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Figure 1.
Loss of Perivascular Aquaporin-4 (AQP4) Localization Is Associated With Alzheimer Disease (AD) Pathology
Loss of Perivascular Aquaporin-4 (AQP4) Localization Is Associated With Alzheimer Disease (AD) Pathology

A, Perivascular AQP4 localization was evaluated by immunofluorescence. In the young cortex, AQP4 was distributed uniformly from the cortical surface to the subcortical white matter boundary. B, High-power confocal imaging showing localization of AQP4 in perivascular astrocytic endfeet (arrowhead). C, In the aged cortex (aged 60-85 years), AQP4 was intensely expressed in a sparse population of cortical astrocytes, while wider AQP4 expression was reduced. D, High-magnification image showing altered AQP4 expression and localization in the aged cortex. E, Quantification of global AQP4-immunoreactivity (IR) showing increased AQP4 expression in the AD cortex compared with the cognitively intact young or aged cortex. F-G, Regression analysis shows that when controlling for the effects of age, increasing AQP4-IR is significantly (P = .01, R2 = 0.30; Huber-White–corrected ordinary least squares) associated with amyloid-β plaque density (F), as is association with Braak stage (P = .029, R2 = 0.20) (G). H, Quantification of perivascular AQP4 localization (ratio of AQP4-IR in perivascular domains to global AQP4-IR) showing reduced perivascular localization in individuals with AD. I-J, Regression analysis shows that when controlling for the effects of age, reduced perivascular AQP4 is significantly associated with both amyloid-β plaque density (age-adjusted P < .001, R2 = 0.14) (I) and Braak stage (age-adjusted P = .004, R2 = 0.12; Huber-White–corrected ordinary least squares) (J). DAPl indicates L-diaminopimelate aminotransferase; GFAP indicates glial fibrillary acidic protein; and PV indicates parenchyma volume.

aP = .03.

bP = .004; Welch-corrected 1-way analysis of variance.

cP = .01.

dP = .003; Welch-corrected 1-way analysis of variance.

Figure 2.
Loss of Perivascular Aquaporin-4 (AQP4) Localization Predicts Alzheimer Disease (AD) Status
Loss of Perivascular Aquaporin-4 (AQP4) Localization Predicts Alzheimer Disease (AD) Status

A, Regression analysis shows that among all individuals, increasing age is significantly associated with increasing AQP4-immunoreactivty (IR) (P = .001, ρ = 0.45, Spearman’s correlation). B, Perivascular AQP4 localization is not significantly associated with age across all individuals; however, among individuals with AD, increasing age was associated with decreasing perivascular localization (P = .02, ρ = −0.57, Spearman’s correlation). Perivascular AQP4 localization among cognitively intact eldest individuals older than 85 years of age (oval) diverged markedly from eldest individuals with AD. Results of White-Huber–corrected logistic regression analysis indicated that AQP4-IR (C) and perivascular AQP4 localization (D) are significant predictors of AD status (P = .01, age-adjusted P = .29; P < .001, age-adjusted P  = .02; respectively). Significance of AQP4 as a covariate predicting cognitive status shown in the figure. PV indicates parenchyma volume.

Figure 3.
Altered Aquaporin-4 (AQP4) Expression in Deep Layers of the Aging Cortex
Altered Aquaporin-4 (AQP4) Expression in Deep Layers of the Aging Cortex

Wide field confocal montages show AQP4 immunoreactivity extending from the cortical surface (dashed line) at the crests of gyri (A-C) and the depths of sulci (D-F). Compared with the uniform AQP4 expression throughout different cortical layers in young cortex (A and D), AQP4 labeling in cortical layers II to IV of cognitively intact aged (60-85 years) individuals (B and E) was reduced, with the appearance of intensely immunoreactive astrocytes throughout these layers. These differences were not observed among cognitively intact eldest individuals (older than 85 years) (C and F).

