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 Aβ_1–42 level, and the more numbers of APOE ε4 are carried, the lower level of Aβ_1–42 is measured. APOE ε4 is associated with elevated NO level, which is linked to the impaired attention and executive function.

Apolipoprotein E (APOE) ε4 is associated with poorer early symptoms of Alzheimer's disease (AD). The more numbers of APOE ε4 are carried, the lower level of β amyloid protein (Aβ)_1–42 is measured. APOE ε4 is associated with elevated nitric oxide (NO) level, which is linked to impaired attention and executive function.

1. INTRODUCTION

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. ⁴ This 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. There are three types of alleles of APOE, including ε2, ε3, and ε4, and APOE ε4 is the highest risk gene for sporadic AD. ⁵ Compared with other APOE alleles, APOE ε4 carriers showed a higher risk of developing sporadic AD, earlier onset age, higher rate of cognitive decline, and poorer cognitive function. ⁵ , ⁶ , 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 , ⁹ Moreover, APOE ε4 carriers had more tau aggregation and a greater extent of somatodendritic tau redistribution in the brain in comparison to other APOE alleles. ¹⁰ In addition, several animal model studies explored that the extent of glial hyperplasia in the brain and levels of TNF‐α, IL‐1β, and IL‐6 of APOE ε4 carriers were greater than that of other APOE alleles. ¹⁰ , ¹¹

Nevertheless, 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.

2. MATERIALS AND METHODS

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 , ¹⁵ The 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. (6) Patients undergone major surgery.

2.2. 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.

2.3. Assessments of cognitive function

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. ¹⁶ , ¹⁷ Language was evaluated by the Verbal Fluency Test (VFT) and the Boston Naming Test (BNT). ¹⁸ , ¹⁹ Attention 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. ²² Visuospatial 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.

2.4. 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 𝜀2, 𝜀3, and 𝜀4, was performed by real‐time Fluorescence Quantitative Polymerase Chain Reaction by using nucleic acid detection reagents (Youzhiyou company).

2.5. 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 JingkeHongda Biotechnology Co., Ltd) and frozen for 0.5 mL per tube at −80°C until the assay. ²³

2.6. Measurements of neuropathological proteins of AD in CSF

Neuropathological proteins of AD, including Aβ_1–42 (CSB‐E10684h kit, CUSABIO Company), total tau (T‐tau) (CSBE12011h kit, CUSABIO Company), P‐tau (T181) (KHB7031 kit, Invitrogen), P‐tau (S199) (KHO0631 kit, Invitrogen), P‐tau (T231) (KHB7041 kit, Invitrogen), and P‐tau (S396) (KhB8051 kit, Invitrogen) were detected by ELISA.

2.7. Measurements of neuroinflammatory factors in CSF

Neuroinflammatory factors, including TNF‐α (Human TNF alpha Uncoated ELISA kit, Invitrogen), IL‐1β (Human IL‐1 beta Uncoated ELISA kit, Invitrogen), IL‐6 (Human IL‐6 Uncoated ELISA kit, Invitrogen), IFN‐γ (Human IFN gamma Uncoated ELISA kit, Invitrogen), sTREM2 (Human TREM2 DuoSet ELISA, R&D Systems), and YKL‐40 (ProcartaPlex™ Immunoassay Kit, Invitrogen) were detected by ELISA. NO (A013‐2‐1 kit, Nanjing Jiancheng Biological Engineering Research Institute) and hydroxy radical (OH) (A018‐1 kit, Nanjing Jiancheng Biological Engineering Research Institute) were measured by chemical colorimetry. ²⁴

2.8. Statistical analysis

Statistical 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.

3. RESULTS

3.1. The frequency of the APOE ε4 in AD patients

A total of 383 AD patients were enrolled in this study, 125 cases (32.6%) carried APOE ε4, among whom four cases (1.0%) carried the APOE ε2/ε4, 96 cases (25.1%) carried the APOE ε3/ε4, and 25 cases (6.5%) carried the APOE ε4/ε4. In addition, 29 cases (7.6%) carried the APOE ε2/ε3, and the APOE ε3/ε3 was the most frequent one (59.8%) (Figure 1).

FIGURE 1.

The frequency of apolipoprotein E (APOE) genotypes in patients with Alzheimer's disease (AD). There are five APOE genotypes in this cohort, including ε2/ε3 (7.6%), ε2/ε4 (1.0%), ε4/ε4 (6.5%), ε3/ε4 (25.1%), and ε3/ε3 (59.8%).

