The roles of apolipoprotein E ε4 on neuropathology and neuroinflammation in patients with Alzheimer's disease

Abstract Aims To explore the roles of apolipoprotein E (APOE) ε4 on the neuropathology and neuroinflammation in Alzheimer's disease (AD) patients. Methods AD patients were divided into the APOE ε4 carrier and the APOE ε4 non‐carrier groups according to APOE genotype. Demographic information, cognitive function, the levels of neuropathological proteins and neuroinflammatory factors in cerebrospinal fluid (CSF) were compared between the two groups, and their correlations were subsequently analyzed. Results β amyloid protein (Aβ)1–42 level from the APOE ε4 carrier group was significantly lower than that from the non‐carrier group (p = 0.023), which was associated with worse cognitive function. The nitric oxide (NO) level was significantly elevated in the APOE ε4 carrier group compared to the non‐carrier group (p = 0.016), which was significantly and positively correlated with the Trail Making Test (TMT)‐A‐time (r = 0.21, p = 0.026) and TMT‐B‐time (r = 0.38, p < 0.01). Conclusion APOE ε4 is associated with poorer cognition, particularly the early symptoms of memory, language, and attention. APOE ε4 is associated with lower Aβ1–42 level, and the more numbers of APOE ε4 are carried, the lower level of Aβ1–42 is measured. APOE ε4 is associated with elevated NO level, which is linked to the impaired attention and executive function.


| INTRODUC TI ON
Alzheimer's disease (AD) is the most common neurodegenerative disease in the elderly, which is characterized by progressive cognitive decline, neuropsychiatric symptoms, and impairments of daily activities.
The pathological hallmark of AD includes neuritic plaques and neurofibrillary tangles, which are composed of β amyloid protein (Aβ) and phosphorylated tau (P-tau), respectively.Aβ and P-tau, as the biomarkers of AD neuropathology, are very pivotal in the development and progression of AD. 1 Neuroinflammation also plays an important role in AD. 2,3 Microglia and astrocytes make up the majority of glial cells, which transform into different subtypes after being stimulated and then play either a pro-inflammatory or anti-inflammatory role. 4This is manifested as overactivation of glial cells, which turn into a pro-inflammatory phenotype, and then produce numerous neuroinflammatory factors, including tumor necrosis factor (TNF)α, interleukin (IL)-1β, IL-6, interferon and nitric oxide (NO), which promote the development of AD pathology and induce progressive degeneration and death of neurons. 2 The human Apolipoprotein E (APOE) encodes the essential lipid transporter APOE in the brain.[7] The roles of APOE ε4 on the neuropathology of AD were investigated.It was reported that APOE ε4 is more potent than other APOE alleles in inducing amyloid precursor protein (APP) transcription, promoting the transformation of Aβ peptide into neurotoxic Aβ oligomer and fibrils, and inhibiting the clearance of Aβ in the brain. 8,9reover, APOE ε4 carriers had more tau aggregation and a greater extent of somatodendritic tau redistribution in the brain in comparison to other APOE alleles. 10In addition, several animal model studies explored that the extent of glial hyperplasia in the brain and levels of TNFα, IL-1β, and IL-6 of APOE ε4 carriers were greater than that of other APOE alleles. 10,11vertheless, there are still many problems remaining with APOE ε4 and AD.Evidence suggests that APOE ε4 is associated with poorer cognitive function than other alleles, however, the association between APOE ε4 and various cognitive domains involved remains unclear.Furthermore, studies related to APOE alleles with pathological mechanisms of AD have predominantly focused on animal models, autopsy studies, or PET studies.It is well established that extrapolating results from animal model studies to clinical patients can occasionally be challenging, and that intracerebral pathological changes shown from autopsy and PET frequently occur later than those detected by CSF.Consequently, the discovery of a link between APOE allele and factor changes in the CSF of AD patients at an earlier stage is currently understudied and particularly crucial.In addition, some recently identified neuroinflammatory factors, including chitinase-3-like protein 1 (CHI3L1, also known as YKL-40) and triggering receptor expressed on myeloid cells 2 (TREM2), were shown alteration in AD. 12,13 However, it is still unclear how these factors are related to APOE alleles in AD.
This study hypothesized that APOE ε4 might promote the processes of AD by accelerating the aggregation of neuropathological proteins and upregulating levels of neuroinflammatory factors.Based on this hypothesis, AD patients enrolled in the study were divided into the APOE ε4 carrier and non-carrier groups.
Demographic information, cognitive function, the levels of neuropathological proteins of AD and neuroinflammatory factors in CSF between the two groups was compared, and the correlations among the above-mentioned variables were subsequently analyzed.

