Association of plasma apolipoprotein B with cerebrospinal fluid Alzheimer’s disease biomarkers in patients with subjective cognitive decline

The preclinical stage of Alzheimer's disease (AD) has become a key target stage for future AD prevention trials. Plasma apolipoprotein, an important lipid-related factor, has been found to be involved in the pathogenesis of AD. This study was to examine whether apolipoprotein B (ApoB), apolipoprotein A-1 (ApoA1) or the ratio of ApoB and ApoA1 (ApoB/A1) were associated with early changes of cerebrospinal fluid (CSF) AD biomarkers in elderly adults with subjective cognitive decline. Methods This study finally included 201 cognitive normal (CN) elderly adults and 101 participants with subjective cognitive decline (SCD) from the Chinese Alzheimer’s Biomarker and Lifestyle (CABLE) database. The One-way analysis of variance, t-test, Kruskall-Wallis test and Chisquare test were applied in the intergroup comparisons. Multiple linear regression models were used to examine the cross-sectional associations of plasma ApoB, ApoA1 and ApoB/A1 levels with CSF AD-related biomarkers. This study is the first to find some protective associations of plasma ApoB, but not ApoA1, with CSF AD core biomarkers in the preclinical stage of AD. This finding indicated that ApoB may play different roles in different stages of AD. Further studies to identify the underlying mechanisms would greatly strengthen the link between ApoB and AD pathogenesis, which may contribute to the discovery of new pathogenic mechanisms and therapeutic targets. apolipoprotein

selected for genotyping with restriction fragment length polymorphism (RFLP) technology, including the two loci related to APOE4 status (rs7412 and rs429358).

Standard protocol approvals, registrations, and patient consents
The CABLE database was conducted in accordance with the Helsinki declaration, and the research program was approved by the Institutional Ethics Committee of Qingdao Municipal Hospital. All study participants or their caregivers provided written informed consent directly.

Statistical analysis
One-way analysis of variance (ANOVA), t-test, Kruskall-Wallis test and Chisquare test were applied in the comparison among groups. Amyloid imaging and neuropathological studies showed that approximately one-third of older adults without objective cognitive impairment had AD pathology in their brains [20][21][22][23]. Therefore, in our study, CSF biomarker positive participants were defined as having CSF Aβ42 levels in the lower onethird of the distribution of participants (A+: ≤ 116.11 pg/mL) or having p-tau (T+: ≥ 39.17 pg/mL) or t-tau (N+: ≥ 180.59 pg/mL) levels in the upper one-third of the distribution. All plasma and CSF variables were log-transformed to normalize the distributions. Multiple linear regression models were used to examine the cross-sectional associations of baseline plasma ApoB, ApoA1 and ApoB/A1 ratio with CSF biomarkers, adjusting for age, sex, education and APOEɛ4 status. Statistical analyses were conducted using R, version 3.5.1. A two-tailed p < 0.05 was considered significant.

Characteristics of participants
For the current study, we analyzed data from 302 participants of the CABLE cohort, including 201 CN and 101 SCD (see Table 1 for participants' characteristics). The average age of participants was 63 years; 111 (63%) participants were male; and 40 (13%) participants were APOEɛ4 positive. We found no significant difference in age, sex distribution or level of education between two subgroups. Frequency of positive APOEε4 status showed an increasing trend in SCD subgroup (CN: 11.94%, SCD: 15.84%), although these results were not statistically significant.

Levels of plasma ApoB and ApoA1 in different diagnostic groups
As for levels of plasma ApoB, in a clinical diagnostic construct, we did not find difference between CN and SCD subgroups (Figure 1a). In an ATN biological construct, all CSF biomarker positive subgroups had lower levels of plasma ApoB compared with CSF biomarker negative subgroups (Figure 1d, e, f). Then, according to the 2018 NIAA research framework, we chose A+T-N-, A+T+N-and A+T+N+ as AD continuum and chose A-T-N-as control group. Results showed that A+T+N+ subgroup showed significantly lower levels of plasma ApoB than A-T-N-(p < 0.0001) and A+T-N-(p = 0.0038) subgroups. Furthermore, in a diagnostic structure combining clinical diagnosis and biomarkers, SCD participants with A+ had significantly lower levels of plasma ApoB than CN participants without pathologic changes in CSF biomarkers (p = 0.0018) ( Figure 1c). As for ApoA1, we didn't find any difference in both clinical construct and biological construct (results were not shown).
Furthermore, we added the interaction between APOEɛ4 status and ApoB into multiple linear regression models. Results showed that these associations between ApoB and Aβrelated biomarkers were independent of APOEε4 status. In these models, both ApoB and APOEɛ4 status were associated with Aβ-related biomarkers, but their interactions were not (see Additional file 1). Moreover, subgroup analyses of APOEɛ4 status also showed that associations between ApoB and Aβ-related biomarkers were similar for different APOEɛ4 statuses (see Additional file 2).

