By performing a two-sample MR analysis, the present study supports the hypothesis that circulating metabolites levels can be causally corelated to risk of AD. We suggest a significant association between higher levels of Gp and higher risk of AD, and genetically predicted glutamine levels are significantly associated with lower risk of AD. Our results also reinforce the idea that circulating lipid-related metabolites may play a role in the in the pathophysiological process of AD. Particularly, we observed robust evidences of causal effects with respect to ApoB, serum-C, three IDL subfractions (i.e., IDL-C, IDL-L, IDL-P), and 13 LDL subfractions (i.e., LDL-C, L-LDL-C, L-LDL-CE, L-LDL-FC, L-LDL-L, L-LDL-P, L-LDL-PL, M-LDL-CE, M-LDL-L, M-LDL-P, M-LDL-PL, S-LDL-C, and S-LDL-P) on higher risk of AD. To our knowledge, this is the most comprehensive MR analysis to examine the causal associations of circulating metabolites and risk of AD.
The measured Gp are mainly α-1-acid glycoprotein (AGP) , also called orosomucoid. Gp is an acute phase plasma α-globulin glycoprotein, involving in many activities including modulating immunity, binding and carrying drugs, maintaining the barrier function of capillary, and mediating the sphingolipid metabolism [29–31]. Gp is associated with AD due to its important role in modulating neuroinflammation . Higher levels of plasma AGP were found in patients with cognitive impairment than in normal subjects . Previous meta-analysis has reported that plasma levels of Gp were associated with increased risk of dementia and lower cognitive function . These results support our findings suggesting a relationship between circulating Gp and AD.
ApoB is synthesized in the liver and circulates in the plasma as the major protein component of LDL, involving in the transport of cholesterol to peripheral tissues . Previous studies have demonstrated that AD group has significantly higher levels ApoB in serum [35, 36] and plasma  than that of the control group, especially in AD subjects with APOE ε4 allele . In AD patients, higher serum levels of ApoB are significantly correlated with higher Aβ42 levels in brain . Additionally, genetic variants in the gene of APOB are strongly associated with early-onset AD , suggesting a link of ApoB to AD risk. However, previous studies of circulating ApoB levels in human are conflicting, with a large population study finding no association between circulating ApoB levels and incident AD . Therefore, our MR results is a significant evidence enhancing the association between circulating ApoB and AD and suggesting it as causality.
It is reasonable of our findings that many biological studies have reported coincident evidences. Plasma ApoB was found co-localized with cerebral amyloid plaque in a transgenic mouse AD model , and was positively correlated with Ab plaque abundance in brain . Overexpressing APOB in a transgenic mouse model induces significant memory impairment and increases Aβ levels compared with wild-type mice, suggesting that increased ApoB levels can contribute to the development of AD-like pathology .
Whereas ApoB is involved in LDL-C metabolism and is regarded as a promising link between cholesterol and AD , many epidemiological evidences of association between LDL-C and AD are consistent with that of ApoB . Observational studies have indicated that LDL-C levels were significantly increased in AD patients [45–47]. Likewise, Zhou et al. suggest that elevated concentration of LDL-C (> 121 mg/dl) may be a potential risk factor for AD . Our MR analysis support these results and suggest a causal effect of high circulating LDL-C levels in increasing risk of AD. Consistent with our findings, another two published MR study also revealed similar effects of LDL-C using different datasets [49, 50], enhancing reliability of the results.
According to the molecular size, LDLs are further categorized as large (L), medium (M), and small (S) LDLs in initial study . Variation in circulating levels and composition of these fractions may have different pathophysiologic significance. Particularly, plasma levels of L-LDL particles were significantly associated with greater cerebral amyloidosis and lower hippocampal volumes independent of LDL-C . Except for LDL-C, our findings suggest that six L-LDL subfractions, four M-LDL subfractions, and two S-LDL subfractions can influence the AD risk, but further investigations are needed to fully understand the molecular mechanisms involved.
