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

Background The preclinical stage of Alzheimer's disease (AD) has become a key target stage for future AD prevention trials. Serum 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 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 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 serum ApoB, ApoA1 and ApoB/A1 levels with CSF AD-related biomarkers. Results Compared with the control group, SCD participants with significant AD biological characteristics had lower ApoB levels. In the total participants, higher level of serum ApoB was associated with increases in CSF Aβ42 (p = 0.0009) and Aβ42/40 (p = 0.0038) as well as decreases in CSF t-tau/Aβ42 (p < 0.0001) and p-tau/Aβ42 (p < 0.0001), independent of APOEɛ4 status. In the further subgroup analysis, these associations were much more significant in the SCD participants. In addition, higher levels of serum ApoB were also found associated with decreases in CSF t-tau (p = 0.0224), p-tau (p = 0.0086) and Aβ40 (p = 0.0297) in the SCD subgroup. Furthermore, we found that these protective associations between serum ApoB and CSF AD core biomarkers were much more significant in the overweight participants. Results showed no association between ApoA1 and all CSF biomarkers in either total participants or subgroups. 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.


Levels of serum ApoB and ApoA1 in different diagnostic groups
As for levels of serum 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 serum 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 serum ApoB than A-T-N-(p = 0.0009) ( Figure 1b). Furthermore, in a diagnostic structure combining clinical diagnosis and biomarkers, SCD participants with A+ had significantly lower levels of serum ApoB than CN participants without pathologic changes in CSF biomarkers (p = 0.0093) ( Figure 1c). As for ApoA1, we didn't find any difference in both clinical construct and biological construct (results were not shown).

Associations of ApoB, ApoA1, and ApoB/A1 with CSF AD core biomarkers in total participants without objective cognitive impairment
The results on associations of ApoB, ApoA1, and ApoB/A1 with CSF biomarkers in total participants without objective cognitive impairment were shown in Table 2. Results showed that higher levels of serum ApoB were significantly associated with increased levels of CSF Aβ42 (β = 0.34, p = 0.0009) and Aβ42/40 (β = 0.35, p = 0.0038) but not with Aβ40 (Table 2, Figure 2). Although no association of ApoB was found with t-tau and p-tau, higher levels of serum ApoB were significantly associated with decreased levels of CSF t-tau/Aβ42 (β = -0.47, p < 0.0001) and p-tau/Aβ42 (β = -0.41, p < 0.0001) (Table 2, Figure 2). All results remained significant after Bonferroni correction. Similar associations were found of ApoB/A1 with these Aβ-related biomarkers (Aβ42, Aβ42/40, t-tau/Aβ42 and p-tau/Aβ42) (Table 2), but these associations seemed less significant than those of ApoB (Table 2). 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). Furthermore, we added the interaction between APOEɛ4 status and ApoB into multiple linear regression models. Results showed that these associations between ApoB and Aβ-related biomarkers were independent of APOEε4 status. In these models, both ApoB and APOEɛ4 status were associated with Aβ-related biomarkers, but their interactions (ApoB×APOEε4) were not (see Additional file 1).
Moreover, subgroup analyses of APOEɛ4 status also showed that associations between ApoB and Aβrelated biomarkers were similar for different APOEɛ4 statuses (see Additional file 2).
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 serum 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 serum 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 occurred 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 serum 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 serum 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 serum 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 late 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 serum 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 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 serum 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 serum ApoB on CSF AD core biomarkers may be the potential mechanism. In other words, the serum 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 serum 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.
However, a recent liquid chromatography-tandem mass spectrometry based proteomics study highlighted plasma ApoA1 as a biomarker in Alzheimer's disease and Aβ burden, even in cognitively unimpaired individuals [36]. So the effects of ApoA1 on the early pathological changes in population without objective cognitively impairment still need to be tested in more studies.
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 was the first to find some protective associations of serum 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.   Bold indicated that the results were statistically significant.
Multiple linear regression models were used to examine the crosssectional associations between baseline serum 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; p-tau, phosphorylated tau protein; ApoB, Apolipoprotein B Multiple linear regression models were used to examine the cross-sectional associations between baseline serum 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; p-tau, phosphorylated tau protein; ApoB, Apolipoprotein B Multiple linear regression models were used to examine the crosssectional associations between baseline serum ApoB levels with CSF biomarkers, adjusting for age, sex, education and APOEɛ4 status.