Plaque-Associated Local Toxicity Increases over the Clinical Course of Alzheimer Disease
2015; Elsevier BV; Volume: 186; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2015.10.010
ISSN1525-2191
AutoresAlberto Serrano‐Pozo, Rebecca A. Betensky, Matthew P. Frosch, Bradley T. Hyman,
Tópico(s)Dementia and Cognitive Impairment Research
ResumoAmyloid (senile) plaques, one of the two pathologic hallmarks of Alzheimer disease (AD), are associated with dystrophic neurites and glial responses, both astrocytic and microglial. Although plaque burden remains relatively stable through the clinical course of AD, whether these features of local plaque toxicity continue to worsen over the course of the disease is unclear. We performed an unbiased plaque-centered quantification of SMI312+ dystrophic neurites, GFAP+ reactive astrocytes, and IBA1+ and CD68+ activated microglia in randomly selected dense-core (Thioflavin-S+) plaques from the temporal neocortex of 40 AD subjects with a symptom duration ranging from 4 to 20 years, and nine nondemented control subjects with dense-core plaques. Dystrophic neurites (Kendall τ = 0.34, P = 0.001), reactive astrocytes (Kendall τ = 0.30, P = 0.003), and CD68+ (Kendall τ = 0.48, P < 0.0001), but not IBA1 microglia (Kendall τ = 0.045, P = 0.655), exhibited a significant positive correlation with symptom duration. When excluding control subjects, only the positive association between CD68+ microglia and symptom duration remained significant (Kendall τ = 0.39, P = 0.0003). The presence of the APOEε4 allele did not affect these results. We conclude that plaques exert an increasing toxicity in the surrounding neuropil over the clinical course of AD, thereby potentially contributing to cognitive decline. Amyloid (senile) plaques, one of the two pathologic hallmarks of Alzheimer disease (AD), are associated with dystrophic neurites and glial responses, both astrocytic and microglial. Although plaque burden remains relatively stable through the clinical course of AD, whether these features of local plaque toxicity continue to worsen over the course of the disease is unclear. We performed an unbiased plaque-centered quantification of SMI312+ dystrophic neurites, GFAP+ reactive astrocytes, and IBA1+ and CD68+ activated microglia in randomly selected dense-core (Thioflavin-S+) plaques from the temporal neocortex of 40 AD subjects with a symptom duration ranging from 4 to 20 years, and nine nondemented control subjects with dense-core plaques. Dystrophic neurites (Kendall τ = 0.34, P = 0.001), reactive astrocytes (Kendall τ = 0.30, P = 0.003), and CD68+ (Kendall τ = 0.48, P < 0.0001), but not IBA1 microglia (Kendall τ = 0.045, P = 0.655), exhibited a significant positive correlation with symptom duration. When excluding control subjects, only the positive association between CD68+ microglia and symptom duration remained significant (Kendall τ = 0.39, P = 0.0003). The presence of the APOEε4 allele did not affect these results. We conclude that plaques exert an increasing toxicity in the surrounding neuropil over the clinical course of AD, thereby potentially contributing to cognitive decline. Amyloid plaques and neurofibrillary tangles are the core features of Alzheimer disease (AD). Among amyloid plaques, the subset of dense-core plaques defined by positive staining with dyes selective for β-pleated sheet structure such as Congo red and Thioflavin-S are considered more toxic and correlate more specifically with the presence of dementia. Dense-core plaques have a number of associated features that include dystrophic neurites, reactive astrocytes, and activated microglial cells. Dystrophic neurites are described as spheroids, swellings, and