Figure 4.
Amyloid-β (Aβ) Plaques Are Associated With Aquaporin-4 (AQP4) Mislocalization
Amyloid-β (Aβ) Plaques Are Associated With Aquaporin-4 (AQP4) Mislocalization

Large wide-field confocal montages extending from the crests of sulci to the base of gyri were generated. A, AQP4-immunoreactivity (IR) in the young brain is uniform through the depth of the cortex to the gray matter−white matter (WM) boundary. Insets depict a region of the montage at higher magnification. B, In the cognitively intact aged cortex (60-85 years), AQP4-IR is reduced in deeper cortical layers (blue arrowhead), while a band of intense IR runs along the gray matter−WM boundary (white arrowhead) within the cortex at the crest of the gyrus but not in the cortex in the depth of the sulcus. C, In individuals with Alzheimer disease, AQP4-IR in deeper cortical layers is increased compared with aged individuals, owing to the presence of AQP4-IR associated with Aβ plaques (dashed circles in inset). D, High-power confocal microscopy shows that Aβ plaques are surrounded by foci of AQP4-IR. E, These foci are features of Aβ plaque-associated reactive astrocytes (glial fibrillary acidic protein).

Table.  
Cohort Demographic, Cognitive, and Pathological Features
Cohort Demographic, Cognitive, and Pathological Features
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Mawuenyega  KG, Sigurdson  W, Ovod  V,  et al.  Decreased clearance of CNS beta-amyloid in Alzheimer’s disease.  Science. 2010;330(6012):1774.PubMedGoogle ScholarCrossref
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Patterson  BW, Elbert  DL, Mawuenyega  KG,  et al.  Age and amyloid effects on human central nervous system amyloid-beta kinetics.  Ann Neurol. 2015;78(3):439-453.PubMedGoogle ScholarCrossref
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Tarasoff-Conway  JM, Carare  RO, Osorio  RS,  et al.  Clearance systems in the brain-implications for Alzheimer disease.  Nat Rev Neurol. 2015;11(8):457-470.PubMedGoogle ScholarCrossref
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Zlokovic  BV, Deane  R, Sagare  AP, Bell  RD, Winkler  EA.  Low-density lipoprotein receptor-related protein-1: a serial clearance homeostatic mechanism controlling Alzheimer’s amyloid β-peptide elimination from the brain.  J Neurochem. 2010;115(5):1077-1089.PubMedGoogle ScholarCrossref
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Simon  MJ, Iliff  JJ.  Regulation of cerebrospinal fluid (CSF) flow in neurodegenerative, neurovascular and neuroinflammatory disease.  Biochim Biophys Acta. 2016;1862(3):442-451.PubMedGoogle ScholarCrossref
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Yang  J, Lunde  LK, Nuntagij  P,  et al.  Loss of astrocyte polarization in the tg-ArcSwe mouse model of Alzheimer’s disease.  J Alzheimers Dis. 2011;27(4):711-722.PubMedGoogle Scholar
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Original Investigation
January 2017

Association of Perivascular Localization of Aquaporin-4 With Cognition and Alzheimer Disease in Aging Brains

Author Affiliations
  • 1Department of Anesthesiology and Perioperative Medicine, Oregon Health and Science University, Portland
  • 2Department of Neurology, Oregon Health and Science University, Portland
  • 3Department of Pathology, Oregon Health and Science University, Portland
  • 4Knight Cardiovascular Research Institute, Oregon Health and Science University, Portland
 

Copyright 2016 American Medical Association. All Rights Reserved.

JAMA Neurol. 2017;74(1):91-99. doi:10.1001/jamaneurol.2016.4370
Key Points

Question  Is the expression or localization of the astroglial water channel aquaporin-4 altered in patients with advanced age or with Alzheimer disease?

Findings  In this postmortem analysis, aquaporin-4 protein expression and localization in the cortex of a series of aged cognitively intact individuals and patients with Alzheimer disease revealed statistically significant associations between aquaporin-4 expression and aging. Loss of aquaporin-4 protein localization to perivascular astrocytic endfeet was strongly associated with Alzheimer disease status and pathology.

Meaning  Increasing aquaporin-4 expression is a feature of the aging human brain, and mislocalization of aquaporin-4 is related to the development of Alzheimer disease pathology.

Abstract

Importance  Cognitive impairment and dementia, including Alzheimer disease (AD), are common within the aging population, yet the factors that render the aging brain vulnerable to these processes are unknown. Perivascular localization of aquaporin-4 (AQP4) facilitates the clearance of interstitial solutes, including amyloid-β, through the brainwide network of perivascular pathways termed the glymphatic system, which may be compromised in the aging brain.