3.2. Association of APOE ε4 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 non‐carrier group. The APOE ε4 carrier group had a significantly higher frequency of AD‐D than the APOE ε4 non‐carrier group (p < 0.001). (Table 1).

TABLE 1.

Clinical features of the APOE ε4− and APOE ε4+ groups.

	APOE ε4− group (n = 258)	APOE ε4+ group (n = 125)	p
Female [n (%)]	149 (57.75)	76 (60.80)	0.617
Age [years, median (quartile)]	66.00 (60.00, 74.00)	68.00 (62.00, 74.00)	0.150
Age of onset [years, median (quartile)]	62.00 (55.00, 70.00)	65.00 (58.00, 72.00)	0.143
Education level [n (%)]			0.109
Primary school and below	54 (20.93)	27 (21.60)
Middle and high school	126 (48.84)	60 (48.00)
Bachelor's degree and above	78 (30.23)	38 (30.40)
Smoking [n (%)]	61 (23.64)	26 (21.00)	0.519
Drinking [n (%)]	53 (20.54)	24 (19.20)	0.792
BMI [median (quartile)]	23.89 (21.89, 26.11)	23.40 (21.30, 24.90)	0.087
Resting heart rate [Times/minute, median (quartile)]	72.00 (69.50, 78.00)	72.00 (69.00, 76.00)	0.344
Systolic blood pressure [mmHg, median (quartile)]	132.00 (122.00, 143.00)	133.50 (124.75, 148.50)	0.195
Diastolic blood pressure [mmHg, median (quartile)]	81.00 (75.00, 87.75)	83.00 (75.75, 90.00)	0.336
History
Hypertension [n (%)]	102 (39.53)	43 (34.40)	0.404
Hyperlipidemia [n (%)]	49 (18.99)	29 (23.20)	0.489
Myocardial infarction [n (%)]	3 (1.16)	1 (0.80)	0.892
Atrial fibrillation [n (%)]	2 (0.78)	2 (2.00)	0.512
Diabetes mellitus [n (%)]	49 (18.99)	17 (13.60)	0.253
Hyperhomocysteinemia [n (%)]	3 (1.16)	16 (12.80)	0.897
Cerebrovascular disease [n (%)]	37 (14.34)	10 (10.00)	0.119
Sleep apnea syndrome [n (%)]	14 (5.42)	6 (4.80)	0.954
Depression [n (%)]	16 (6.20)	11 (8.80)	0.686
Disease duration [years, median (quartile)]	24.00 (12.00, 48.00)	24.00 (12.00, 48.00)	0.730
Stage of disease [n (%)]
MCI	150 (58.14)	50 (40.00)	0.001**
Dementia	108 (41.86)	75 (60.00)
Cognitive function
Global cognitive function
MMSE [points, median (quartile)]	22.00 (15.00, 26.00)	19.00 (11.00, 24.50)	0.005**
MoCA (points, mean ± SD)	15.01 ± 7.44	12.93 ± 7.41	0.011*
Individual cognitive domain
Memory
AVLT N1‐3 (points, mean ± SD)	11.46 ± 6.04	8.98 ± 5.67	<0.001**
AVLT N4 [points, median (quartile)]	1.00 (0.00, 4.00)	0.00 (0.00, 2.00)	<0.001**
AVLT N5 [points, median (quartile)]	0.00 (0.00, 4.00)	0.00 (0.00, 2.00)	<0.001**
RFT delayed recall [points, median (quartile)]	3.00 (0.00, 10.00)	0.00 (0.00, 8.00)	0.027*
Language
VFT (points, mean ± SD)	33.05 ± 16.64	26.88 ± 16.06	<0.001**
BNT [points, median (quartile)]	23.00 (18.00, 26.00)	21.00 (14.00, 25.00)	0.023*
Attention
SDMT (points, mean ± SD)	21.09 ± 15.27	19.83 ± 21.41	0.571
TMT‐A time (seconds, mean ± SD)	105.99 ± 72.95	128.59 ± 76.28	0.012*
SCWT‐A time [seconds, median (quartile)]	40.00 (30.00, 54.00)	49.89 (27.00, 60.00)	0.925
SCWT‐B time [seconds, median (quartile)]	53.00 (39.00, 72.50)	55.50 (40.43, 80.00)	0.620
Visuospatial ability
RFT imitation [points, median (quartile)]	27.25 (8.75, 34.00)	22.00 (2.00, 33.00)	0.255
Executive function
SCWT‐C time (seconds, mean ± SD)	107.97 ± 73.25	102.81 ± 61.18	0.554
TMT‐B‐time [seconds, median (quartile)]	207.00 (122.00, 240.00)	240.00 (161.00, 240.00)	0.050

*p < 0.05, **p < 0.01.

