections from the brains of wild type, APP/PS1, and APP/PS1/eNOS-/- animals were immunolabeled with anti-Cdk5 (with an anti-rabbit IgG FITC secondary) and anti-NeuN Alexa Fluor 647 conjugated primary. 4’,6’-diamiddino-2-phenylindole dilactate (DAPI) to visualize nuclei. Representative images of the cortex are shown. Magnification 40x; bar is representative of 20 µm.

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Online Figure III

Online Figure III. Protein and phosphorylation levels of GSK3β are unaltered in the brains of APP/PS1/eNOS-/- mice. A, Brain tissue from 4 month old wild type, APP/PS1, and APP/PS1/eNOS-/- mice was analyzed by Western blot. A representative image is shown. Densitometric analysis for B, pGSK3β, C, GSK3β, and D, pGSK3β/GSK3β ratio, is shown. Data is presented as relative mean O.D. ± SEM (n=9-11 animals per background).

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Online Figure IV

Online Figure IV. APP is increased in the brains of APP/PS1 and APP/PS1/eNOS-/- mice while BACE1 is higher in only APP/PS1/eNOS-/- mice as compared to wild type. A, Brain tissue from 4 month old wild type, APP/PS1, and APP/PS1/eNOS-/- mice was analyzed by Western blot. A representative image is shown. Densitometric analysis for B, APP, and C, BACE1 is shown. Data is presented as relative mean O.D. ± SEM (n=8 animals per background, *P<0.05).

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Online Figure V

Online Figure V. iNOS and nNOS are unchanged in APP/PS1/eNOS-/- mice. A, Brain tissue from 4 month old wild type, APP/PS1, and APP/PS1/ eNOS-/- mice was analyzed by Western blot. A representative image is shown. Densitometric analysis for B, eNOS, C, iNOS, and D, nNOS is shown. Data is presented as relative mean O.D. ± SEM (n=10-14 animals per background, ***P<0.001 from wild type, $P<0.001 from APP/PS1).

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Online Figure VI

Online Figure VI. Antioxidant enzyme levels from brain tissue of wild type, APP/PS1, and APP/PS1/eNOS-/- are not different. A, Brain tissue from 4 month old wild type, APP/PS1, and APP/PS/ eNOS-/- mice was analyzed by Western blot. A representative image is shown. Densitometric analysis for B, CuZn SOD, C, catalase, D, EC SOD, and E, Mn SOD is shown. Data is presented as relative mean O.D. ± SEM (n=3-6 animals per background). F, Brain tissue, cut into small pieces, was incubated with 50 µmol/L dihydroethidium at 37° for 15 minutes. Brain tissue was homogenized in methanol and intracellular superoxide anions quantified by HPLC-based fluorescence. Intracellular superoxide anions were normalized to mg protein of each sample (n=6). Data are represented as mean ± SEM.

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Online Figure VII

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Online Figure VII. Several microglial markers are unaltered in brains of APP/PS1/eNOS-/- mice. A, Brain tissue from 4 month old wild type, APP/PS1, and APP/PS1/eNOS-/- mice was analyzed by Western blot. A representative image is shown. Densitometric analysis for B, CD68, C, MHC II, and D, Iba-1 is shown. Data is presented as relative mean O.D. ± SEM (n=8-10 animals per background). E, Levels of IL-1α in brain tissue lysates from 5-6 mice per background were measured via a commercially available ELISA. Data is presented as mean ± SEM.