Objectives  To determine whether alterations in AQP4 expression or loss of perivascular AQP4 localization are features of the aging human brain and to define their association with AD pathology.

Design, Setting, and Participants  Expression of AQP4 was analyzed in postmortem frontal cortex of cognitively healthy and histopathologically confirmed individuals with AD by Western blot or immunofluorescence for AQP4, amyloid-β 1-42, and glial fibrillary acidic protein. Postmortem tissue and clinical data were provided by the Oregon Health and Science University Layton Aging and Alzheimer Disease Center and Oregon Brain Bank. Postmortem tissue from 79 individuals was evaluated, including cognitively intact “young” individuals aged younger than 60 years (range, 33-57 years), cognitively intact “aged” individuals aged older than 60 years (range, 61-96 years) with no known neurological disease, and individuals older than 60 years (range, 61-105 years) of age with a clinical history of AD confirmed by histopathological evaluation. Forty-eight patient samples (10 young, 20 aged, and 18 with AD) underwent histological analysis. Sixty patient samples underwent Western blot analysis (15 young, 24 aged, and 21 with AD).

Main Outcomes and Measures  Expression of AQP4 protein, AQP4 immunoreactivity, and perivascular AQP4 localization in the frontal cortex were evaluated.

Results  Expression of AQP4 was associated with advancing age among all individuals (R2 = 0.17; P = .003). Perivascular AQP4 localization was significantly associated with AD status independent of age (OR, 11.7 per 10% increase in localization; z = −2.89; P = .004) and was preserved among eldest individuals older than 85 years of age who remained cognitively intact. When controlling for age, loss of perivascular AQP4 localization was associated with increased amyloid-β burden (R2 = 0.15; P = .003) and increasing Braak stage (R2 = 0.14; P = .006).

Conclusions and Relevance  In this study, altered AQP4 expression was associated with aging brains. Loss of perivascular AQP4 localization may be a factor that renders the aging brain vulnerable to the misaggregation of proteins, such as amyloid-β, in neurodegenerative conditions such as AD.

Introduction

Advancing age is the strongest risk factor for the development of neurodegenerative disorders such as Alzheimer disease (AD).1 A common feature of these diseases is the age-associated accumulation of protein aggregates, including senile plaques comprised of amyloid-β (Aβ) 1-42 in AD, α-synuclein within Lewy bodies in Parkinson disease and Lewy body dementia, and hyperphosphorylated tau within neurofibrillary tangles in AD and chronic traumatic encephalopathy,2 yet the upstream changes that render the aging brain vulnerable to the misaggregation of proteins remain unknown.

Since 2013, we have defined a brain wide perivascular pathway, termed the glymphatic system, that facilitates the recirculation of cerebrospinal fluid (CSF) through the brain parenchyma and supports the clearance of interstitial solutes including Aβ and tau.3-7 Perivascular exchange of CSF and interstitial fluid is dependent on the astroglial water channel aquaporin-4 (AQP4), which is localized to perivascular astrocytic endfeet that ensheathe the cerebral vasculature.7 We demonstrated that perivascular CSF recirculation and Aβ clearance are impaired in the aging mouse brain, impairment that was associated with the loss of perivascular AQP4 localization. Prior studies in postmortem human tissue show that AQP4 is up regulated8,9 and that localization of AQP4 to the cerebral vasculature is disrupted in the AD cortex.10 This suggests that age-related mislocalization of AQP4 may slow glymphatic function and promote protein aggregation and neurodegeneration. In this study, we assessed AQP4 expression and perivascular localization in brain samples from a human case series including individuals of different ages and with different cognitive and neuropathological AD profiles.

Methods
Human Participants and Tissues

This study consisted of 79 patients from the Oregon Health and Science University Layton Aging and Alzheimer Disease Center and associated postmortem tissue repository, the Oregon Brain Bank. The Oregon Health and Science University institutional review board approved the study. Volunteers signed written informed consent. All aged participants were community-dwelling individuals with no known neurological disease (control individuals) or with a clinical history of AD as established by neurologic evaluation in the Layton Aging and Alzheimer Disease Center as previously described11 and in accordance with established consensus criteria.12 Brain autopsy was performed on all participants after consent was obtained from the next of kin and in accordance with Oregon Health and Science University guidelines.