| Collection of demographic information
Demographic information was collected, including gender, age, age of onset, education level, drinking, smoking, body mass index (BMI), resting heart rate, blood pressure, history of hypertension, history of hyperlipidemia and history of diabetes mellitus, etc.  16,17 Language was evaluated by the Verbal Fluency Test (VFT) and the Boston Naming Test (BNT). 18,19Attention was evaluated by the Symbol Digit Modalities Test (SDMT), 20 the Trail Making Test (TMT)-A, 21 as well as the Stroop Color-Word Test (SCWT)-A and SCWT-B. 22suospatial ability was evaluated by RFT.Executive function was evaluated by the SCWT-C and the TMT-B.The detailed description of these scales was provided in the Appendix S1.

| Detection of APOE genotypes
The venous blood samples of enrolled patients were collected from the median elbow under fasting condition the next morning after admission, and then sent to the clinical laboratory of Beijing Tiantan Hospital.
Genotyping for APOE single nucleotide variants (rs429358 C/T and rs7412C/T), which define the APOE 2, 3, and 4, was performed by real-time Fluorescence Quantitative Polymerase Chain Reaction by using nucleic acid detection reagents (Youzhiyou company).

| Collections of CSF samples
All patients in our study were first-time visitors and had not used cognitive-improving drugs such as cholinesterase inhibitors before admission, thus excluding the influence of drugs on the results.Lumbar puncture was conducted and CSF samples were collected under fasting condition through lumbar puncture, followed by being immediately centrifuged at 4°C with 3000 r/min for 10 min.Each CSF sample was then allocated into separate Nunc cryotubes (Beijing Jin-gkeHongda Biotechnology Co., Ltd) and frozen for 0.5 mL per tube at −80°C until the assay.

| Association of APOE ε 4 with clinical features
In this study, 125 cases (32.6%) were the APOE ε4 carrier group, in which 76 cases (60.8%) were female and the median age was 68.00 (62.00, 74.00) years.The data showed no significant differences in demographic variables between the two groups (Table 1).The median disease duration was 24.00 (12.00, 48.00) months in the APOE ε4 carrier group, which was not significantly different from the noncarrier group.The APOE ε4 carrier group had a significantly higher frequency of AD-D than the APOE ε4 non-carrier group (p < 0.001).
Cognitive function was also compared between the two groups.
As far as global cognitive function, the scores of MMSE (p = 0.005) and MOCA scales (p = 0.011) were all significantly decreased in the APOE ε4 carrier group compared with that in the non-carrier group.Besides, each individual cognitive domain between the two groups was also conducted.Firstly, in terms of the memory, the scores of AVLT N1-3 (p < 0.001), AVLT N4 (p < 0.001), AVLT N5 (p < 0.001), and RFT-delayed recall (p = 0.027) in the APOE ε4 carrier group were all significantly lower than those in the non-carrier group.Secondly, in terms of the language, the APOE ε4 carrier group had significantly decreased scores of VFT (p < 0.001) and BNT scales (p = 0.023) compared with the noncarrier group.As far as the attention, the APOE ε4 carrier group spent more time on the TMT-A test (p = 0.012) and the TMT-B test (p = 0.050) than the non-carrier group did.There was no significant difference in executive function and visuospatial ability between the two groups.

| Neuropathological proteins between the APOE ε4 carrier and the non-carrier groups
Aβ 1-42 level in CSF from the APOE ε4 carrier group was significantly lower than that from the non-carrier group (p = 0.023) (Table 2).There were no statistical differences in the levels of P-tau (T181), P-tau (S199), P-tau (T231), P-tau (S396) and T-tau in CSF from the APOE ε4 carrier group than that from the non-carrier group.Multiple

| Correlations between Aβ 1-42 level and cognition
In the aspect of overall cognitive function, Aβ 1-42 level in CSF was significantly and positively correlated with the scores of MMSE (r = 0.32, p < 0.001) and MoCA (r = 0.36, p < 0.001) scales (Figure 3).  the brain was significantly correlated with the dramatically declined overall cognitive function and multiple cognitive domains of memory, language, attention, and visuospatial ability in APOE ε4 carriers.