distorted neurites (dendrites and axons) that are embedded within dense-core plaques or in their close vicinity and exhibit immunoreactivity for neurofilament proteins and hyperphosphorylated tau.1Serrano-Pozo A. Frosch M.P. Masliah E. Hyman B.T. Neuropathological alterations in Alzheimer disease.Cold Spring Harb Perspect Med. 2011; 1: a006189Crossref Scopus (1952) Google Scholar Reactive astrocytes and activated microglial cells also cluster within and around dense-core plaques. Therefore, the microenvironment of dense-core neuritic plaques is thought to recapitulate all of the steps of the amyloid cascade hypothesis.2Selkoe D.J. The molecular pathology of Alzheimer's disease.Neuron. 1991; 6: 487-498Abstract Full Text PDF PubMed Scopus (2188) Google Scholar, 3Hardy J. Selkoe D.J. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.Science. 2002; 297: 353-356Crossref PubMed Scopus (10962) Google Scholar With the use of quantitative postmortem measures, we previously showed that amyloid both plaque burden and plaque size remain relatively constant throughout the clinical course of AD.4Ingelsson M. Fukumoto H. Newell K.L. Growdon J.H. Hedley-Whyte E.T. Frosch M.P. Albert M.S. Hyman B.T. Irizarry M.C. Early Abeta accumulation and progressive synaptic loss, gliosis, and tangle formation in AD brain.Neurology. 2004; 62: 925-931Crossref PubMed Scopus (523) Google Scholar, 5Serrano-Pozo A. Mielke M.L. Gómez-Isla T. Betensky R.A. Growdon J.H. Frosch M.P. Hyman B.T. Reactive glia not only associates with plaques but also parallels tangles in Alzheimer's disease.Am J Pathol. 2011; 179: 1373-1384Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 6Serrano-Pozo A. Mielke M.L. Muzitansky A. Gómez-Isla T. Growdon J.H. Bacskai B.J. Betensky R.A. Frosch M.P. Hyman B.T. Stable size distribution of amyloid plaques over the course of Alzheimer disease.J Neuropathol Exp Neurol. 2012; 71: 694-701Crossref PubMed Scopus (31) Google Scholar Thanks to the body of evidence accumulated with the use of fibrillar amyloid positron emission tomography in human subjects, there is now consensus that amyloid plaque deposition occurs for the most part before onset of cognitive deficits and plateaus soon after.7Jack Jr., C.R. Knopman D.S. Jagust W.J. Shaw L.M. Aisen P.S. Weiner M.W. Petersen R.C. Trojanowski J.Q. Hypothetical model of dynamic biomarkers of the Alzheimer's pathological cascade.Lancet Neurol. 2010; 9: 119-128Abstract Full Text Full Text PDF PubMed Scopus (3186) Google Scholar, 8Jack Jr., C.R. Knopman D.S. Jagust W.J. Petersen R.C. Weiner M.W. Aisen P.S. Shaw L.M. Vemuri P. Wiste H.J. Weigand S.D. Lesnick T.G. Pankratz V.S. Donohue M.C. Trojanowski J.Q. Tracking pathophysiological processes in Alzheimer's disease: an updated hypothetical model of dynamic biomarkers.Lancet Neurol. 2013; 12: 207-216Abstract Full Text Full Text PDF PubMed Scopus (2641) Google Scholar, 9Bateman R.J. Xiong C. Benzinger T.L. Fagan A.M. Goate A. Fox N.C. Marcus D.S. Cairns N.J. Xie X. Blazey T.M. Holtzman D.M. Santacruz A. Buckles V. Oliver A. Moulder K. Aisen P.S. Ghetti B. Klunk W.E. McDade E. Martins R.N. Masters C.L. Mayeux R. Ringman J.M. Rossor M.N. Schofield P.R. Sperling R.A. Salloway S. Morris J.C. Dominantly Inherited Alzheimer NetworkClinical and biomarker changes in dominantly inherited Alzheimer's disease.N Engl J Med. 2012; 367: 795-804Crossref PubMed Scopus (2389) Google Scholar However, whether plaque-associated toxicity, represented by surrounding dystrophic neurites, reactive astrocytes, and activated microglial cells, is stable or worsens during disease progression remains unclear. Two models could be proposed. At one extreme, plaques could cause local damage to the neuropil as they deposit, but then remain relatively static lesions10Meyer-Luehmann M. Spires-Jones T.L. Prada C. Garcia-Alloza M. de Calignon A. Rozkalne A. Koenigsknecht-Talboo J. Holtzman D.M. Bacskai B.J. Hyman B.T. Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer's disease.Nature. 2008; 451: 720-724Crossref PubMed Scopus (806) Google Scholar as the disease pathologic process becomes increasingly dominated by nonplaque pathologies, including tau-associated lesions such as neuropil threads and tangles, neuronal and synaptic loss, and non–plaque-associated glial reactions.5Serrano-Pozo A. Mielke M.L. Gómez-Isla T. Betensky R.A. Growdon J.H. Frosch M.P. Hyman B.T. Reactive glia not only associates with plaques but also parallels tangles in Alzheimer's disease.Am J Pathol. 2011; 179: 1373-1384Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar At the other extreme, plaques could increasingly contribute to local neural system destruction over the entire course of the disease. Therefore, we tested the hypothesis that the microenvironment in the vicinity of plaques becomes more and more toxic as the disease advances. Specifically, we investigated whether the plaque-associated features of neuritic changes and reactive glia have already reached a plateau at dementia onset or parallel the progression of dementia and continue to accrue over time. Although cross-sectional postmortem studies are not able to unambiguously distinguish the prior history of the brain during life, we reasoned that examining specimens from individuals who had known durations of illness would provide insight into these questions. With the use of quantitative neuropathologic measures in postmortem specimens and robust statistical methods, we demonstrate that these markers of plaque toxicity change throughout the clinical course of AD, leading to an overall increase in local plaque-associated damage as time goes on. The temporal accrual of features of plaque-associated injury is independent of the APOEε4 allele. Paraffin-embedded sections from the temporal neocortex (BA 38) of 40 AD subjects and nine nondemented control (CTRL) subjects were obtained from the Massachusetts General Hospital Alzheimer Disease Research Center. Next of kin for study subjects provided informed consent to donate their brain, and the study was approved by the Institutional Review Board at Massachusetts General Hospital. All AD subjects met the clinical11McKhann G.M. Knopman D.S. Chertkow H. Hyman B.T. Jack Jr., C.R. Kawas C.H. Klunk W.E. Koroshetz W.J. Manly J.J. Mayeux R. Mohs R.C. Morris J.C. Rossor M.N. Scheltens P. Carrillo M.C. Thies B. Weintraub S. Phelps C.H. The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease.Alzheimers Dement. 2011; 7: 263-269Abstract Full Text Full Text PDF PubMed Scopus (9233) Google Scholar, 12McKhann G. Drachman D. Folstein M. Katzman R. Price D. Stadlan E.M. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease.Neurology. 1984; 34: 939-944Crossref PubMed Google Scholar and neuropathologic diagnostic13Hyman B.T. Phelps C.H. Beach T.G. Bigio E.H. Cairns N.J. Carrillo M.C. Dickson D.W. Duyckaerts C. Frosch M.P. Masliah E. Mirra S.S. Nelson P.T. Schneider J.A. Thal D.R. Thies B. Trojanowski J.Q. Vinters H.V. Montine T.J. National Institute on Aging-Alzheimer's Association guidelines for the neuropathologic assessment of Alzheimer's disease.Alzheimers Dement. 2012; 8: 1-13Abstract Full Text Full Text PDF PubMed Scopus (1512) Google Scholar, 14Consensus recommendations for the postmortem diagnosis of Alzheimer's disease. The National Institute on Aging, and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimer's Disease.Neurobiol Aging. 