Neuropathological Evaluation and Evaluation of AQP4 Expression and Localization

Brains in the Oregon Brain Bank underwent neuropathological evaluation for Aβ plaque density, neurofibrillary pathology, and vascular pathology. Expression of AQP4 and localization was evaluated by Western blot and immunofluorescence in frozen and fixed frontal cortical tissue, respectively. Detailed descriptions of these methods are provided in the eMethods in the Supplement.

Statistical Evaluation

A detailed description of statistical approaches is provided in the eMethods in the Supplement. Multiple comparisons corrected P values, means, and standard errors of the mean are indicated in the article. All statistical analyses were carried out using Prism 6 (GraphPad) and R 3.2.1 (R Foundation).13

Results
Demographic and Histopathological Characteristics

Expression of AQP4 and localization were evaluated from a cohort of 79 individuals including “young” control individuals younger than 60 years of age (range, 33-57 years) without a history of neurological disease, cognitively intact (Clinical Dementia Rating14 = 0) “aged” individuals older than 60 years of age (range, 61-96 years), and individuals with histopathologically confirmed AD older than 60 years of age (range, 61-105 years) . Among these, 48 (10 young, 20 aged, and 18 with AD) underwent histological analysis. Sixty underwent Western blot analysis (15 young, 24 aged, and 21 with AD) of AQP4. Twenty-nine individuals (11 aged and 18 with AD) underwent both histological and Western blot analysis. The Table shows sex balance and median and interquartile ranges of age, years of schooling, Aβ plaque density, and Braak stage for all participants. Most recent Mini-Mental State Examination and Clinical Dementia Ratings are provided for a subset of aged individuals and individuals with AD who underwent cognitive testing at the Layton Aging and Alzheimer Disease Center. No significant differences between groups were observed in sex balance (χ2 = 0.51; P = .78), years of schooling (t = 0.39; P = .70), or postmortem interval (F = 2.37; P = .10). Linear regression showed no association between postmortem interval and global AQP4-immunoreactivity (IR) or perivascular AQP4 localization assessed by immunofluorescence (eFigure 1A in the Supplement, R2 = 0.01; P = .44; eFigure 1B in the Supplement, R2 = 0.02; P = .29; Pearson correlation) or postmortem interval and AQP4 expression evaluated by Western blot (eFigure 1C in the Supplement, R2 = 0.01; P = .39; Pearson correlation). The mean (SE) interval between age at most recent cognitive testing and death did not differ between aged individuals (0.9 [0.2] years) and individuals with AD (1.9 [0.5] years) (P = .13, independent samples t test). Histopathological examination revealed greater Aβ plaque density in AD compared with young or aged individuals (AD vs young: Dunn’s z = 6.64; adjusted P <.001; AD vs old: Dunn’s z = 5.27; adjusted P <.001; Kruskal-Wallis test). Individuals with AD had higher Braak scores compared with aged or young individuals (AD vs young: Dunn’s z = 7.54; adjusted P <.001; AD vs old: Dunn’s z = 4.58; adjusted P <.001; Kruskal-Wallis test); however, aged individuals exhibited significantly higher Braak scores than young individuals (Dunn’s z = 3.59; adjusted P = .005; Kruskal-Wallis test).

General cerebrovascular pathology (VIS) was rated on the degree of arteriolosclerosis, perivascular hemosiderin leakage, perivascular space dilation and myelin loss from the frontal cortex, and cortical amyloid angiopathy in the occipital cortex according to criteria published be Deramecourt et al.15 Median and interquartile ranges for VIS and subscores are shown in the eTable in the Supplement. As shown in eFigure 2A in the Supplement, VIS was significantly greater in the aged individuals and individuals with AD compared with young individuals (young vs old: Dunn’s z = −2.24; adjusted P = .04; young vs AD: Dunn’s z = −3.99; adjusted P <.001, respectively; Kruskal-Wallis test). Evaluation of the relationship between age and VIS indicated a significant linear association (eFigure 2B in the Supplement; R2 = 0.36; P < .001, Pearson correlation).