Abbreviations: APOE ε4−, APOE ε4 non‐carriers; APOE ε4+, APOE ε4 carriers; APOE, apolipoprotein E; AVLT, Auditory Verbal Learning Test; BMI, body mass index; BNT, Boston Naming Test; MCI, mild cognitive impairment; MMSE, Mini‐Mental State Examination; MoCA, Montreal Cognitive Assessment; RFT, Rey‐Osterrieth Complex Figure Test; SCWT, The Stroop Color and Word Test; SDMT, Symbol Digit Modalities Test; TMT, Trail Making Test; VFT, Verbal Fluency Test.

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 non‐carrier 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.

3.3. Association among APOE ε4, neuropathological proteins in CSF, and cognition in AD patients

3.3.1. 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 linear regression analyses showed APOE ε4 was negatively associated with Aβ_1–42 level after adjusting for age, gender, and disease duration [β, −1.13; 95% CI (−2.15, −0.11); p = 0.031].

TABLE 2.

Levels of neuropathological proteins between the APOE ε4− and APOE ε4+ groups.

	APOE ε4− group	APOE ε4+ group	p
Aβ1–42 [ng/mL, median (quartile)]	0.79 (0.44, 1.09)	0.63 (0.35, 1.00)	0.023*
P‐tau (T181) [ng/mL, median (quartile)]	58.25 (31.94, 89.84)	72.30 (37.94, 90.47)	0.737
P‐tau (S199) [ng/mL, median (quartile)]	6.43 (4.29, 11.34)	7.21 (4.11, 11.77)	0.932
P‐tau (T231) [ng/mL, median (quartile)]	81.79 (64.74, 107.60)	86.10 (59.45, 112.16)	0.239
P‐tau (S396) (ng/mL, mean ± SD)	66.38 ± 27.47	60.21 ± 28.17	0.949
T‐tau [ng/mL, median (quartile)]	89.12 (72.62, 122.93)	94.83 (66.18, 123.83)	0.365

*p < 0.05.

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.

Aβ_1–42 level from the APOE ε4−/−, the APOE ε4+/−, and APOE ε4+/+ groups were compared (Figure 2). In comparison to the APOE ε4−/− group, Aβ_1–42 level in CSF was reduced in the APOE ε4+/− group (p = 0.879) and significantly decreased in the APOEε4+/+ group (p = 0.011). In addition, compared with the APOE ε4+/− group, Aβ_1–42 level was markedly declined in the APOEε4+/+ group (p = 0.009).

FIGURE 2.

Aβ_1–42 level between the groups of APOE ε4−/−, APOE ε4+/− and APOE ε4+/+. APOE ε4+/−, single APOEε4 allele carriers; APOE ε4+/+, double APOE ε4 alleles carriers; APOE, apolipoprotein E; Aβ, β amyloid protein; APOE ε4−/−, APOE ε4 allele non‐carrier. APOE ε4−/− versus APOE ε4+/+, *p < 0.05; APOE ε4+/− versus APOE ε4+/+, **p < 0.01.

3.4. 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).

FIGURE 3.

Heat map of correlations between neuroinflammatory factors, Aβ_1–42 and cognition in AD patients. AVLT, Auditory Verbal Learning Test; Aβ, β amyloid protein; BNT, Boston Naming Test; IFN‐γ, interferon‐γ; IL‐1β, interleukin‐1β; IL‐6, interleukin‐6; MMSE, Mini‐Mental State Examination; MoCA, Montreal Cognitive Assessment; NO, nitric oxide; OH, hydroxy radical; RFT, Rey‐Osterrieth Complex Figure Test; SDMT, Symbol Digit Modalities Test; SCWT, The Stroop Color and Word Test; sTREM‐2, triggering receptor expressed on myeloid cells‐2; TMT, Trail Making Test; TNF‐α, tumor necrosis factor‐α; VFT, Verbal Fluency Test; YKL‐40, Tyr‐Lys‐Leu‐40. *p < 0.05, **p < 0.01.