1997; 18: S1-S2Abstract Full Text PDF PubMed Scopus (289) Google Scholar, 15Montine T.J. Phelps C.H. Beach T.G. Bigio E.H. Cairns N.J. Dickson D.W. Duyckaerts C. Frosch M.P. Masliah E. Mirra S.S. Nelson P.T. Schneider J.A. Thal D.R. Trojanowski J.Q. Vinters H.V. Hyman B.T. National Institute on Aging, Alzheimer's AssociationNational Institute on Aging-Alzheimer's Association guidelines for the neuropathologic assessment of Alzheimer's disease: a practical approach.Acta Neuropathol. 2012; 123: 1-11Crossref PubMed Scopus (1512) Google Scholar criteria of AD. CTRL subjects had no clinical or neuropathologic evidence of any neurodegenerative disease. AD subjects were selected on the basis of disease duration from symptom onset as assessed by their neurologist at the patient's first clinical encounter with Massachusetts General Hospital Alzheimer Disease Research Center Neurology team (≤5 years, n = 10; 6 to 10 years, n = 10; 11 to 15 years, n = 10, and >15 years, n = 10). The demographic and clinical characteristics of AD and CTRL subjects and of AD subgroups by APOEε4 status are depicted in Table 1. The temporal neocortex was chosen because it is a region with abundant and early amyloid deposition and because our prior quantitative neuropathologic studies in this area have revealed marked glial responses.5Serrano-Pozo A. Mielke M.L. Gómez-Isla T. Betensky R.A. Growdon J.H. Frosch M.P. Hyman B.T. Reactive glia not only associates with plaques but also parallels tangles in Alzheimer's disease.Am J Pathol. 2011; 179: 1373-1384Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 16Serrano-Pozo A. Muzikansky A. Gómez-Isla T. Growdon J.H. Betensky R.A. Frosch M.P. Hyman B.T. Differential relationships of reactive astrocytes and microglia to fibrillar amyloid deposits in Alzheimer disease.J Neuropathol Exp Neurol. 2013; 72: 462-471Crossref PubMed Scopus (135) Google Scholar, 17Serrano-Pozo A. Gómez-Isla T. Growdon J.H. Frosch M.P. Hyman B.T. A phenotypic change but not proliferation underlies glial responses in Alzheimer disease.Am J Pathol. 2013; 182: 2332-2344Abstract Full Text Full Text PDF PubMed Scopus (109) Google ScholarTable 1Demographic and Clinical Characteristics of the Study SubjectsCharacteristicCTRL subjectsAD subjectsPAD APOEε4 carriersAD APOEε4 noncarriersPSubjects, n9402119Sex, female, n (%)4 (44.4)26 (65.0)0.2816 (76.2)10 (52.6)0.18Age at death, mean ± SD (years)80.3 ± 14.477.6 ± 8.60.1275.8 ± 8.779.6 ± 8.20.16Age at onset, mean ± SD (years)66.9 ± 10.264.4 ± 10.669.6 ± 9.30.07Symptom duration, mean ± SD (years)10.7 ± 5.011.3 ± 4.910.0 ± 5.10.39APOEε4 carriers, n (%)3 (33.3)21 (52.5)0.46APOEε4 alleles, n (%)3 (16.7)25 (31.2)0.75Postmortem interval, mean ± SD (hours)17.4 ± 11.414.1 ± 6.20.5314.5 ± 5.813.7 ± 6.70.68Postmortem interval was not available for two CTRL subjects. Comparisons were performed with U-test, unpaired t-test, or Fisher's exact test as appropriate.AD, Alzheimer disease; CTRL, control. Open table in a new tab Postmortem interval was not available for two CTRL subjects. Comparisons were performed with U-test, unpaired t-test, or Fisher's exact test as appropriate. AD, Alzheimer disease; CTRL, control. Eight-micron–thick paraffin sections were cleared with xylenes, rehydrated with decreasing concentrations of ethanol, and subjected to a standard antigen retrieval procedure (microwave for 20 minutes at 95°C in boiling citrate buffer 0.01 mol/L with 0.05% Tween 20, pH 6.0) before immunohistochemistry. Primary antibodies and concentrations used were mouse anti-SMI312 (dilution 1:1000; Covance, Princeton, NJ; catalog no. SMI312R), rabbit anti-GFAP (dilution 1:1000; Sigma-Aldrich, St. Louis, MO; catalog no. G6296), rabbit anti-IBA1 (dilution 1:250; Wako, Osaka, Japan; catalog