Original manuscript number 18-0171

Responses to review (2)

We thank the reviewer for their constructive comments and suggestions. Below is a response

to the point raised:

Reviewer 2

Thank you for the opportunity to review this revised manuscript. The authors have addressed

the comments made resulting in a stronger submission.

Minor comment -

The concluding sentence on page 17 should be revised. As it reads currently a causal

relationship is inferred between genotype and neuropathology/ longevity whereas the

observational nature of the study only allows for a significant association to be determined.

Response to Reviewer 2

We have amended the manuscript accordingly, taking into consideration the point raised by

the reviewer (page 17).

Pathological correlates of cognitive impairment in The University of Manchester

Longitudinal Study of Cognition in Normal Healthy Old Age

Andrew C Robinson1, Yvonne S Davidson1, Michael A Horan1, Neil Pendleton*1, David MA

Mann*1.

* These authors contributed equally to the study.

1 Faculty of Biology, Medicine and Health, School of Biological Sciences, Division of Neuroscience & Experimental Psychology, University of Manchester, Salford Royal Hospital, Salford, M6 8HD, UK

Running title

Pathological changes in an ageing cohort

Correspondence to: Dr Andrew Robinson (address as above)

Email: andrew.c.robinson@manchester.ac.uk; Tel. +44 (0) 161-206-4408.

Abstract1

The neuropathological changes responsible for cognitive impairment and dementia remain

incompletely understood. Longitudinal studies with a brain donation end point allow the

opportunity to examine relationships between cognitive status and neuropathology. We report

on the first 97 participants coming to autopsy with sufficient clinical information from The

University of Manchester Longitudinal Study of Cognition in Normal Healthy Old Age. This

study began in 1983 and recruited 6542 healthy individuals between 1983 and 1994, 312 of

whom consented to brain donation.

Alzheimer-type pathology was common throughout the cohort and generally correlated well

with cognitive status. However, there was some overlap between cognitive status and

measures of Alzheimer pathology with 26% of cognitively intact participants reaching either

CERAD B or C, 11% reaching Thal phase 4 or 5 and 29% reaching Braak stage III – VI.

Cerebral amyloid angiopathy (CAA), α-synuclein and TDP-43 pathology was less common,

but when present correlated well with cognitive status. Possession of APOE ε4 allele(s) was

associated with more severe Alzheimer-type and CAA pathology and earlier death, whereas

possession of APOE ε2 allele(s) had no effect on pathology but was more common in

cognitively intact individuals.

The University of Manchester Longitudinal Study of Cognition in Normal Healthy Old Age

cohort is pathologically representative when compared with similar studies. Cognitive

impairment in life correlates strongly with all pathologies examined and the APOE status of

an individual can affect pathology severity and longevity.

Keywords: Neuropathology, Cohort Studies, Longitudinal Studies, Dementia, Cognitive

Dysfunction, Alzheimer’s Disease, alpha-Synuclein, APOE

Introduction

2

Longitudinal studies that are community or population-based and have brain donation end

points offer an opportunity to examine correlations between pathology and cognitive

impairment, and are therefore fundamentally important for the field of dementia research.

However, true unbiased community-based longitudinal studies are rare as it is common

practice for epidemiological studies of dementia to use cohorts selected according to their

cognitive status, age, gender or ethnicity. Nonetheless, there have been several longitudinal

studies of brain ageing and dementia that include brain autopsy end points: The Medical

Research Council Cognitive Function and Ageing Studies (CFAS) [1, 2], the Nun Study [3],

the Religious Orders Study [4], the Baltimore Longitudinal Study of Ageing (BLSA) [5], the

Cambridge City over 75 Cohort Study (CC75C) [6], the Honolulu-Asia Aging Study (HAAS)

[7], the Oxford Project to Investigate Memory and Aging samples (OPTIMA) [8], the Vantaa

85+ Study [9] and the Adult Changes in Thought Study (ACT) [10]. Demographic

characteristics of these studies are shown in Table 1. Most commenced in the late 1980’s

early 1990’s and were based either on healthy volunteers of all ages from local communities,

or selected cohorts based either on age specification or particular lifestyle which included

both cognitively normal and cognitively impaired individuals. Cohort size has ranged from a

few hundred to many thousand individuals with brain donations ranging from 180-500 (at

time of last publication). Most are ongoing, though the Honolulu-Asia and Optima studies

have now closed.

The present report is based on The University of Manchester Longitudinal Study of

Cognition in Normal Healthy Old Age [11]. This study began in 1983 and recruited, via local

advertisement, 6542 healthy individuals aged between 42 and 92 years. People with evidence

of cognitive decline/dementia at the time of recruitment were not eligible for the study.

Hence, this study represents one of the longest running studies in which cognitively healthy

individuals at the outset have been followed up for periods of 30 years or more. From 2003,

3

312 of the surviving individuals consented to brain donation of which 100+ have

subsequently died and their brains have become available for investigation. Here, we report

neuropathological findings on the first 97 cases coming to donation.