Evaluation of Perivascular AQP4 Localization by Immunofluorescence

When AQP4 expression and localization were evaluated by immunofluorescence double-labeling, we observed that in the young cortex, AQP4 expression was uniform through the cortical layers spanning from the glia limitans to the subcortical white matter (Figure 1A). Although expressed in fine processes throughout the neuropil, AQP4-IR was greatest in perivascular endfeet surrounding the cerebral vasculature (Figure 1B, arrowhead). Expression and localization of AQP4 were dramatically altered in the aged brain. While intense AQP4 expression was maintained near the cortical surface, AQP4 expression became discontinuous below cortical layer II (Figure 1C) where AQP4-IR remained intense within a sparse subpopulation of astrocytes scattered throughout the deeper cortical layers (Figure 1D). When compared between groups, the mean (SD) AQP4-IR was significantly increased among individuals with AD (31.57 [2.84] arbitrary units) compared with young individuals (20.21 [0.99] arbitrary units; P  = .004; Welch-corrected 1-way ANOVA) or aged individuals (23.78 [1.31] arbitrary units; adjusted P = .029; Welch-corrected 1-way analysis of variance [ANOVA]) (Figure 1E). Multiple linear regression analysis showed that when correcting for the influence of age, increasing AQP4-IR was significantly associated with increasing Aβ plaque density (Figure 1F; unadjusted P = .006; R2 = 0.21; adjusted for age P = .01; R2 = 0.30; Huber-White–corrected ordinary least squares [OLS]). In addition, increasing AQP4-IR also tended to be associated with increasing Braak stage, a relationship persisting even when adjusting for the effects of age (Figure 1G; unadjusted P = .002; R2 = 0.15; adjusted for age P = .03; R2 = 0.20; Huber-White–corrected OLS).

In parallel with increasing global AQP4-IR, mean (SE) perivascular AQP4 localization was reduced in individuals with AD (1.095 [0.007]) compared with young individuals (1.160 [0.019]; adjusted P = 0.01; Welch-corrected 1-way ANOVA) or aged individuals (1.161 [0.016]; P  = .003; Welch-corrected 1-way ANOVA; Figure 1H). Regression analysis showed that when correcting for the influence of age, reduced perivascular AQP4 localization was associated with increasing Aβ plaque density (Figure 1I; unadjusted P < .001; R2 = 0.15; adjusted for age P < .001; R2= 0.14; Huber-White–corrected OLS) and increasing Braak stage (Figure 1J; unadjusted P = .002; R2 = 0.14; adjusted for age P = .004; R2 = 0.12; Huber-White–corrected OLS).

We next evaluated whether altered AQP4 expression or localization are general features of the aging brain. Regression analysis showed a significant association between increasing age and increasing AQP4-IR (Figure 2A; P = .001; ρ = 0.45, Spearman’s correlation), while no significant association was evident between age and alterations in perivascular AQP4 localization (Figure 2B; P = .15; ρ = −0.21, Spearman’s correlation). Strikingly, although perivascular AQP4 localization values were similar between aged individuals and individuals with AD between 60 and 85 years of age, there was a marked divergence between values among cognitively intact individuals and individuals with AD older than 85 years of age (referred to as “eldest” individuals; Figure 2B, oval). Among individuals with AD, increasing age was significantly associated with reduced perivascular AQP4 localization (P = .02; ρ= −0.57, Spearman’s correlation).

The results of Huber-White–corrected logistic regression analysis modeling AD status (cognitively intact vs AD) with age and perivascular AQP4 localization as independent variables indicated that age was a significant predictor (adjusted P = .02; b = 0.037; z = 2.36) and decreasing perivascular AQP4 localization was significantly associated with AD status (P < .001; b = −24.6; z = −3.58; Figure 2D). In contrast, increasing AQP4-IR was significantly associated with AD status (P = .01; b = 0.13; z = 2.45) in our cohort, while age showed no association (P = .29; b = 0.017; z = 1.07; Figure 2C).