Associations of ApoB, ApoA1, and ApoB/A1 with CSF AD core biomarkers in different diagnostic subgroups
Results from subgroup analyses of different diagnostic groups were shown in Table 3. As for ApoB, these associations between ApoB and Aβ-related biomarkers (Aβ42, Aβ42/40, ttau/Aβ42 and p-tau/Aβ42) still remained significant in both CN and SCD subgroups (Table 3, Figure 3). It is worth noting that these associations were more significant in the SCD subgroup than in the CN subgroup (Table 3). In addition, we found that higher levels of ApoB were also associated with decreased CSF t-tau (β = -0.37, p = 0.0224), p-tau (β = -0.24, p = 0.0086) and Aβ40 (β = -0.43, p = 0.0297) in SCD subgroup, while these associations were not found in the CN subgroup (Table 3, Figure 3). As for ApoB/A1, significant associations between ApoB/A1 and all CSF biomarkers (Aβ42, t-tau, p-tau, Aβ40, Aβ42/40, t-tau/Aβ42 and p-tau/Aβ42) were found only in the SCD subgroup but not in the CN subgroup (Table 3). We did not find any association between ApoA1 and all CSF biomarkers (Aβ42, t-tau, p-tau, Aβ40, Aβ42/40, t-tau/Aβ42 and p-tau/Aβ42).
Given that there may be different metabolic states of apolipoproteins between overweight and normal populations, we further tested our results found above in different weight participants that were classified according to Body Mass Index (BMI) (normal weight participants: BMI < 25, overweight participants: BMI ≥ 25). To our surprise, in both CN subgroup and SCD subgroup, almost all of associations found above existed only in the overweight participants but not in normal weight participants (Table4, Table 5).

Discussion
This study is the first to systematically explore cross-sectional associations of CSF AD core biomarkers with ApoB, ApoA1, and ApoB/A1 in elderly adults without objective cognitive impairment. The main finding of this study was that plasma ApoB may have some protective effects on CSF AD core biomarkers in elderly adults without objective cognitive impairment independently of APOEε4 genotype, especially in the SCD overweight population. This finding was novel and potentially important, because it suggested that levels of plasma ApoB added information beyond APOEε4 status on the level of CSF AD core biomarkers in the preclinical stage, which may contribute to the discovery of new pathogenic mechanisms and therapeutic targets.
First of all, we analyzed the characteristics of SCD participants included in our study.
Results showed that, compared with the CN subgroup, although there was no significant cognitive decline, there were obvious changes in CSF AD core biomarkers (especially Aβ-related biomarkers) in the SCD subgroup, which were consistent with previous studies [8,24]. In fact, accumulating cross-sectional or longitudinal evidence has supported that SCD occured at the preclinical stage of AD and might serve as a symptomatic indicator of preclinical AD [7,8,[24][25][26][27][28][29][30]. Our analysis of population characteristics showed that, in our study, SCD participants had preclinical AD characteristics.
Then we explored the distribution of ApoB and ApoA in the diagnostic and biological constructs. Results showed that SCD participants with significant AD biological characteristics had a lower ApoB level ( Figure 1) suggesting that the level of ApoB may decrease in the preclinical stage of AD.
Finally we detailed the associations of ApoB, ApoA1, and ApoB/A1 with CSF AD core biomarkers in total participants or different diagnostic subgroups and analyzed the factors that might affect these associations. We firstly found that plasma ApoB and ApoB/A1 were associated with CSF AD core biomarkers, especially with the Aβ-related biomarkers (Aβ42, Aβ42/40, t-tau/Aβ42 and p-tau/Aβ42). It is worth noting that these associations were much more significant in SCD participants. Surprisingly, inconsistent with previous studies, above associations showed that plasma ApoB and ApoB/A1 appeared to have some protective effects that may alleviate the early pathological changes of AD. To be precise, higher ApoB and ApoB/A1 were associated with increased CSF Aβ42 and Aβ42/40, as well as with decreased CSF t-tau, p-tau, Aβ40, t-tau/Aβ42 and p-tau/Aβ42. No previous studies have focused on the associations of plasma ApoB and ApoB/A1 with CSF AD core biomarkers in cognitively normal or SCD participant. However, two autopsy studies in AD patients indicated that ApoB may be a risk factor for the late stage of AD. One showed that serum ApoB were positive associated with the amount of Aβ42 in AD brains [14]. The other showed that ApoB immunoreactivity was positively associated with cerebral amyloids [13]. The exact causes of these seemingly contradictory results were still unclear, but we thought that there were several possible reasons as follows.
Firstly, the different stages of the disease might contribute to this contradiction. Previous studies have found that changes in the blood-brain barrier contributed differently to the dynamic changes of CSF AD core biomarkers in different stages of disease. CSF AD core biomarker (especaially Aβ42) may be temporarily elevated and its significant deposition in brain may occur when the blood-brain barrier was significantly impaired [31]. As a possible transporter of CSF biomarkers ApoB may facilitate the processes of early clearance and late deposition of biomarkers in brain. In addition, in the stage of the disease, atherosclerosis and other vascular damages caused by ApoB-LDL may also aggravate the pathological changes, which may not be apparent in the early stage of the disease.
Secondly, genetic factors may also contribute to this contradiction. In this study, we tested the influences of APOEε4 status on above associations. We found that these protective associations were independent of the APOEε4 status, based on the following evidences. a) Both plasma ApoB and APOEε4 status were associated with CSF AD core biomarkers, but their interaction was not associated with these biomarkers. b) The results of the subgroups analysis were similar among population with different APOEε4 status.
This independence on APOEε4 was also seen in a previous study on the association between serum ApoB and amount of Aβ42 in AD brains [14]. Collectively, these data provided evidences that ApoB and APOEε4 status may contribute to the changes of CSF AD core biomarkers, independently of each other. However, a recent study found that rare genetic coding variants of APOB gene were strongly associated with familial AD, which indicated that APOB gene might also harbor protective and deleterious variants in sporadic AD similar to the APOEε2 and APOEε4 alleles [32]. Identifying such a protective coding variant of APOB gene would greatly strengthen the link between ApoB and AD pathogenesis.
In addition, some other factors may also contribute to this contradiction, such as changes involved low density lipoprotein cholesterol metabolism (the low density lipoprotein receptor and proprotein convertase subtilisin/kexin type 9). So given the concept that normal weight populations and overweight populations often have different lipid metabolism status, we further tested our results found above in different weight participants. It is worth noting that these protective effects of plasma ApoB on CSF AD core biomarkers were particularly stronger in overweight participants compared with the normal weight participants. These results indicated that different lipid metabolism status may also influence above associations, which may contribute to the contradiction.
Moreover, growing body of studies suggested that overweight in older age may be a protective factor for AD. Overweight older adults had a lower incidence or a later onset of AD than those of normal weight or low weight [33][34][35]. Our results indicated that the protective effect of plasma ApoB on CSF AD core biomarkers may be the potential mechanism. In other words, the plasma ApoB's ability to alleviate early pathological changes may contribute to slow the progression of the disease in overweight older adults, but not in normal weight older adults.
Overall, we found protective associations of plasma ApoB with CSF AD core biomarkers, these associations may be affected by disease's stages and lipid metabolism status or some other indefinite factors such as genetic factors. In this study we did not find any associations between ApoA1 and CSF AD core biomarkers, which indicated that ApoA1 may not have significant effects on the early pathological changes in population without objective cognitively impairment.
There were some potential limitations in our study. Firstly, this was a cross-sectional study and the results still needed to be tested in larger longitudinal cohort. Secondly, the sample sizes of some subgroups were limited, which may affect the results to some extent.