Materials and Methods

Participants and study design

Participants from The University of Manchester Longitudinal Study of Cognition in Normal

Healthy Old Age [11] were approached (in 2003) for consent to brain donation. From the

original recruited total of 6542 healthy individuals (aged between 42 and 92 years), 312

individuals consented to brain donation. Participants had demographic, education, lifestyle

and health information collected through study-specific self-report questionnaires.

Information regarding educational level were standardised using the International Standard

Classification of Education (ISCED) guidelines [12].

Over five waves between 2004 and 2017, surviving participants underwent assessment by the

modified Telephone Instrument for Cognitive Status (TICSm) which contains 13 questions

testing orientation, concentration, immediate and delayed memory, naming, calculation,

comprehension and reasoning. The TICSm test had a maximum score of 39 [13] and the cut-

off point, which was used to define cognitive impairment in the present study, was a score

below 21 [14].

Cognitive status at death was ascertained using a combination of last TICSm score, patient

notes obtained via participants’ general practitioner and cause of death as recorded on the

death certificate. The first 97 brains donated were accessioned into the present study.

Pathological methods

4

One fresh hemi-brain was fixed in 10% neutral buffered formalin for 3-4 weeks with the

other hemi-brain frozen at -80oC. Standard blocks of frontal, cingulate, temporal (including

superior and middle temporal gyrus), hippocampus, parietal and occipital cortex, amygdala,

corpus striatum, thalamus, midbrain, brainstem and cerebellum were cut from the fixed tissue

and processed into wax blocks. Paraffin sections (6µm) were immunostained for Aβ

(Cambridge Bioscience, clone 4G8, 1:3000), tau proteins phosphorylated at Ser202 and

Thr205 (P-tau) (Innogenetics, clone AT8, 1:750), phosphorylated α-synuclein (#1175) [15]

(1:1000) and TDP-43 (Proteintech, 1:1000). For antigen retrieval, sections were either

immersed in 70% formic acid for 20 minutes (for Aβ only) or microwaved in 0.1M citrate

buffer, pH 6.0 (all other antibodies) prior to incubation with primary antibody.

The presence and severity of Aβ and tau was assessed in all regions examined. A five-point

semi-quantitative grading scale was adopted:

0 – No Aβ/tau pathology present

1 – Rare Aβ/tau pathology present

2 – Mild Aβ/tau pathology present

3 – Moderate Aβ/tau pathology present

4 – Severe Aβ/tau pathology present

A CERAD score [16], Thal phase [17] and Braak stage [18] were also assigned and an

identical five-point semi-quantitative grading scale was used to assess the presence and

severity of cerebral amyloid angiopathy (CAA) throughout the brain.

Vascular pathology was assessed using the Vascular Cognitive Impairment Neuropathology

Guidelines (VCING). The presence of at least one large infarct, moderate to severe small

5

vessel disease and moderate to severe CAA was used to assign low, moderate or high risk of

vascular pathology contributing to cognitive impairment [19].

The presence/absence of TDP-43 pathology was assessed in the temporal cortex and

hippocampus whereas the presence/absence of phosphorylated α-synuclein pathology was

assessed in the cingulate gyrus and the substantia nigra region of the midbrain.

The clinical and neuropathological diagnosis was not known to the examiner (AR), who rated

all cases. Preliminary scoring of the sections was also hidden during subsequent slide re-

examination.

DNA was extracted from frozen brain tissue using REDExtract-N-Amp™ Tissue PCR Kit

(Sigma) or from blood (3 cases). The APOE genotype was determined using routine

polymerase chain reaction (PCR) methods [20]. APOE could not be determined for 2

participants because of lack of blood or frozen brain tissue.

Neuropathological diagnoses

After neuropathological assessment was completed, neuropathological diagnosis was

assigned by Professor David Mann (Professor of Neuropathology).

Cases were assigned to a neuropathological category that accurately described the principal

pathology found. Those considered normal for age included cases that were essentially free

from pathology (pathology score 0) and those with a burden of pathology expected for age

(pathology scores 1 and 2). The category of limited Aβ/tau pathology was assigned to cases

that had some degree of AD pathology (pathology score 3) but insufficient for a full

neuropathological diagnosis of AD (pathology score 4).

Statistical analyses

6

The cohort was split according to severity of pathology with one group representing low

severity (pathology scores 0, 1 and 2) and the other high severity (pathology scores 3 and 4).

Similar groups were established for CERAD, Thal phase, CAA severity, Braak stage,

VCING, presence/absence of TDP-43 pathology and presence/absence of α-synuclein

pathology.

Chi-squared test was used to analyse whether there were differences in severity of pathology

according to cognitive status. Mann-Whitney test assessed differences in educational level

between cognitive status groups. A p value of < 0.05 was considered significant.

Similarly, when assessing age at death, the cohort was split according to age at death into two

groups: Under 90 years and 90 years and over.

Chi-squared test was used to analyse whether there were differences in frequencies of APOE

ε2 or ε4 alleles according to age group at death. A p value of < 0.05 was considered

significant.