In terms of individual cognitive domains, Aβ
There were no marked correlations between Aβ 1-42 level and executive function (Figure 3).

Multiple linear regression analyses further illustrated that lower
Aβ 1-42 level was associated with worse overall cognitive function and individual cognitive domains, including memory, language, and attention, which was independent of age, disease duration, and education level (Table S1).3).

| Correlations between neuroinflammatory factors and cognition
Correlations between the levels of neuroinflammatory factors in CSF and the scores of cognitive rating scales in AD patients were displayed (Figure 3).The NO level was significantly and positively corre-  S2-S5).The results showed that the higher NO level was also associated with longer time of TMT-A [β,  S3).In addition, YKL-40 level was significantly and negatively associated with the score of BNT [β, −1.30E-4; 95% CI (−2.26E-4, 3.30E-5); p = 0.011], which was independent of age, disease duration, and education level (Table S5).
The predictive value of sTREM2 for VFT was not found (Table S4).

| Frequency of the APOE ε 4 in AD patients
In this study, 32.6% of total AD patients carried APOE ε4, among whom, 6.5% carried double APOE ε4.The frequency of APOE ε4 in this study was higher than that in Hispanic (24.0%) and Asian (28.0%) populations reported by previous studies, 25,26 which might be due to the different ethnic and national backgrounds of the enrolled subjects.

| APOE ε 4 aggravated cognitive impairment of AD patients
In this study, no difference in demographic variables, including age of onset were found between the two groups, which differed from  28,29 which was possibly because APOE ε4 was associated with temporal lobe atrophy (especially hippocampus) and dysfunction in the default mode network. 29,30Furthermore, we applied multiple cognitive tests to comprehensively evaluate individual domains and found that AD patients carrying APOE ε4 were markedly impaired in language and attention in addition to memory.It is well known that memory, language, and attention are the cognitive domains involved in the early stage of AD, suggesting that APOE ε4 might play a key role in the early stage of AD.

| APOE ε 4 aggravated cognitive impairment via neuropathological proteins of AD
In this study, Aβ 1-42 level in CSF was significantly and positively correlated with both overall cognitive function and multiple cognitive domains, including memory, language, and attention, suggesting that the higher Aβ burden in the brains of AD patients, the worse the cognitive function.
In a previous animal model study, APOE ε4-targeted replacement familial AD (EF4AD) transgenic mice had more Aβ deposition in brain than E3FAD mice had. 31A longitudinal study based on Aβ-PET imaging showed that AD patients with APOE ε4 had diffusely increased accumulation of Aβ pathology through the cortex. 32The clearance of soluble Aβ in brain depended on the transport of lowdensity lipoprotein receptor-related protein 1 (LRP1), hence, the binding of LRP1 to APOE protein blocked the clearance of soluble Aβ.APOE4 protein encoded by APOE ε4 had the highest binding ability to LRP1, and thus had the strongest blocking effect on Aβ clearance. 9In addition, APOE ε4 had a greater effect than other APOE alleles on enhancing APP transcription, promoting transformation of Aβ peptide into neurotoxic Aβ oligomer and fibrils, prolongating half-life of Aβ and inhibiting enzymatic degradation of Aβ in the brain of AD animal models 33,34 and AD patients. 8 This study observed no obvious correlation between APOE ε4 and tau pathology in AD patients.6][37] Nevertheless, there is still a lack of studies on the relationship between APOE alleles and tau pathology in CSF.The subjects included in this study were mainly in the early stage of AD (the median disease duration was 24 months), which might be the reason that the correlation between APOE ε4 and tau level in CSF was not found.