no. 019-19741), and mouse anti-CD68 (dilution 1:100; Dako, Glostrup, Denmark; catalog no. M0814). Cyanine 3-conjugated anti-rabbit or anti-mouse secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA) and used at a 1:200 concentration. Sections were counterstained with Thioflavin S (Sigma-Aldrich) 0.05% in 50% ethanol for 8 minutes and differentiated in 80% ethanol for 30 seconds before being coverslipped with Vectashield mounting media with DAPI (Vector Labs, Burlingame, CA; catalog no. H-1200). Single paraffin-embedded temporal neocortex sections were randomly sampled with the use of a stereology microscope-computer system, and 100 dense-core (Thioflavin-S+) amyloid plaques per subject were selected from randomly sampled microscopic fields by a trained observer (A.S.-P.). Briefly, sections were placed on a motorized stage of an upright Olympus BX51 epifluorescence microscope controlled by a computer with the computer-assisted stereological toolbox software version 2.3.1.5 (Olympus, Tokyo, Japan). Reactive (GFAP+) astrocytes and activated (IBA1+ or CD68+) microglia within 50 μm from the edge of the dense-core plaques were manually counted as previously described.16Serrano-Pozo A. Muzikansky A. Gómez-Isla T. Growdon J.H. Betensky R.A. Frosch M.P. Hyman B.T. Differential relationships of reactive astrocytes and microglia to fibrillar amyloid deposits in Alzheimer disease.J Neuropathol Exp Neurol. 2013; 72: 462-471Crossref PubMed Scopus (135) Google Scholar, 18Serrano-Pozo A. William C.M. Ferrer I. Uro-Coste E. Delisle M.-B. Maurage C.-A. Hock C. Nitsch R.M. Masliah E. Growdon J.H. Frosch M.P. Hyman B.T. Beneficial effect of human anti-amyloid-beta active immunization on neurite morphology and tau pathology.Brain. 2010; 133: 1312-1327Crossref PubMed Scopus (125) Google Scholar This boundary was selected on the basis of our previous animal and human postmortem quantitative studies.5Serrano-Pozo A. Mielke M.L. Gómez-Isla T. Betensky R.A. Growdon J.H. Frosch M.P. Hyman B.T. Reactive glia not only associates with plaques but also parallels tangles in Alzheimer's disease.Am J Pathol. 2011; 179: 1373-1384Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 16Serrano-Pozo A. Muzikansky A. Gómez-Isla T. Growdon J.H. Betensky R.A. Frosch M.P. Hyman B.T. Differential relationships of reactive astrocytes and microglia to fibrillar amyloid deposits in Alzheimer disease.J Neuropathol Exp Neurol. 2013; 72: 462-471Crossref PubMed Scopus (135) Google Scholar, 18Serrano-Pozo A. William C.M. Ferrer I. Uro-Coste E. Delisle M.-B. Maurage C.-A. Hock C. Nitsch R.M. Masliah E. Growdon J.H. Frosch M.P. Hyman B.T. Beneficial effect of human anti-amyloid-beta active immunization on neurite morphology and tau pathology.Brain. 2010; 133: 1312-1327Crossref PubMed Scopus (125) Google Scholar, 19Knowles R.B. Wyart C. Buldyrev S.V. Cruz L. Urbanc B. Hasselmo M.E. Stanley H.E. Hyman B.T. Plaque-induced neurite abnormalities: implications for disruption of neural networks in Alzheimer's disease.Proc Natl Acad Sci U S A. 1999; 96: 5274-5279Crossref PubMed Scopus (205) Google Scholar, 20Le R. Cruz L. Urbanc B. Knowles R.B. Hsiao-Ashe K. Duff K. Irizarry M.C. Stanley H.E. Hyman B.T. Plaque-induced abnormalities in neurite geometry in transgenic models of Alzheimer disease: implications for neural system disruption.J Neuropathol Exp Neurol. 2001; 60: 753-758Crossref PubMed Scopus (82) Google Scholar, 21D'Amore J.D. Kajdasz S.T. McLellan M.E. Bacskai B.J. Stern E.A. Hyman B.T. In vivo multiphoton imaging of a transgenic mouse model of Alzheimer disease reveals marked thioflavine-S-associated alterations in neurite trajectories.J Neuropathol Exp Neurol. 