The odds ratio of age group at death according to presence of APOE ε2 or ε4 alleles was

ascertained using a binary logistic regression model. Age group at death was the dependent

variable and presence of APOE ε2 allele(s), APOE ε4 allele(s) and cognitive impairment were

the covariates. A p value of < 0.05 was considered significant.

Results

The first brain donation accrued into the study occurred on 11/03/2005 with the last brain

donation for the study taking place on 21/11/2016. The mean time between final TICSm test

and death was 42 months (±30) with a range of 134 months. The ratio of women to men in

the study was approximately 2:1. The median age at death for the 97 participants was 89

(range 72 to 104 years) and there were no differences in age group at death between men and

7

women (χ2 = 2.04; p = 0.15). The proportion of participants with cognitive impairment was

41% with no differences in cognitive status when comparing sex (χ2 = 0.62; p = 0.43), age

group (χ2 = 0.28; p = 0.60) or education level (U = 1006.5; p = 0.38). APOE ε4 allele(s) were

more common in cognitively impaired individuals (40%) than cognitive intact individuals

(25%), this difference being marginally significant (χ2 = 3.25; p = 0.07). However,

cognitively normal individuals were more likely to carry APOE ε2 alleles(s) (χ2 = 3.80; p =

0.05) when compared with cognitively impaired participants (Table 2).

The principal neuropathological diagnosis varied throughout the cohort (Table 3). Those

considered pathologically normal for age were more likely to be cognitively intact (χ2 =

10.87; p < 0.001) as were those with only limited Aβ/tau pathology (χ2 = 5.29; p = 0.02).

However, a proportion of cognitively intact individuals exhibited sufficient pathology to meet

current neuropathological criteria for AD (8%) or DLB (3%). The proportion of individuals

with incipient AD pathology was similar between the cognitive groups (χ2 = 0.21; p = 0.65).

Individuals with pathologically confirmed AD (χ2 = 12.18; p < 0.001) and DLB/PD (χ2 =

6.91; p = 0.01) were more likely to be cognitively impaired. Conversely, a proportion (10%)

of cognitively impaired individuals had only limited Aβ/tau pathology or had pathology

considered normal for age. Individuals exhibiting more than one pathology were more likely

to be considered cognitively impaired than those with only one principal pathology (χ2 =

12.89; p < 0.001) and those considered pathologically normal for age (χ2 = 33.23; p < 0.001).

Four cognitively intact individuals over the age of 90 years at death (mean 93.5 ±1.7 years)

were noted to have very little to no pathological changes, being CERAD score 0, Thal phase

0, Braak stage I or less and low VCING stage, with no TDP-43 or α-synuclein pathology. No

APOE ε4 alleles were present in this subset of individuals.

8

Aβ pathology was common across all participants (Table 4). Within the neocortex,

cognitively impaired individuals were more severely affected in frontal (χ2 = 3.89; p = 0.05),

occipital (χ2 = 5.38; p = 0.02) and parietal (χ2 = 5.30; p = 0.02) regions. However, Aβ

pathology was of comparable severity in the temporal region (χ2 = 2.47; p = 0.12) when

comparing cognitive status groups. In the limbic region, cognitively impaired individuals

were more likely to exhibit moderate to severe Aβ pathology in the cingulate (χ2 = 4.26; p =

0.04) but not the amygdala (χ2 = 2.76; p = 0.10) or hippocampus (χ2 = 2.43; p = 0.12). In

addition, cognitively impaired individuals showed greater severity of Aβ pathology in the

midbrain (χ2 = 8.07; p = 0.004) and corpus striatum (χ2 = 8.25; p = 0.004).

Moderate to severe tau pathology was less common than Aβ pathology but still correlated

well with cognitive status (Table 5). Within all regions of the neocortex, individuals

considered cognitively impaired were more severely affected by tau pathology: frontal (χ2 =

13.28; p < 0.001), occipital (χ2 = 11.00; p = 0.001), parietal (χ2 = 8.53; p = 0.004), temporal

(χ2 = 7.86; p = 0.005). The limbic regions showed differences in severity of tau pathology

between cognitive groups in the cingulate (χ2 = 10.56; p = 0.001) and amygdala (χ2 = 5.20; p

= 0.02) but not the hippocampus (χ2 = 3.06; p = 0.08). Analysis of other brain areas showed

cognitively impaired individuals had greater severity of tau pathology in the midbrain (χ2 =

6.63; p = 0.01) and corpus striatum (χ2 = 7.01; p = 0.01).

Only low levels of vascular pathology (as measured by VCING) were found in 74%

individuals. Moderate to high levels of vascular pathology were equally likely in both

cognitively impaired and cognitively intact individuals (χ2 = 1.61; p = 0.20) (Table 6).

Cognitively intact individuals exhibiting significant AD pathology were more frequently

found to have low levels of vascular pathology than cognitively impaired individuals with

significant AD pathology when applying Thal phase (83% vs 60%) or Braak stage (69% vs

9

64%) criteria. However, neither of these proportional differences reached statistical

significance.