In terms of individual cognitive domains, Aβ_1–42 level was also significantly and positively correlated with the scores of AVLT N1‐3 (r = 0.26, p = 0.003), AVLT N4 (r = 0.25, p = 0.006), AVLT N5 (r = 0.20, p = 0.026), RFT‐delayed recall (r = 0.27, p = 0.005), VFT (r = 0.30, p < 0.001), SDWT (r = 0.34, p < 0.001), and RFT imitation (r = 0.25, p = 0.009). These results suggested that more Aβ_1–42 deposition in 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. 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).

3.5. Association among APOE ε4, neuroinflammatory factors in CSF, and cognitive function

3.5.1. Association between APOE ε4 and the levels of neuroinflammatory factors

Multiple linear regression analyses were conducted to explore associations between APOE ε4 and levels of neuroinflammatory factors, including TNF‐α, IL‐1β, IL‐6, IFN‐γ, NO, OH, sTREM2, and YKL‐40 in CSF. It was found that APOE ε4 was markedly associated with elevated NO level in CSF after adjusting for age, gender and disease duration [β, 2.24; 95% CI (0.18, 4.30); p = 0.033] (Table 3).

TABLE 3.

Association between levels of neuroinflammatory factors and APOE ε4 in AD patients.

	Unadjusted		Adjusted
	β (95%CI)	p	β (95%CI)	p
TNF‐α (pg/mL)	2.08 (−0.17,4.33)	0.070	2.14 (−0.22, 4.50)	0.075
IL‐1β (pg/mL)	0.24 (−0.69, 1.16)	0.618	0.16 (−0.81, 1.14)	0.744
IL‐6 (pg/mL)	0.02 (−0.43, 0.48)	0.918	−0.02 (−0.49, 0.44)	0.920
IFN‐γ (pg/mL)	−0.11 (−1.99,1.77)	0.906	−0.27 (−2.26, 1.72)	0.787
NO (μmol/L)	2.24 (0.18, 4.30)	0.033*	2.58 (0.40, 4.77)	0.021*
OH (U/mL)	−42.85 (−101.50, 5.81)	0.151	−52.03 (−113.04, 8.98)	0.094
STREM‐2 (pg/mL)	−72.30 (−240.23, 95.69)	0.395	−82.51 (−246.54, 81.51)	0.321
YKL‐40 (pg/mL)	5764.47 (−6400.52, 17929.44)	0.347	3564.20 (−9368.21, 16496.62)	0.583

Age, gender, disease duration, and education level were adjusted. AD, Alzheimer's disease; APOE ε4‐, APOE ε4 non‐carriers; APOE ε4+, APOE ε4 carriers; APOE, apolipoprotein E; IFN‐γ, interferon‐γ; IL‐1β, interleukin‐1β; IL‐6, interleukin‐6; NO, nitric oxide; OH, hydroxy radical; sTREM‐2, triggering receptor expressed on myeloid cells‐2; TNF‐α, tumor necrosis factor‐α; YKL‐40, Tyr‐Lys‐Leu‐40. *p < 0.05.

3.6. 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 correlated 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 S2‐S5). The results showed that the higher NO level was also associated with longer time of TMT‐A [β, 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 (Table S2). 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 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).

4. DISCUSSION

4.1. Frequency of the APOE ε4 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.

4.2. APOE ε4 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 previous study showing that APOE ε4 carriers had an earlier onset age of AD compared with other APOE alleles carriers. ²⁷ The differences might be related to the different race, age, and duration of disease of the population between our and other investigations. Large‐scale epidemiological study from China is required to clarify the link between APOE ε4 and demographic variables, such as age, age of onset, and gender, etc.

In this study, APOE ε4 was obviously associated with poorer cognitive function. AD patients with APOE ε4 had considerably worse overall cognitive function than those without APOE ε4. Previous studies demonstrated that AD patients with APOE ε4 had poorer cognitive function, particularly the significantly severe memory impairment, ²⁸ , ²⁹ which was possibly because APOE ε4 was associated with temporal lobe atrophy (especially hippocampus) and dysfunction in the default mode network. ²⁹ , ³⁰ Furthermore, 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.

