Association of Cerebrovascular and Alzheimer Disease Biomarkers With Cholinergic White Matter Degeneration in Cognitively Unimpaired Individuals

Background and Objectives Several pathologic processes might contribute to the degeneration of the cholinergic system in aging. We aimed to determine the contribution of amyloid, tau, and cerebrovascular biomarkers toward the degeneration of cholinergic white matter (WM) projections in cognitively unimpaired individuals. Methods The contribution of amyloid and tau pathology was assessed through CSF levels of the Aβ42/40 ratio and phosphorylated tau (p-tau). CSF Aβ38 levels were also measured. Cerebrovascular pathology was assessed using automatic segmentations of WM lesions (WMLs) on MRI. Cholinergic WM projections (i.e., cingulum and external capsule pathways) were modeled using tractography based on diffusion tensor imaging data. Sex and APOE ε4 carriership were also included in the analysis as variables of interest. Results We included 203 cognitively unimpaired individuals from the H70 Gothenburg Birth Cohort Studies (all individuals aged 70 years, 51% female). WM lesion burden was the most important contributor to the degeneration of both cholinergic pathways (increase in mean square error [IncMSE] = 98.8% in the external capsule pathway and IncMSE = 93.3% in the cingulum pathway). Levels of Aβ38 and p-tau also contributed to cholinergic WM degeneration, especially in the external capsule pathway (IncMSE = 28.4% and IncMSE = 23.4%, respectively). The Aβ42/40 ratio did not contribute notably to the models (IncMSE<3.0%). APOE ε4 carriers showed poorer integrity in the cingulum pathway (IncMSE = 21.33%). Women showed poorer integrity of the external capsule pathway (IncMSE = 21.55%), which was independent of amyloid status as reflected by the nonsignificant differences in integrity when comparing amyloid-positive vs amyloid-negative women participants (T201 = −1.55; p = 0.123). Discussion In cognitively unimpaired older individuals, WMLs play a central role in the degeneration of cholinergic pathways. Our findings highlight the importance of WM lesion burden in the elderly population, which should be considered in the development of prevention programs for neurodegeneration and cognitive impairment.

Ingmar Skoog: Drafting/revision of the manuscript for content, including medical writing for content; Major role in the acquisition of data Eric Westman: Drafting/revision of the manuscript for content, including medical writing for content; Study concept or design; Analysis or interpretation of data   [ 2 ] All Cerebrovascular disease/Stroke, [ 25 ] All Cognitive Disorders/Dementia, [ 26 ] Alzheimer's disease, [ 54 ] Cohort studies, [ 118 ] All Imaging

INTRODUCTION
The cholinergic neurons located in the nucleus basalis of Meynert (NBM) provide the major cholinergic input to the cerebral cortex and are essential to cognitive functioning 1 . Postmortem studies have traced two principal cholinergic projection pathways from the NBM to the neocortex: the medial and the lateral pathways 1 . The medial pathway advances through the white matter (WM) axons of the rectus gyrus, bends at the rostrum of the corpus callosum and enters the cingulum bundle, projecting to the paraolfactory, cingulate and restrosplenial cortices. The lateral pathway advances both through the claustrum and the extreme capsule (i.e., perisylvian division), projecting to the frontoparietal operculum, insula, and superior temporal gyrus; as well as through the external capsule and uncinate fasciculus (i.e., capsular division), projecting to the remaining parts of the frontal, parietal, and temporal neocortex. Recent diffusion tensor imaging (DTI)-based tractography studies have examined these pathways [2][3][4][5] , providing the opportunity to study the integrity of the cholinergic system and its potential association with cognitive performance and pathophysiological processes in vivo.
The strategic location of the NBM and its connective circuitry to the cortex results in increased vulnerability to brain pathology. For example, cholinergic neurons are affected in early stages of Alzheimer's disease (AD)-related tauopathy due to their proximity to heavily affected basotemporal regions, which likely also alters their connective circuitry to the cortex 1 . Further, other age-related pathologies can also impact the integrity of the cholinergic system. WM lesions (WML), which are thought to be a marker of cerebrovascular disease, are commonly found on magnetic resonance images (MRI) in the elderly 6 . A recent study showed that WML are associated with worse integrity of the cholinergic projections in cognitively unimpaired older individuals 4 , and cholinergic projections influenced cognitive performance 4 . Interestingly, despite the association of WML with the integrity of the cholinergic projection system, neither WML burden itself nor NBM volume contributed to cognitive performance 4 . These findings raised the question of whether other age-associated pathologies apart from WML might be affecting the integrity of the cholinergic projections in cognitively unimpaired individuals.
In this study, we investigated the contribution of amyloid and tau pathology in combination with cerebrovascular disease towards the degeneration of cholinergic WM projections in cognitively unimpaired individuals. It is important to address these research questions to assess whether and how other pathologies apart from cerebrovascular disease may affect the integrity of cholinergic projections in cognitively unimpaired individuals.