Other pathologies were less common (Table 6). Moderate to severe CAA was found in 27%

of participants and was more likely to occur in cognitively impaired individuals (χ2 = 8.55; p

= 0.003). Similarly, the presence of α-synuclein pathology in the form of LBs and LNs was

more common in the cingulate (χ2 = 6.44; p = 0.01) and midbrain (χ2 = 4.86; p = 0.03) for

those considered cognitively impaired. In addition, cognitively impaired individuals were

more likely to present with TDP-43 pathology in the temporal lobe than those considered

cognitively intact (χ2 = 10.56; p = 0.001).

Established protocols for grading AD pathology correlated well with cognitive status (Figure

1). There were strong positive correlations between CERAD score (rs = 0.34, p = 0.001), Thal

phase (rs = 0.34, p = 0.001) and Braak stage (rs = 0.37, p < 0.001) and cognitive impairment.

However, there was a degree of overlap between cognitive status and these measures with

26% of cognitively intact participants reaching either CERAD B or C, 11% reaching Thal

phase 4 or 5 and 29% reaching Braak stage III – VI.

The presence of APOE ε2 allele(s) was more common in cognitively normal participants (χ2 =

3.80; p = 0.05). However, there was a comparable distribution of APOE ε4 allele(s) between

the cognitive groups. Pathologically, there were no differences in the proportions of APOE ε2

allele(s) between any of the measures analysed. Conversely, APOE ε4 allele(s) were more

commonly found in individuals exhibiting CERAD B – C (χ2 = 3.81; p = 0.05), Thal phase 4

– 5 (χ2 = 8.15; p = 0.004), Braak stage III – VI (χ2 = 4.65; p = 0.03) and moderate to severe

CAA (χ2 = 8.22; p = 0.004) (Table 7). When considering age at death, there were no

differences in the distribution of APOE ε2 allele(s) between the age groups, but APOE ε4

allele(s) were more likely to be found in those individuals whose age at death was before they

10

reached 90 years of age (χ2 = 5.47; p = 0.02) (Figure 2). Specifically, the odds of an

individual living beyond 90 years of age significantly decreased if they carried one or more

APOE ε4 allele(s) (OR = 0.30, 95% CI: 0.11 – 0.80) even when controlling for cognitive

status and presence of APOE ε2 allele(s).

Discussion

The present study reports the neuropathological findings of the first 97 brains collected from

The University of Manchester Longitudinal Study of Cognition in Normal Healthy Old Age

and relates these findings to cognitive impairment. Although community and population-

based studies with brain donation end-points are becoming more common, the present study

avoids many of the selection criteria maintained by other studies such as cognitive status, age,

gender or ethnicity. With this in mind, there are several limitations to the study. Brain

donation was not in the original scope of the longitudinal study and was only introduced in

2004 and consequently a number of potential donors were lost due to early death or

withdrawal from the study. Our protocol for assessing cognitive status uses the TICSm test; a

telephone administered task for general cognition. Particular limitations of this test are that it

consists of a single task rather than battery assessment and that there is a lack of physical

clinical assessment. However, TICS is becoming widely used in longitudinal ageing research,

including the Health and Retirement Survey family of international studies [21] and has been

validated against comprehensive cognitive assessment [22, 23]. In addition, the geographical

areas covered (Greater Manchester and Newcastle) may not reflect society as a whole and the

fact that the cohort was self-selected may indicate that the study sample may not be

representative of the general population.

Aβ plaque pathology

11

In the neocortex and limbic regions, Aβ plaque pathology was common in the cohort as a

whole, mirroring findings from previous cohort studies [1, 24, 25] but contrary to other,

smaller, studies [26]. Approximately half of the cohort also had significant Aβ plaque

pathology in the corpus striatum, which corroborates findings from other studies; namely that

diffuse Aβ plaques can be located in subcortical regions (such as the striatum) of cognitively

intact individuals as well as those with AD [27].

Similar to previous work [28], the majority of the cohort had CERAD scores indicative of

low risk of AD. This was also the case for Thal staging. However, a proportion of cognitively

intact individuals exhibited levels of Aβ plaque pathology which would warrant a diagnosis

of possible or probable AD; a common finding in other similar studies [28, 29]. Despite this

overlap, Aβ plaque pathology was still related strongly to cognitive function [30 – 38] as was

CERAD score and Thal phase [29].

Tau pathology

Tau pathology was much less common than Aβ plaque pathology in the cohort as a whole.

The majority of brain regions showed only a low burden of tau pathology and moderate to

severe tau pathology was only prevalent in the temporal cortex (51% of cases), amygdala

(56%) and, to a lesser extent, the hippocampus (48%). The involvement of moderate to

severe tau pathology in the limbic region and hippocampus has been described previously [1,

25, 26] and correlates well with the present study. It has previously been shown that

significant tau burden is more likely to be found in cognitive impaired individuals than

cognitively normal individuals [39 – 43] and the present study shows similar findings.