Conclusions
This study is the first to find some protective associations of plasma ApoB, but not ApoA1, with CSF AD core biomarkers in the preclinical stage of AD. This finding indicated that ApoB may play different roles in different stages of AD. Further studies to identify the underlying mechanisms would greatly strengthen the link between ApoB and AD pathogenesis, which may contribute to the discovery of new pathogenic mechanisms and therapeutic targets.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study was approved by the local ethics committee, and all individuals gave written informed consent to participate.

Consent for publication
Not applicable.   Abbreviations: CN, cognitively normal participants; SCD, participants with subjective cognitive decline; CSF, cerebrospinal fluid; Aβ, amyloid-β; t-tau, total tau protein; p-tau, Bold indicated that the results were statistically significant.
Multiple linear regression models were used to examine the crosssectional associations between baseline plasma ApoB, ApoA1 and ApoB/A1 levels with CSF biomarkers, adjusting for age, sex, education and APOEɛ4 status. Bold indicated that the results were statistically significant.
Multiple linear regression models were used to examine the crosssectional associations between baseline plasma ApoB, ApoA1 and ApoB/A1 levels with CSF biomarkers, adjusting for age, sex, education and APOEɛ4 status.  Bold indicated that the results were statistically significant.
Multiple linear regression models were used to examine the crosssectional associations between baseline plasma ApoB, ApoA1 and ApoB/A1 levels with CSF biomarkers, adjusting for age, sex, education and APOEɛ4 status.  Associations between ApoB and CSF AD biomarkers in total participants Abbreviations: CSF, cerebrospinal fluid; Aβ, amyloid-β; t-tau, total tau protein; ptau, phosphorylated tau protein; ApoB, Apolipoprotein B Multiple linear regression models were used to examine the cross-sectional associations between baseline plasma ApoB levels with CSF biomarkers, adjusting for age, sex, education and APOEɛ4 status.

Figure 3
Associations between ApoB and CSF AD biomarkers in SCD participants Abbreviations: CSF, cerebrospinal fluid; Aβ, amyloid-β; t-tau, total tau protein; ptau, phosphorylated tau protein; ApoB, Apolipoprotein B Multiple linear regression models were used to examine the crosssectional associations between baseline plasma ApoB levels with CSF biomarkers, adjusting for age, sex, education and APOEɛ4 status.

Supplementary Files
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