| Neuroinflammation exacerbated cognitive impairment of AD patients
In this study, the increased levels of multiple neuroinflammatory factors in CSF were associated with the compromised cognitive function of AD patients.In detail, the enhanced NO level in CSF was associated with worsened attention and executive function, the elevated IL-1β level in CSF was linked to the impaired language, attention, visuospatial ability, and executive functions, and the increased YKL- predicted the faster cognitive decline in the early stage of AD. 12,39 In this study, high level of YKL-40 in CSF was associated with compromised language function, one of the early impaired cognitive domains in AD, suggesting that overactivation of astroglia might occur in the early stage and was one of the upstream mechanisms of AD.
1][42] Nevertheless, the correlation between sTRME2 level in CSF and cognitive function of AD patients was not understood, and the roles of sTREM2 in the different stages of AD patients remain unclear.Research derived from AD mouse models demonstrated that TREM2 might play a deleterious role in the earlier stage and a protective role in the later stage of AD. 43 In this study, we found that higher sTREM2 level in CSF was associated with poorer language function.The median disease duration of patients included in this study was 24 months, indicating that sTREM2 might exert a negative effect on cognitive function in the early stage of AD.

| APOE ε 4 aggravated cognitive impairment of AD patients via neuroinflammation
The current study showed that APOE ε4 independently predicted the higher NO level in CSF from AD patients.As one of the critical redox active species and a free radical participating in both oxidative and nitrosative reactions, the NO level is increased because of inflammatory activation and oxidative stress in AD. 44 Previous studies on AD mouse models discovered that APOE ε4 significantly drove microglia to transform into the classic activated type (M1 type) and release magnitude of proinflammatory factors, including NO, compared with other APOE alleles. 44,45However, whether APOE ε4 can affect NO level in CSF of AD patients and the precise mechanism by which APOE ε4 promotes NO production are not yet known.Our study was innovative in finding an association between APOE ε4 and NO level in CSF from AD patients, whereas the underlying mechanisms need to be further explored.
In this study, APOE ε4 was not significantly associated with levels of YKL-40 and sTREM2 in CSF from AD patients.A previous study found the relationships between APOE ε4 and YKL-40, 46 the mechanisms underlying their relationships were still unclear.We speculate that the differences in the results among individual investigations may be due to the different race and disease duration of the enrolled patients.Although the positive correlation between APOE and TREM2 was found in animal models of AD, 47 investigations on the relationships between APOE ε4 and sTREM2 in CSF from AD patients are still lacking.Further studies are needed to explore the association of APOE ε4 with YKL-40 and sTREM2 in CSF from AD patients.

| Limitations
This study has the following limitations.Measurement of variables in CSF is one of the most objective ways reflecting the pathophysiological changes in the brains of AD patients.However, multiple factors make it challenging to acquire CSF from the elderly, particularly from people in normal cognitive condition, and we will increase CSF samples from AD patients and collect CSF samples from cognitively normal controls in the future.In addition, this is a cross-sectional study, thus, further longitudinal studies with large samples will be performed to explore the dynamic influence and underlying mechanism of APOE ε4 on neuropathology and neuroinflammation in AD patients.

ACK N O WLE D G E M ENTS
We wish to acknowledge the staff and participants of our study for their contribution.

2 |
MATERIAL S AND ME THODS 2.1 | Subjects Patients who were diagnosed with AD according to the National Institute of Aging and Alzheimer's Association (NIA-AA) criteria were consecutively enrolled in this cross-sectional study from the Center for Cognitive Neurology, Department of Neurology, Beijing Tiantan Hospital, Capital Medical University. 14,15The exclusion criteria were as follows: (1) Patients with neurological diseases that might affect cognition besides AD, including frontotemporal dementia, dementia with Lewy bodies, corticobasal degeneration, Parkinson's disease, multiple sclerosis, and epilepsy, etc. (2) Patients with severe systematic diseases, including heart failure, pulmonary diseases, and kidney failure, etc. (3) Patients with active systemic infections, including pulmonary infection and urinary tract infection, etc. (4) Patients with chronic infectious diseases, malignancy, autoimmune disease or are being treated with steroids, etc. (5) Patients suffered traumatic brain injury recently.
Global cognitive function of patients was assessed by the Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA) scales.In terms of functions of individual cognitive domains, verbal memory was evaluated by the Auditory Verbal Learning Test (AVLT) and visual delayed memory was evaluated by the Rey-Osterreithm Complex Figure Test (RFT)-delayed recall.