The majority of individuals in the cohort exhibited Braak stages indicative of low risk of AD.

Braak stages of 0 – II were considered to be a low risk of AD with Braak II being the most

frequently found. A previous population-based study of individuals over the age of 75

12

showed that Braak stage III is most commonly found [28]. However, the comparative

differences between Braak stage II and III are minor. There was a considerable degree of

overlap when comparing Braak stage and cognitive status with 29% of cognitively normal

individuals reaching Braak stage III – VI; a finding similar to one previously reported [29].

However, it is of note that the majority of cognitively normal individuals in the Braak III - VI

group were found to be at Braak III which is not sufficient for a probable AD diagnosis.

Despite this overlap, there was a strong relationship between Braak stage and cognitive

impairment with those impaired more likely to be at Braak III-VI; a conclusion also found by

a number of other studies [29, 44].

VCING

Whilst there are no pathological criteria for vascular dementia that are fully agreed upon by

all neuropathologists, the recent study by Skrobot, et al [19] attempted to use consensus

guidelines to create an acceptable protocol. Their most successful model incorporated

infarct(s) of more than 10mm, moderate to severe CAA in the occipital lobe and moderate to

severe arteriosclerosis to predict whether cognitive impairment was due to vascular problems.

Previous studies have shown that the presence of vascular disease may exacerbate the effects

of dementia attributed to Aβ plaques and tangles [45]. This finding may help to explain why a

proportion of individuals in the present cohort remained cognitively intact despite exhibiting

high levels of Aβ plaque and tau pathology, since the majority of these individuals scored

‘low’ on VCING criteria, such that the combined effects of vascular disease, and the amount

of Alzheimer or other neurodegenerative pathology present were insufficient to overcome a

threshold to cognitive impairment.

CAA pathology

13

In previous studies, the presence of CAA has been found to be highly prevalent in the elderly

[46] and more likely to be found in cognitively impaired individuals, possibly by interacting

with other pathologies that may be present [47]. The present study also found CAA to be

fairly common with 27% of cases showing moderate to severe CAA in the regions examined.

There was a strong relationship between severity of CAA and cognitive status. Similar to

previous findings [29], there was a proportion of cognitively normal individuals (16%) who

showed moderate to severe CAA. It is possible that these individuals have sufficient

compensatory mechanisms and are therefore currently pre-symptomatic.

α-synuclein pathology

The incidence of cortical and subcortical α-synuclein pathology in clinico-pathological or

population based cohorts of aged and very aged individuals has been estimated between 16 –

25% [25, 28, 48] although has been described as a rare occurrence in some studies [49].

When examining specific regions, such as the amygdala, the incidence of α-synuclein

pathology in individuals over the age of 85 increases to 33% [50]. These findings compare

well with the 13% incidence of α-synuclein in the areas examined in the present study.

Further examination of α-synuclein in the amygdala of individuals in the present study may a

reveal similar incidence of that previously described. Presence of α-synuclein pathology was

more likely to occur in cognitively impaired individuals, when compared with cognitively

normal individuals. This finding is confirmed by a number of previous studies [48, 51].

TDP-43 pathology

In cohorts of individuals that are very aged, TDP-43 pathology has been found to occur very

rarely [25] but in less aged cohorts, TDP-43 has been identified in 27 – 46% of individuals

[52, 53]. TDP-43 pathology was found in the temporal cortex and hippocampus in 18% of

14

cases which reflects the age range of the cohort. Where present, TDP-43 pathology was more

likely to occur in individuals exhibiting cognitive impairment [52, 53].

APOE genotype

Those individuals with APOE ε2 allele(s) were more likely to be cognitively intact. However,

there were no differences in the severity of any of the pathological measures when comparing

APOE ε2 groups suggesting that carrying APOE ε2 allele(s) has no protective effect against

amyloid deposition or tau tangle formation. Although this contradicts some previous studies

[54, 55] it supports findings from other studies [56]. The relatively small number of APOE ε2

carriers in the present study may under-represent this group and mask possible changes in

pathology. Therefore, more work and larger sample sizes are needed to clarify these findings.

In agreement with previous studies [57, 58], those individuals carrying APOE ε4 allele(s)

were more likely to exhibit moderate to severe AD and CAA pathology. Although APOE ε4

allele(s) were more likely to occur in those cognitively impaired, this was not significantly so

and, statistically, there was a comparable distribution of APOE ε4 allele(s) across the

cognitive groups. It is possible that a more extensive battery of cognitive tests may have

uncovered cognitive impairment in a number of individuals who were considered cognitively

normal but carrying APOE ε4 allele(s). Another explanation may lie with greater cognitive

reserve in those considered cognitively intact and this may explain why they were able to

carry APOE ε4 allele(s) without succumbing to cognitive impairment.