Participants
The study sample belongs to the Gothenburg H70 Birth Cohort Studies 7 . Every 70year-old listed in the Swedish Population Registry as a resident in Gothenburg (Sweden) was invited to a comprehensive examination on aging and age-related factors 7 . A total of 1203 individuals born in 1944 (response rate 72.2%; mean age 70.5 years) agreed to participate, of whom 430 consented to a lumbar puncture (response rate 35.8%). Lumbar puncture was considered as contraindicated in participants under anticoagulant therapy, immune-modulated therapy, and cancer therapy. After excluding participants not suitable for a lumbar puncture, the cerebrospinal fluid (CSF) extraction was conducted in 322 (26.8%) individuals. Every participant was also invited to take part in a brain MRI examination of which 792 individuals (response rate 65.8%) underwent MRI conducted at Aleris in Gothenburg.
The MRI examination was conducted within 3 months from the initial study visit. The lumbar puncture was conducted within 2 months from the MRI examination. The general examinations and other procedures have previously been described in detail 7 .
General cognitive status was measured using the Mini-Mental State Examination (MMSE) and the Clinical Dementia Rating (CDR) scale.
For the current study, inclusion criteria were: (1) a CDR score of 0; (2) MMSE >24; (3) availability of CSF biomarkers; and (4) availability of MRI data, yielding a final sample of 203 individuals (51% female). has previously been shown that hypointense and hyperintense WML are strongly correlated 6 . Previous findings revealed that hypointense WML might represent necrotic damage closer to accumulated cerebrovascular pathology 10 , while hyperintense WML might also represent acute damage including peri-inflammatory processes 11 . Due to the aim of the current study, we focused on hypointense WML, but all the analyses were replicated using hyperintense WML and are reported in supplementary material. MRI data management and processing was done using theHiveDB 12 database system. WML volumes in milliliters (ml) were adjusted by TIV to account for variability in head size 13 .

MRI data acquisition, image processing, and assessment of WML
Previously established ROI masks for the cholinergic WM pathways (i.e., cingulum and external capsule pathways) were used 4 . Briefly, the masks were created using probabilistic diffusion-based fiber tracking of the NBM WM projections. These ROI masks of the cholinergic WM pathways were transferred from MNI standard space to Copyright

Complementary MRI markers of cerebrovascular disease and vascular risk factors
In addition to the automated measure of WML, we assessed cerebral microbleeds,

CSF sampling and biomarker analysis
Lumbar puncture for CSF sampling and determination of APOE e4 carriership were conducted following standard procedures 7 . CSF biomarker levels were determined by a commercially available assay 7  to determine its association with AD biomarkers as well as cerebrovascular disease in the general population.

Statistical analysis
Statistical analyses were conducted using the R statistical software (http://www-Rproject.org). A p-value <0.05 (two-tailed) was deemed significant in all the analyses.
We used random forest (RF) regression models to assess the differential contributions of the different pathology-specific biomarkers towards the integrity of NBM A conditional importance score is computed for each tree in RF analysis. This is done by measuring the change in the prediction error when the values of a certain variable are permuted within a grid defined by the included covariates. Then this conditional score is averaged across the entire ensemble. These conditional importance scores are designed to reduce the undesirable effects of collinearity among predictor variables.
The final importance of each predictor denotes its contribution to the model. Importance values below or equal to zero denote no contribution. A conditional regression tree is produced as a graphical representation of the model. The RF was comprised of 5000 conditional inference trees. R2 was computed to assess the quality of the RF models. Although aging is associated with WM neurodegeneration and greater WML volumes 4,28 , age was not included as a covariate in the models since it was controlled from the design (i.e. all participants were 70 years old). For completeness of information, we also report Pearson correlation coefficients among the predictor variables included in the random forest models and independent sample t-tests for categorical variables that resulted important in the RF analysis. The randomForest 29 and Party packages 30 were used for these analyses.

Standard Protocol Approvals, Registrations, and Patient Consents.
The H70 study was approved by the Regional Ethical Review Board in Gothenburg Helsinki declaration and its later amendment.

Data availability statement
The authors state that anonymized data on which the article is based will be shared by request from any qualified investigator. The RF models showed that WML volume was the most important predictor for the average MD of the cingulum pathway (see Figure 1). P-tau, Aβ 38  Five groups were distinguished at the end of the tree (see Figure 3). Copyright   The NBM is well-known for its key role in cognitive functioning and its deterioration is linked to cognitive impairment in AD 1 . It is important to determine the pathological processes contributing towards degeneration of the cholinergic system as it has previously been demonstrated to be associated with cognitive impairment in advanced aging 4 . In this sample of cognitively unimpaired aged individuals, we demonstrated that WML were the most important contributor towards the degeneration of the studied cholinergic pathways, followed by CSF Aβ 38 and p-tau levels. Conversely, the Aβ 42/40 ratio did not show a substantial contribution.

Demographic
The integrity of the cholinergic system is crucial for proper cognitive functioning 1 .
The cholinergic hypothesis of cognitive aging postulates that age-related memory decline and other cognitive problems may arise due to declining cholinergic activity 31,32 . In a previous study, we demonstrated that the WM integrity of cholinergic projections was closely associated with attention and memory performance in an independent aging cohort of cognitively unimpaired individuals 4  The cholinergic circuitry is highly vulnerable to brain pathology. In our study, we Regarding tau pathology, our results showed a negative association between p-tau and degeneration of cholinergic WM projections (i.e. a poorer integrity of WM projections was associated with lower levels of CSF p-tau). This counterintuitive finding might be the result of a selection bias in our sample. All our participants were cognitively unimpaired 70-year-olds, and only 6.4% had abnormal CSF p-tau levels.
It is important to take into consideration that the combination of abnormal levels of ptau with other brain pathologies such as WML will most probably result in cognitive impairment and therefore those individuals may have been excluded from our study.
Whether increased CSF p-tau levels are associated with degeneration of cholinergic WM projections needs to be further tested in more diverse populations of older individuals, including patients with cognitive impairment.
The data provided by this study describes the contribution of CSF Aβ 42/40 ratio, Aβ 38 and p-tau levels in combination with WML burden towards the degeneration of the cholinergic system in cognitively unimpaired elderly from a population-based cohort 7      Background of significant correlations (p < 0.05) was colored according to the value of the correlation coefficient and shaped accordingly to the association distribution.

ACKNOWLEDGMENTS
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