2. 8 |
Statistical analysisStatistical analyses were performed by SPSS Statistics 21.0 (IBM Corporation).Statistical significance was defined as a two-sided p < 0.05.Data were tested for normal distribution using the Kolmogorov-Smirnov test.Demographic variables, cognitive function, the levels of neuropathological proteins of AD and neuroinflammatory factors between the APOE ε4 carrier and non-carrier groups were compared.Continuous variables conforming to normal distribution were presented as means ± standard deviations (SD) and compared by two-tailed t test, while non-normal distributed measurement variables were presented as median (quartile) and compared by nonparametric test, and categorical variables were presented as number (percentage) and compared by Chi-Squared test.The correlation of cognitive function with neuropathological proteins of AD and neuroinflammatory factors in CSF were performed by Spearman's correlation analysis and presented by heat maps.Multiple linear regression analysis were further performed to adjust for confounding variables.
lated with TMT-A-time (r = 0.21, p = 0.026) and TMT-B-time (r = 0.38, p < 0.01), demonstrating that higher NO level was significantly correlated with worse attention and executive function.Moreover, IL-1β level in CSF was significantly and negatively correlated with the scores of VFT (r = −0.18,p = 0.043) and RFT-imitation scales (r = −0.21,p = 0.025), while it was significantly and positively correlated with the time spent on SCWT-A (r = 0.22, p = 0.017), SCWT-B (r = 0.23, p = 0.013), and SCWT-C (r = −0.29,p < 0.01) in AD patients, indicating that the elevated IL-1β level was markedly correlated with impaired language, visuospatial ability, attention, and executive function.Additionally, the levels of sTREM2 and YKL-40 were all significantly and negatively correlated with worse language function (p < 0.05).Associations between neuroinflammatory factors in CSF and cognitive function of AD were further validated by linear regression analysis and adjusted for possible confounding factors, including age, disease duration, and education level (Tables 40 level was related to compromised language function.The above data suggested that neuroinflammation exacerbated the functions of overall cognition and multiple cognitive domains of AD patients.Neuroinflammation featured by the overactivation of glial cells, robustly produced a body of neuroinflammatory factors, including TNFα, IL-6, IL-1β and NO, etc., and subsequently led to neuronal damage and cognitive impairment. 38We found that the elevations of neuroinflammatory factors exacerbated the cognitive impairment of AD patients, and different neuroinflammatory factors were involved in different cognitive domains, indicating that these neuroinflammatory factors might play distinct roles in different stages of AD.Recently, a variety of novel neuroinflammatory factors, such as astroglia-expressed YKL-40 and microglia-expressed TREM2, have been discovered.It was found that the increased YKL-40 level in CSF Clinical features of the APOE ε4− and APOE ε4+ groups.
TA B L E 1 Levels of neuropathological proteins between the APOE ε4− and APOE ε4+ groups.
3.24; 95% CI (0.00, 6.47); p = 0.050] and TMT-B [β, 5.24; 95% CI (1.11, 9.37); p = 0.013] after adjusting for the above confounding factors Abbreviations: APOE ε4−, APOE ε4 non-carriers; APOE ε4+, APOE ε4 carriers; APOE, apolipoprotein E; Aβ, β amyloid protein; P-tau, phosphorylated tau; T-tau, total tau.(TableS2).Furthermore, the higher IL-1β level was associated with longer time of SCWT-A [β, 8.10; 95% CI (2.51, 13.68); p = 0.005] and SCWT-C [β, 6.61; 95% CI (0.27, 12.94); p = 0.041], which were independent of age, disease duration, and education level (Table Totally 32.6% AD patients carry APOE ε4.APOE ε4 is associated with poorer cognitive function of AD, particularly the early symptoms of memory, language, and attention.APOE ε4 is associated with lower Aβ 1-42 level in CSF, and the more numbers of APOE ε4 are carried, the lower level of Aβ 1-42 is measured.APOE ε4 is associated with the elevated NO level in CSF, which is linked to the impaired cognitive domains of attention and executive function.Results from this investigation help understand the mechanism underlying the pathogenesis of AD and cast a new light in terms of the pivotal roles of APOE ε4 on neuropathology and neuroinflammation in patients with AD.