Participants who were 90 years of age or older at death were much less likely to carry APOE

ε4 allele(s), probably due to the fact that those who did carry APOE ε4 allele(s) succumbed to

the increase pathological burden at an earlier age. However, contrary to previous studies [59],

those carrying APOE ε2 allele(s) did not exhibit increased longevity. Again, the relatively

small numbers of APOE ε2 carriers in the present study may provide a simple explanation for

15

this result. Alternatively, the discrepancy may lie with the location of the present cohort

(Manchester and Newcastle). The main, large studies showing correlations between APOE ε2

and longevity were based in Spain, Italy and Japan where cognitive activity, lifestyle, diet

and environment may have enhanced longevity via gene-environment interactions. Similar

interactions have previously been shown to occur with APOE ε4 and lifetime cognitive

activity [60].

The subset of four individuals that were over 90 years old at death but remained cognitively

intact and essentially pathology-free are of particular interest. It has generally been

considered that the prevalence of moderate or severe AD-type pathology increases with age

in people without dementia, and that the strength of the association between AD-type

pathology and dementia is at its weakest in the oldest-old [61] although this has been

contested in other studies [25]. Therefore, very aged individuals may show many Aβ neuritic

plaques and tau tangles but they do not necessarily show the cognitive impairment expected

from such a pathology load. In the present study, we highlight a number of cognitively intact

individuals who reached very old age but remained virtually free from neurodegenerative or

vascular pathology. Further study of the genetic profile and lifestyle of these individuals may

shed light on possible factors that could promote the chances of pathology-free, healthy

cognitive ageing.

Within the study, there were a number of findings which indicated conflicts between

cognitive status in life and pathological lesions found after death. Namely, that there were a

number of cognitive intact individuals who exhibited more severe AD-like pathology than

would be expected for age. This could be explained in a number of ways. It is possible that

these cognitively normal individuals may have had a greater degree of cognitive impairment

than first thought which more rigorous cognitive testing may have uncovered. Our methods

for concluding cognitive impairment, whilst robust, are not as extensive as those in the CFAS

16

[62] and Framingham [63] studies where extensive cognitive testing decreased the likelihood

of errors in classification. Another possibility may relate to the lack of vascular lesions which

are known to exacerbate cognitive impairment in those meeting the pathological criteria for

AD [64]. The majority of cognitively intact individuals with significant levels of AD

pathology had low levels of vascular pathology, which collectively were insufficient to

generate the clinical symptoms of dementia.

In conclusion, the characterisation of the clinical and neuropathological findings from the

first 97 donated brains of The University of Manchester Longitudinal Study of Cognition in

Normal Healthy Old Age correlated well with the established literature and affirmed that the

cohort is typical and representative when compared with community–based, population-

based and clinico-pathological cohorts. Although, in general, cognitive impairment in life

correlated strongly with most pathologies found at post-mortem, it was notable that

individuals exhibiting more than one pathology were more likely to be cognitively impaired

than those with only one principal pathology and those considered pathologically normal for

age. Such data suggest that the presence of dementia in very old subjects is represented by the

cumulative tissue effects of several ‘lower grade’ pathologies. In themselves, each would be

insufficient to bring about significant cognitive impairment, but when present in combination

can overcome threshold levels of pathology necessary to bring about clinical change. While

the presence of APOE ε4 allele was associated with increased the severity of all pathologies

examined and seemingly reduced the chances of living past 90 years of age, possession of

APOE ε2 allele was not associated with a reduction in did not reduce the severity of any of

the pathologies measured nor did it increase longevity.

17

Acknowledgements/Funding

Longitudinal Cognitive studies were funded by Medical Research Council, Economic and

Social Research Council, The Wellcome Trust and Unilever PLC. The work of Manchester

Brain Bank is supported by Alzheimer’s Research UK and Alzheimer’s Society through the

Brains For Dementia Research (BDR) Programme, which also kindly allowed AR to

undertake a PhD studentship.

Author Contributions

David Mann and Neil Pendleton devised and designed the study and helped with writing the

paper. DM finalised neuropathological diagnosis. NP finalised clinical cognitive impairment

diagnosis.

Andrew Robinson helped to devise and design the study, performed immunohistochemistry,

microscopic assessments, genetic analysis and data/statistical analysis and wrote the paper.

Yvonne Davidson helped with immunochemistry.

Michael Horan helped to finalise clinical cognitive impairment diagnosis, provided clinical

data and assisted with preparation of the manuscript.

Declaration of Interest

The authors have no conflict of interest to report.

Research Ethics Committee Approval

The study was approved by Manchester Brain Bank Management Committee (REC reference

09/H0906/52+5). Under conditions agreed with the Research Ethics Committee, The

Manchester Brain Bank can supply tissue or data to researchers, without requirement for

researchers to apply individually to the REC for approval.