2003; 62: 137-145Crossref PubMed Scopus (86) Google Scholar, 22Spires T.L. Meyer-Luehmann M. Stern E.A. McLean P.J. Skoch J. Nguyen P.T. Bacskai B.J. Hyman B.T. Dendritic spine abnormalities in amyloid precursor protein transgenic mice demonstrated by gene transfer and intravital multiphoton microscopy.J Neurosci. 2005; 25: 7278-7287Crossref PubMed Scopus (473) Google Scholar Only glial cells with a DAPI+ visible nucleus were counted. To prevent double counting of cells in areas rich in plaques, individual astrocytes and microglia that were close to two or more plaques were split among those plaques (ie, 0.5 cells if close to two plaques, 0.33 if close to three plaques, 0.25 if close to four plaques, etc.). The number of SMI312+ axonal swellings/spheroids and distorted neurites >2.5 μm embedded within the Thioflavin-S+ area or in contact with its edges were manually counted as described before.18Serrano-Pozo A. William C.M. Ferrer I. Uro-Coste E. Delisle M.-B. Maurage C.-A. Hock C. Nitsch R.M. Masliah E. Growdon J.H. Frosch M.P. Hyman B.T. Beneficial effect of human anti-amyloid-beta active immunization on neurite morphology and tau pathology.Brain. 2010; 133: 1312-1327Crossref PubMed Scopus (125) Google Scholar Figure 1 shows examples of these quantitative methods. Analyses were conducted blinded to clinical diagnosis, symptom duration, and APOEε4 status. After completing the above quantifications, each of the 40 AD subjects yielded a histogram distribution with 100 values for each of the neuropathologic quantitative measures. For some CTRL subjects, the number of dense-core plaques was lower than the goal of 100 plaques, despite sampling 100% of the cortex included in the section. CTRL subjects were excluded from analyses of measures for which they had <100 plaques. Data for SMI312+ dystrophic neurites were not available for one CTRL subject. We used symptom duration as a surrogate of progression because i) duration of illness and clinical severity are generally correlated; ii) duration of illness overcomes biases inherent to formal neuropsychological testing in advanced dementia patients, including floor effect of psychometric measures, loss to follow-up, impact of intercurrent illnesses (eg, cataracts, medications) on test performance, and variable period of time between last follow-up and death; and iii) in prior studies we have found correlation between duration from symptom onset and variables of progression such as number of neurons and tau-related pathology,23Gómez-Isla T. Hollister R. West H. Mui S. Growdon J.H. Petersen R.C. Parisi J.E. Hyman B.T. Neuronal loss correlates with but exceeds neurofibrillary tangles in Alzheimer's disease.Ann Neurol. 1997; 41: 17-24Crossref PubMed Scopus (1119) Google Scholar cortical atrophy,5Serrano-Pozo A. Mielke M.L. Gómez-Isla T. Betensky R.A. Growdon J.H. Frosch M.P. Hyman B.T. Reactive glia not only associates with plaques but also parallels tangles in Alzheimer's disease.Am J Pathol. 2011; 179: 1373-1384Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar and glial responses.4Ingelsson M. Fukumoto H. Newell K.L. Growdon J.H. Hedley-Whyte E.T. Frosch M.P. Albert M.S. Hyman B.T. Irizarry M.C. Early Abeta accumulation and progressive synaptic loss, gliosis, and tangle formation in AD brain.Neurology. 2004; 62: 925-931Crossref PubMed Scopus (523) Google Scholar, 5Serrano-Pozo A. Mielke M.L. Gómez-Isla T. Betensky R.A. Growdon J.H. Frosch M.P. Hyman B.T. Reactive glia not only associates with plaques but also parallels tangles in Alzheimer's disease.Am J Pathol. 