Although both AQP4-IR and perivascular AQP4 localization were associated with vascular pathology (P = .05; R2 = 0.07 and P = .05; R2 = 0.07, respectively), this association disappeared when the effects of age were controlled for (P = .83; R2 = 0.17 and P = .17; R2 = 0.05, respectively; Huber-White–corrected OLS). These results suggest that increasing vascular pathology and increasing global AQP4-IR are general features of the aging brain with the preservation of perivascular AQP4 localization a significant predictor of preserved cognitive function, even in individuals who remain cognitively intact late into life.

Regional Patterns of AQP4 Localization

When evaluating the spatial pattern of AQP4 expression in the cortex, we observed that compared with the uniform distribution of AQP4 through layers II to VI of the young cortex (Figure 3A and E), AQP4 localization was dramatically altered in the cortex of aged individuals (Figure 3B and F). Interestingly, different layer-specific patterns of AQP4 expression were evident among aged individuals at the crests of gyri vs the depths of sulci. At the crests of gyri of aged individuals, AQP4 expression was lost between layers II and IV, while a band of AQP4-IR remained in layers V to VI at the gray matter–white matter boundary (Figure 3A-B). At the depths of sulci of aged individuals, an intense band of AQP4-IR remained in cortical layer II, while AQP4 expression within deeper layers was reduced (Figure 3D-E). Figure 4A-B shows representative multifield montages depicting the effects of age on AQP4 expression along the length of whole gyri from young and aged individuals. Such regional patterns of AQP4-IR were observed in whole-gyrus images from 6 to 7 individuals per group.

Among cognitively intact eldest individuals (older than 85 years), AQP4 expression in the middle layers of the cortex was maintained (Figure 3C). Within the depths of the sulci, the intense band of AQP4 immunofluorescence observed superficially in layer II of aged individuals was absent in the cognitively intact eldest individuals (Figure 3F). Thus, the regional patterns of altered AQP4 expression and localization that are features of the aged brain are absent among the cognitively intact eldest individuals.

Evaluation of AQP4 Expression by Western Blot

Expression of AQP4 in the frontal cortex was evaluated by Western blot. As shown in eFigure 3A in the Supplement, no significant differences in total AQP4 expression were observed between young individuals, aged individuals, or individuals with AD (P = .23, 1-way ANOVA). The discrepancy between these findings and the age-related differences in AQP4-IR (Figure 2A-B) may stem from the fact that our immunofluorescence analysis specifically omitted analysis of cortical layer I and the glial limitans, which exhibit pronounced AQP4-IR in the aged and AD cortex (Figure 3 and Figure 4). Aquaporin-4 is expressed in the human brain as 2 alternative isoforms, AQP4-M1 and AQP4-M23, the relative expression of which has been proposed to drive perivascular localization.16 When we evaluated expression of AQP4-M1 and AQP4-M23 separately, we did not observe any specific differences in expression of either isoform between groups. However, when the ratio of AQP4-M1 to AQP4-M23 expression was evaluated, we observed that the relative expression of AQP4-M1 (the AQP4-M1 to AQP4-M23 ratio) significantly declined in aged cortex (mean [SD], 0.10 [0.09]; adjusted P = 0.05, 1-way ANOVA) and AD cortex (mean [SD], 0.13 [0.02]; adjusted P  = .01, 1-way ANOVA) (eFigure 3B in the Supplement).

Differences in AQP4 Localization Associated With Senile Plaques

Participants with AD exhibited increased AQP4-IR in cortex (Figure 1E) in addition to reduced perivascular AQP4 localization (Figure 1H). Wide field immunofluorescence imaging showed that in contrast to the pattern of AQP4-IR in the aged cortex (Figure 4B), AQP4-IR in the AD cortex remained high through all cortical depths (Figure 4C). Confocal microscopy showed that this was attributable to intense AQP4-IR associated within glial fibrillary acidic protein–positive reactive astrocytes surrounding cortical senile plaques (Figure 4C-E).