18

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Study Commenced Cohort size Recruitment age Cognitive status Demographics Donations

To dateMRC CFAS 1989 18000 65 + Normal/C.I. Population-based 500 +Nun study 1986 678 75 + Random Selected (women) 221 +

Religious Orders Study 1994 1000 + Aged Normal Selected (Roman Catholic clergy) 180 +

BLSA 1958 1400 20 – 90 Normal Community 335 +CC75C 1985 2600 ≤ 75 Normal/C.I. Population-based 213 +

HAAS 1991-2012 3734 71 – 93 Random Population-based (Japanese-American men) 443 +

OPTIMA 1988-2008 1100 + Aged (65 +) Normal/C.I. Community 300

Vantaa 85+ 1991 601 85 + Random Population-based (South Finland) 304

ACT 1994 2581 65 + Random Community (King’s County, WA) 232 +

University of Manchester 1983 6542 42 – 92 Normal Community (Manchester and Newcastle) 107 +

Table 1 – Overview of demographic characteristics from longitudinal studies of brain ageing and dementia that include a brain donation end

point (C.I. – Cognitive impairment).

29

Characteristic No cognitive impairment (n = 57) Cognitive impairment (n = 40) Total (n = 97)n % n % n %

SexMale 20 35 11 27 31 32Female 37 65 29 73 66 68

Age at deathUnder 90 years 33 58 21 53 54 5690 years and over 24 42 19 47 43 44Median age at death (range) 89 (26) 89 (31) 89 (32)

Education (ISCED yrs. equiv)Mean (± s.d) 15.1 (± 3.4) 15.7 (± 3.8) 15.4 (± 3.6)

Geneticsa

Presence of APOE ε4 allele(s) 14 25 16 40 30 31Presence of APOE ε2 allele(s) 11 19 2 5 13 13

Table 2 – Characteristics of The University of Manchester Longitudinal Study of Cognition in Normal Healthy Old Age cohort stratified by

cognitive status (a Genetic data not available for two cases)

30

Principal neuropathological

diagnosis

No cognitive impairment

(n = 57)

Cognitive impairment

(n = 40)

Total

(n = 97)n % n % n %

Normal for age 21 37 3 8 24 25Limited Aβ/tau pathology 10 18 1 2 11 11Incipient AD 9 16 5 13 14 15AD 4 8 14 36 18 19DLB/PD 2 3 8 20 10 10Cerebrovascular disease 2 3 5 13 7 7CAA 3 5 1 2 4 4Haemorrhage 2 3 1 2 3 3CBD 1 2 1 2 2 2AGD 2 3 0 0 2 2ARTAG 1 2 0 0 1 1PART 0 0 1 1 1 1

Table 3 – Distributions of principal neuropathological diagnosis in The University of

Manchester Longitudinal Study of Cognition in Normal Healthy Old Age cohort stratified by

cognitive status

31

Brain region Severity

No cognitive impairment

(n = 57)

Cognitive impairment

(n = 40)

Total

(n = 97)n % n % n %

FrontalNone to mild 27 47 11 27 38 39Moderate to severe 30 53 29 73 59 61Missing 0 0 0 0 0 0

OccipitalNone to mild 35 61 15 37 50 51Moderate to severe 22 39 25 63 47 49Missing 0 0 0 0 0 0

ParietalNone to mild 29 51 11 27 40 41Moderate to severe 28 49 29 73 57 59Missing 0 0 0 0 0 0

TemporalNone to mild 23 40 10 25 33 34Moderate to severe 34 60 30 75 64 66Missing 0 0 0 0 0 0

HippocampusNone to mild 47 82 29 72 76 78Moderate to severe 8 14 11 28 19 20Missing 2 4 0 0 2 2

AmygdalaNone to mild 29 51 13 32 42 43Moderate to severe 26 46 24 60 50 52Missing 2 4 3 8 5 5

CingulateNone to mild 29 51 11 27 40 41Moderate to severe 25 44 24 60 49 51Missing 3 5 5 13 8 8

Corpus striatum

None to mild 34 60 12 30 46 47Moderate to severe 22 38 27 68 49 51Missing 1 1 1 2 2 2

MidbrainNone to mild 52 91 26 65 78 80Moderate to severe 5 9 12 30 17 18Missing 0 0 2 5 2 2

MedullaNone to mild 52 91 33 82 85 88Moderate to severe 0 0 0 0 0 0Missing 5 9 7 18 12 12

CerebellumNone to mild 56 98 37 92 93 96Moderate to severe 1 2 2 5 3 3Missing 0 0 1 3 1 1

Table 4 – Presence, distribution and severity of Aβ plaques in selected brain regions from

individuals in The University of Manchester Longitudinal Study of Cognition in Normal

Healthy Old Age cohort stratified by cognitive status

32

Brain region Severity

No cognitive impairment

(n = 57)

Cognitive impairment

(n = 40)

Total

(n = 97)n % n % n %

FrontalNone to mild 51 90 23 57 74 76Moderate to severe 6 10 17 43 23 24Missing 0 0 0 0 0 0

OccipitalNone to mild 54 28 95 70 82 85Moderate to severe 3 12 5 30 15 15Missing 0 0 0 0 0 0