2011; 179: 1373-1384Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar Because histograms are multidimensional, to investigate the correlation between each of the plaque-associated features and symptom duration, we used the signed Kolmogorov-Smirnov (K-S) measure of distance between two histograms and estimated its association with symptom duration with the use of Kendall τ (nonparametric rank) correlation. The K-S distance is the maximum absolute distance between subjects' empirical distribution functions, across all values of the variable. The signed version of K-S distance inherits the sign (positive or negative) of the distance that yields the K-S distance; therefore, a larger positive distance between subject i's histogram and subject j's histogram suggests that subject j has larger values than subject i. To investigate whether the strength of the association between each plaque-associated feature and symptom duration remains constant throughout the clinical course of the disease or changes over time, we plotted Kendall τ correlation coefficients that were calculated for subsets of subjects defined by moving windows of symptom duration. Cross-correlations between plaque-associated features (ie, CD68+ microglia versus SMI312+ dystrophic neurites) were estimated with Kendall τ test with the use of the medians of the distributions. Finally, the numbers of SMI312+ dystrophic neurites per plaque were compared between APOEε4 carriers and noncarriers with the use of a two-sided Wilcoxon rank-sum test that accommodates clustered data.24Datta S. Satten G.A. Rank-sum tests for clustered data.J Am Stat Assoc. 2005; 100: 908-915Crossref Scopus (119) Google Scholar All analyses were conducted including and excluding CTRL subjects. Statistical significance was set at a level of P < 0.05. Analysis and graphs were performed with the statistical software package R version 3.2 (http://www.r-project.org/about.html), except for the two-sided clustered Wilcoxon rank-sum test, which was run in SAS version 9.3 (SAS Institute, Cary, NC). We have shown before that amyloid plaque burden and plaque size remain relatively constant throughout the clinical course of AD. Here, we investigated whether plaque-associated features also remain constant or, by contrast, continue to accrue as the disease advances. To this goal, we counted the number of SMI312+ axonal swellings and spheroids, GFAP+ astrocytes, IBA1+ microglial cells, and CD68+ microglial cells per plaque in 100 randomly selected dense-core Thioflavin-S+ amyloid plaques from the temporal cortex of 40 AD subjects with a symptom duration that ranged between 4 and 20 years and up to nine CTRL subjects. Counts were performed without knowledge of clinical history or APOE genotype. These analyses yielded up to 49 histograms with 100 values per subject. Histograms can be summarized with the use of central measures (ie, mean for normal distributions and median for nonnormal distributions) and dispersion measures (ie, interquartile range), but these descriptive measures are limited because they can still be influenced by outliers, and they reduce the multidimensional information to a single number. To avoid these limitations, we compared the histograms among pairs of subjects with the use of the K-S test and correlated their signed K-S difference (or distance) with their difference in symptom duration. A positive correlation between the K-S distance from distribution histograms of pairs of subjects for a certain plaque-associated feature and their difference in symptom duration indicates an accrual of that plaque-associated feature over time. Figure 2 contains plots of the histogram distance versus the difference in disease duration for each pair of subjects. It is seen that the number of SMI312+ dystrophic neurites (Figure 2A), GFAP+ astrocytes (Figure 2B), and CD68+ microglia (Figure 2D) but not IBA1+ microglia (Figure 2C) per plaque increase with increasing duration of illness. As expected from these graphs, a positive correlation was found between symptom duration and number of SMI312+ dystrophic neurites (Kendall τ = 0.34, P = 0.001), GFAP+ astrocytes (Kendall τ = 0.30, P = 0.003), and CD68+ (Kendall τ = 0.48, P < 0.0001) but not IBA1+ microglia (Kendall τ = 0.045, P = 0.655). When CTRL subjects were removed from the analyses, only the association between CD68+ microglia and symptom duration remained significant (Kendall τ = 0.39, P = 0.0003) (Supplemental Figure S1 and Supplemental Table S1). No significant correlation was found between any pair of plaque-associated features, either in AD subjects or in all (AD and CTRL) subjects (Table 2).Table 2Summary of Study Results for All SubjectsAll subjectsSymptom durationSMI312+ dystrophic neuritesGFAP+ astrocytesIBA1+ microgliaCD68+ microgliaKendall τPKendall τPKendall τPKendall τPKendall τPSymptom duration0.34∗Statistically significant results.0.001∗Statistically significant results.0.30∗Statistically significant results.0.003∗Statistically significant results.0.0450.6550.48∗Statistically significant results.<0.0001∗Statistically significant results.SMI312+ dystrophic neurites0.34∗Statistically significant results.0.001∗Statistically significant results.−0.0280.7350.0060.9430.0080.929GFAP+ astrocytes0.30∗Statistically significant results.0.003∗Statistically significant results.−0.0280.735−0.1370.064−0.0140.849IBA1+ microglia0.0450.6550.0060.849−0.1370.0640.1090.151CD68+ microglia0.48∗Statistically significant results.<0.0001∗Statistically significant results.0.0080.929−0.0140.8490.1090.151Depicted are correlations between the Kolmogorov-Smirnov distances of pairs of distribution histograms of a given plaque-associated feature and the difference in symptom duration of that pair of subjects, and the correlations between the median values of the distribution histograms for a given pair of plaque-associated features (ie, GFAP+ astrocytes versus CD68+ microglia). Data refer to all subjects, that is, 40 AD and 9 CTRL subjects (8 for SMI312+ dystrophic neurites).AD, Alzheimer disease; CTRL, control.∗ Statistically significant results. Open table in a new tab Depicted are correlations between the Kolmogorov-Smirnov distances of pairs of distribution histograms of a given plaque-associated feature and the difference in symptom duration of that pair of subjects, and the correlations between the median values of the distribution histograms for a given pair of plaque-associated features (ie, GFAP+ astrocytes versus CD68+ microglia). Data refer to all subjects, that is, 40 AD and 9 CTRL subjects (8 for SMI312+ dystrophic neurites). AD, Alzheimer disease; CTRL, control. The APOEε4 allele is the strongest known genetic risk factor for the development of sporadic AD, and it does so by promoting the accumulation of amyloid β (Aβ) peptide and its deposition in plaques (reviewed in Holtzman et al25Holtzman D.M. Herz J. Bu G. Apolipoprotein E and apolipoprotein E receptors: normal biology and roles in Alzheimer disease.Cold Spring Harb Perspect Med. 2012; 2: a006312Crossref Scopus (534) Google Scholar), but whether plaques from APOEε4 carriers are more toxic to the surrounding neuropil than plaques from APOEε4 noncarriers remains unclear. There were 21 APOEε4 carriers and 19 APOEε4 noncarriers among the 40 AD subjects and 3 APOEε4 carriers and 6 APOEε4 noncarriers among the nine CTRL subjects in this sample. With the use of this same data set, we have previously reported that dense-core plaques from AD APOEε4 carriers and noncarriers do not differ in the magnitude of glial resp
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