Discussion

We have evaluated the association between differences in AQP4 expression and localization and Alzheimer pathology across a wide range of aged human samples, including those from cognitively intact individuals and individuals with AD. We discovered that AQP4 expression increases in the aging brain and that loss of perivascular AQP4 localization is associated with worsening AD pathology. We observed that AQP4 expression increased with age across all individuals. Regional patterns of AQP4 expression and localization were dramatically altered in the aged cortex (60-85 years of age) yet were preserved among cognitively intact individuals older than 85 years. Perivascular AQP4 localization was reduced among individuals with AD and reduced localization was strongly associated with increased neurofibrillary and Aβ pathology, even when controlling for the effect of age on AD pathology. In line with these findings, logistic regression analysis revealed that perivascular AQP4 localization was strongly associated with AD status independent of age.

Analysis of Aβ kinetics within the CSF of clinical participants showed that Aβ clearance is impaired among participants with late-onset AD, suggesting that slowing Aβ clearance rather than increasing Aβ production underpins Aβ plaque deposition.17 A similar analysis in 2015 demonstrated that Aβ clearance slows with increasing age, including in individuals prior to the deposition of Aβ plaques as detected by Pittsburgh compound B positron emission tomographic scans.18 The changes in the aging human brain that underlie the slowing of Aβ clearance have not yet been defined.

Amyloid β clearance from the brain appears to involve several different processes, including uptake and degradation by brain cells such as microglia; efflux across the blood-brain barrier by transporters, such as low-density lipoprotein receptor-related protein-1; and clearance by bulk flow along perivascular pathways.19-21 Because each takes place within the perivascular compartment, it appears likely that processes such as perivascular bulk clearance and blood-brain barrier efflux interact to support Aβ clearance from the brain.4,22 The precise nature and extent of this interaction, and how these relationships may change as the brain ages, remain unknown and are an important subjects for future research.

In the rodent brain, perivascular CSF–interstitial fluid exchange and interstitial Aβ clearance are markedly reduced in the absence of the astroglial water channel AQP4,7 while in a transgenic mouse model of AD, deletion of the Aqp4 gene increased Aβ plaque formation and worsened cognitive impairment.23 In the aging mouse cortex, impairment of perivascular CSF–interstitial fluid exchange and interstitial Aβ clearance was associated with reduced perivascular AQP4 localization.4 These experimental findings suggest that loss of perivascular localization in the aging rodent brain slows Aβ clearance and promotes Aβ deposition. Our findings here demonstrate that similar age-related changes to astroglial AQP4 localization are occurring in the human as in the rodent brain, and suggest that AQP4 mislocalization may be 1 of the factors underlying the slowing of Aβ clearance and promoting Aβ plaque formation in the aging human brain.

Our findings are consistent with prior studies carried out in human postmortem cortical tissue showing that while AQP4 expression evaluated by Western blot does not significantly change in the AD cortex,24 AQP4 immunoreactivity is substantially increased in the AD cortex.8,9 Additionally, Wilcock et al10 reported that AQP4 localization to the microvasculature is reduced among individuals with AD. In prior studies, disruption of AQP4 expression and localization have been reported to be associated with both Aβ plaques and vascular Aβ deposits,9,24,25 suggesting that changes in AQP4 expression and localization are features of the aging brain. This study extends these previous findings to include what is, to our knowledge, the largest clinical case series to date to assess the relationship between changes in AQP4 expression and localization, aging, and AD pathology. Importantly, to our knowledge, this is the first study to explicitly quantify perivascular AQP4 localization and to evaluate whether changes in this feature, which is believed to underlie perivascular Aβ clearance along the glymphatic pathway,4,5,7,26 are significantly associated with AD status or AD pathology as measured by Braak stage or Aβ plaque density.

We did not observe evidence of Aβ plaque–associated differences in AQP4 expression or localization in cognitively intact young or aged individuals. The relationship between increasing AQP4-IR and advancing age and between differences in AQP4 localization and increasing Aβ plaque deposition and neurofibrillary pathology observed even among cognitively intact individuals argues that the loss of perivascular AQP4 localization is not the result of advancing AD pathology. However, we cannot rule out the possibility that subclinical Aβ plaque burden or neurofibrillary pathology, which is present both among cognitively intact individuals and those with mild cognitive impairment,27 contributed to differences in AQP4 expression or localization in this study. However, the absence of a significant association between cerebrovascular pathology and differences in AQP4 expression or localization when controlling for effects of age suggest that these effects are likely not driven by vascular injury.