4.3. APOE ε4 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. ³¹ A longitudinal study based on Aβ‐PET imaging showed that AD patients with APOE ε4 had diffusely increased accumulation of Aβ pathology through the cortex. ³² The clearance of soluble Aβ in brain depended on the transport of low‐density 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. ⁹ In 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 These data further demonstrated that APOE ε4 played an important role on AD pathology in the early stage of disease. Nevertheless, clinical studies on the relationship between APOE ε4 and Aβ in CSF were insufficient, although the alterations of neuropathological protein in CSF generally precede imaging changes.

Hence, we particularly focused on the association between APOE ε4 and Aβ_1–42 level in CSF in AD patients, and found that APOE ε4 was markedly and negatively correlated with the significantly decreased Aβ_1–42 level in CSF, and innovatively demonstrated that quantitative dependence of APOE ε4 on Aβ_1–42 level in CSF. That is, the more numbers of APOE ε4 that AD patients carried, the heavier brain burdens of Aβ_1–42 that AD patients had.

This study observed no obvious correlation between APOE ε4 and tau pathology in AD patients. Previous studies revealed that APOE ε4 carriers had greater tau aggregation in the brain of AD animal models ³⁵ , ³⁶ and AD patients ³⁷ compared to other APOE allele carriers. ³⁵ , ³⁶ , 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.

4.4. 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‐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 TNF‐α, IL‐6, IL‐1β and NO, etc., and subsequently led to neuronal damage and cognitive impairment. ³⁸ We 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 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. In addition, results from AD mouse models suggested that TREM2 deficiency increased the volume of neuritic plaques in brain, induced tau hyperphosphorylation, promoted neuroinflammation, and exacerbated cognitive impairment. ⁴⁰ , ⁴¹ , 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.

4.5. APOE ε4 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 , ⁴⁵ However, 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.

4.6. 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.

5. CONCLUSION

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.

AUTHOR CONTRIBUTIONS

Mingyue He contributed to the conception, design, and data statistics of the study and paper writing; Tenghong Lian, Peng Guo, Weijiao Zhang, Huiying Guan, Jinghui Li, Dongmei Luo, Weijia Zhang, Wenjing Zhang, Jing Qi, and Hao Yue contributed to the acquisition and collation of data; Yue Huang, Yanan Zhang, Gaifen Liu, and Xiaomin Wang contributed to direct paper writing and data statistics; Wei Zhang contributed to the conception and design of the study and the supervision of paper writing.

FUNDING INFORMATION

This research was supported by The National Key R&D Program of China‐European Commission Horizon 2020 (2017YFE0118800‐779,238); The National Key Research and Development Program of China (2016YFC1306300, 2016YFC1306000); The National Natural Science Foundation of China (81,970,992, 81,571,229, 81,071,015, 30,770,745, 82,201,639); Capital's Funds for Health Improvement and Research (CFH) (2022–2‐2048); The Key Technology R&D Program of Beijing Municipal Education Commission (kz201610025030); The Key Project of Natural Science Foundation of Beijing, China (4161004); The Natural Science Foundation of Beijing, China (7082032); Project of Scientific and Technological Development of Traditional Chinese Medicine in Beijing (JJ2018‐48); Capital Clinical Characteristic Application Research (Z121107001012161); High Level Technical Personnel Training Project of Beijing Health System, China (2009‐3‐26); Project of Beijing Institute for Brain Disorders (BIBD‐PXM2013_014226_07_000084); Excellent Personnel Training Project of Beijing, China (20071D0300400076); Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges Under Beijing Municipality (IDHT20140514); Beijing Healthcare Research Project, China (JING‐15‐2); Basic‐Clinical Research Cooperation Funding of Capital Medical University, China (2015‐JL‐PT‐X04, 10‐JL‐49, 14‐JL‐15); Natural Science Foundation of Capital Medical University, Beijing, China (PYZ2018077); Youth Research Funding, Beijing Tiantan Hospital, Capital Medical University, China (2015‐YQN‐14, 2015‐YQN‐15, 2015‐YQN‐17).

CONFLICT OF INTEREST STATEMENT

The authors report no competing interests.

Supporting information

Appendix S1

He M, Lian T, Guo P, et al. The roles of apolipoprotein E ε4 on neuropathology and neuroinflammation in patients with Alzheimer's disease. CNS Neurosci Ther. 2024;30:e14440. doi: 10.1111/cns.14440

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the first author or the corresponding author upon reasonable request.