ParietalNone to mild 50 25 88 62 75 77Moderate to severe 7 15 12 38 22 23Missing 0 0 0 0 0 0

TemporalNone to mild 35 61 13 32 48 49Moderate to severe 22 39 27 67 49 51Missing 0 0 0 0 0 0

HippocampusNone to mild 32 56 16 40 48 50Moderate to severe 23 40 24 60 47 48Missing 2 4 0 0 2 2

AmygdalaNone to mild 28 49 10 25 38 39Moderate to severe 27 47 27 68 54 56Missing 2 4 3 7 5 5

CingulateNone to mild 48 84 23 58 71 73Moderate to severe 5 9 14 35 19 20Missing 4 7 3 7 7 7

Corpus striatum

None to mild 54 95 31 78 85 88Moderate to severe 2 4 8 20 10 10Missing 1 1 1 2 2 8

MidbrainNone to mild 52 91 27 67 79 81Moderate to severe 5 9 11 28 16 17Missing 0 0 2 5 2 2

MedullaNone to mild 49 86 30 75 79 81Moderate to severe 3 5 3 8 6 6Missing 5 9 7 17 12 12

CerebellumNone to mild 57 100 38 96 95 98Moderate to severe 0 0 1 2 1 1Missing 0 0 1 2 1 1

Table 5 – Presence, distribution and severity of tau pathology in selected brain regions from

individuals in The University of Manchester Longitudinal Study of Cognition in Normal

Healthy Old Age cohort stratified by cognitive status

33

Pathology Severity/Presence

No cognitive impairment

(n = 57)

Cognitive impairment

(n = 40)

Total

(n = 97)n % n % n %

VCINGNone to Low 45 79 27 67 72 74Moderate to High 12 21 13 33 25 26Missing 0 0 0 0 0 0

CAANone to mild 48 84 23 57 71 73Moderate to severe 9 16 17 43 26 27Missing 0 0 0 0 0 0

TDP-43 in temporal lobe

Absent 53 93 27 67 80 82Present 4 7 13 33 17 18Missing 0 0 0 0 0 0

α-synuclein in cingulate

Absent 54 95 31 77 85 88Present 3 5 9 23 12 12Missing 0 0 0 0 0 0

α-synuclein in midbrain

Absent 53 93 31 77 84 87Present 4 7 9 23 13 13Missing 0 0 0 0 0 0

Table 6 – Presence and severity of other pathologies in individuals from The University of

Manchester Longitudinal Study of Cognition in Normal Healthy Old Age cohort stratified by

cognitive status.

34

Pathology Severity/Presence

APOE ε2 allele(s) absent

(n = 82)

APOE ε2 allele(s) present(n = 13)

APOE ε4 allele(s) absent

(n = 65)

APOE ε4 allele(s) present(n = 30)

n % n % n % n %

CERAD score

0 – A 45 55 10 77 42 65 13 43B – C 37 45 3 23 23 35 17 57Missing 0 0 0 0 0 0 0 0

Thal phase0 – 3 63 77 11 85 56 86 18 604 – 5 19 23 2 15 9 14 12 40Missing 0 0 0 0 0 0 0 0

Braak stage0 – II 48 59 10 77 44 68 14 47III – VI 32 39 3 23 19 29 16 53Missing 2 2 0 0 2 3 0 0

VCINGNone – Low 61 74 10 77 50 77 21 70Moderate – High 21 26 3 23 15 23 9 30Missing 0 0 0 0 0 0 0 0

CAANone to mild 60 73 9 69 53 81 16 53Moderate to severe 22 27 4 31 12 19 14 47Missing 0 0 0 0 0 0 0 0

TDP-43 in temporal lobe

Absent 68 83 12 92 57 88 23 77Present 14 17 1 8 8 12 7 23Missing 0 0 0 0 0 0 0 0

α-synuclein in cingulate

Absent 70 85 13 100 58 89 25 83Present 12 15 0 0 7 11 5 17Missing 0 0 0 0 0 0 0 0

α-synuclein in midbrain

Absent 69 84 13 100 57 88 25 83Present 13 16 0 0 8 12 5 17Missing 0 0 0 0 0 0 0 0

Table 7 – Presence of APOE ε2 and ε4 alleles in individuals from The University of

Manchester Longitudinal Study of Cognition in Normal Healthy Old Age cohort stratified by

pathology type (Two cases were not genotyped for APOE due to lack of fresh frozen tissue).

35

36

Figure legends37

Figure 1 – Distribution of CERAD, Thal and Braak staging in individuals from The

University of Manchester Longitudinal Study of Cognition in Normal Healthy Old Age

without cognitive impairment (black) and with (white) cognitive impairment.

Figure 2 – Distribution of APOE ε4 (panel A) and APOE ε2 (panel B) alleles in individuals

from The University of Manchester Longitudinal Study of Cognition in Normal Healthy Old

Age stratified by age group. Those without any APOE ε4 (panel A) or APOE ε2 (panel B)

alleles are shown in black and those with the relevant allele(s) are shown in white.

38