Age-related cognitive decline in the absence of frank dementia is commonly viewed as an inherent concomitant of normal aging.28 Yet among the nondemented aging population, an important subset harbor a high prevalence of the neuropathologies that are commonly associated with frank dementia.29 The substrate of this resistance to age-related pathologies and cognitive decline is not well understood but has been attributed to aspects of greater brain reserve or resilience.30 In this study, we observed loss of perivascular AQP4 localization with increasing age among individuals with AD, while perivascular AQP4 localization was preserved in individuals older than 85 years of age. Similarly, regional differences in AQP4 expression and localization observed in aging individuals 60 to 85 years of age were generally absent among these cognitively intact individuals older than 85 years of age. Whether preservation of perivascular AQP4 localization throughout the aging process provides protection or a reserve against the development of AD pathology or whether other factors contribute independently to both reduced AD pathology and increased perivascular AQP4 localization cannot be ascertained from this data.

Limitations

In experimental studies in rodents, the role of AQP4 in glymphatic system function has been best characterized in the cortex, while the spread of amyloid and neurofibrillary pathology throughout the frontal cortex is believed to play a key role in the development of cognitive decline in the progression of AD. Therefore, this initial study was limited to analysis of the expression and localization of AQP4 in the frontal cortex. Whether these age-associated effects also occur in other brain areas relevant to cognitive decline, such as the hippocampus and entorhinal cortex, is an important consideration. Because this study is reliant on postmortem tissue, it is impossible from these data to verify whether AQP4 mislocalization is itself the causative feature rendering the aging brain susceptible to neurodegenerative processes or merely a parallel consequence of an independent upstream pathological process. Additionally, whether preservation of perivascular AQP4 localization throughout the aging process provides protection or a reserve against development of AD pathology or whether other factors contribute independently to both reduced AD pathology and increased perivascular AQP4 localization cannot be ascertained from these data. Further inquiry into these questions is warranted.

Conclusions

These histological findings are consistent with the notion that loss of perivascular AQP4 localization is one of the factors that renders the aging brain vulnerable to Aβ aggregation and neurodegeneration. If substantiated, this would potentially place age-related changes in perivascular AQP4 localization mechanistically upstream of processes of protein misaggregation and would suggest that targeting AQP4 localization may provide a therapeutic approach to intervene in the AD pathogenesis upstream of Aβ plaque or neurofibrillary tangle formation.

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Article Information

Corresponding Author: Jeffrey J. Iliff, PhD, Department of Anaesthesiology and Perioperative Medicine, Oregon Health and Science University, 3181 SW Sam Jackson Park Rd, Mail Code L459, Portland, OR 97239 (iliffj@ohsu.edu).

Accepted for Publication: September 9, 2016.

Published Online: November 28, 2016. doi:10.1001/jamaneurol.2016.4370

Author Contributions: Dr Iliff had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Concept and design: Zeppenfeld, Haswell, Iliff.

Acquisition, analysis, or interpretation of data: All Authors.

Drafting of the manuscript: Zeppenfeld, Haswell, D'Abreo, Murchison, Grafe, Iliff.

Critical revision of the manuscript for important intellectual content: Zeppenfeld, Simon, Murchison, Quinn, Grafe, Woltjer, Kaye, Iliff.

Statistical analysis: D'Abreo, Murchison, Iliff.

Administrative, technical, or material support: Zeppenfeld, Simon, Grafe, Woltjer, Kaye, Iliff.

Study supervision: Zeppenfeld, Grafe, Iliff.

Conflict of Interest Disclosures: Dr Iliff reports serving as a consultant for Shire and receiving honoraria from Shire Pharmaceuticals and Genentech. No other disclosures are reported.

Funding/Support: This work was supported by funding from the American Heart Association grant 12SDG11820014 (Dr Iliff), the Oregon Partnership for Alzheimer’s Research (Dr Iliff), grants from the Research and Development Office of the Department of Veterans Affairs (Merit Review Grant, Dr Kaye), and grant NS089709 from the National Institutes of Health (Dr Iliff), including Alzheimer’s Disease Center grant AG08017 from the National Institute on Aging that supported the longitudinal follow-up and subsequent brain autopsies providing the human brain samples used in this study.

Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

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