In results of observational studies, the effects of serum-C on risk of AD were highly heterogeneous . With respect to serum-C, several meta-analyses revealed non-significant effect on AD . While other epidemiological studies reported that serum-C levels were significantly increased in AD patients [45–47]. The significance of serum TC differs between mid-life and older adults . Several studies state that high mid-life serum TC levels represent a risk factor for subsequent AD , but that there are no detectable differences in serum TC levels at older ages . Additionally, except for long-term average serum TC levels, higher TC variability is significantly associated with increased risk of all-cause dementia and AD in the general population, independent of mean TC levels . Thus, the in-coincident results between serum-C studies may be explained by the variations in total cholesterol levels and the disease progression. Taking the advantage of not being affected by unmeasured confounders inherent in observational studies , our MR results are more robust, suggesting the high serum-C levels may have a causal effect in increasing AD risk.
Many epidemiological evidences suggest a protective association of circulating HDL cholesterol (HDL-C) levels against AD risk . While we found HDL-C levels are not associated with AD risk with enough power (see Additional file 3), concordant with a large population study . In our study, four very large HDL subfractions (i.e., XL-HDL-C, XL-HDL-CE, XL-HDL-FC, and XL-HDL-P) yield inverse effects in AD risk, however, these sensitivity results showed inconsistent effect estimates against IVW. Investigations are needed to further clarify whether the relationship between HDL and AD are causal.
Our study also reported several metabolic pathways that might be involved in the pathogenesis of AD, in which the D-glutamine and D-glutamate metabolism has been reported to be associated with AD [4, 59]. Observational study showed that glutamine concentrations in plasma is positively correlated with that in posterior cingulate cortex , which is associated with cognitive impairment in AD . We found consistent results that genetically determined circulating glutamine show a protective effect against AD. Nevertheless, a cohort study found higher glutamine levels were associated with lower cognitive function and higher risk of dementia . Whereas observational studies are prone to reverse causation and confounding bias, an MR analysis with balanced horizontal pleiotropy is more credible . Consistent with our results, a published two-sample MR study came to a similar conclusion of glutamine using a different AD dataset . Furthermore, by conducting a series of rigorous sensitivity, pleiotropy, and validation analyses, our results are more comprehensive and robust. Moreover, there also exist biological evidences of this result. Anderson et al. have observed that reduced glutamine metabolism, reduced TCA activity, and impaired oxidative glutamine metabolism precede amyloid plaque formation in AD mouse model compared to controls . And glutamine is proved to protect against oxidative stress-induced injury that is intimately related to AD in AD mice model .
Citrate is key constituent of the TCA cycle, serves as a substrate in the cellular energy metabolism cycle involved in the fatty acid synthesis, glycolysis, and gluconeogenesis . There are very few researches exploring the relationship between citrate and AD. However, our current analysis found a protective effect of citrate in AD risk at significant level. Although additional evidence is needed, it might provide valuable information to help understand the underlying biological mechanisms in the pathogenesis of AD.
Our study also has several limitations. First, a general challenge of MR is the persistent possibility of horizontal pleiotropic associations between exposure and outcome. In the present study, we conducted up-to-date analyses to detect and correct the potential pleiotropy. One limitation is that the Q-test and MR-PRESSO global test is significant in some metabolites. Nevertheless, MR-PRESSO outlier test was further performed to correct for horizontal pleiotropy and returned an unbiased causal estimate. Second, some metabolites yield opposite direction of effect estimates across sensitivity analysis and IVW. It is generally recommended that the emphasis of sensitivity analysis should be laid on the direction of point estimates among the IVW and sensitivity analyses, rather than just the P values. Although this standard ruled out several metabolites from robust results, a serious screening protocol ensure the reliability of our results. For instance, the Gp and glutamine showed the most robust casualty. However, we didn’t have enough evidence that those “non-robust” metabolites are not associated with AD. Third, we used a proxy-AD GWAS dataset to verify our analysis. Hence, the phenotypes used for validation were different from that used in primary analysis, resulting in smaller effect sizes. However, the validation is an independent replication analysis for that there is no overlap between primary AD dataset and validation dataset.
Despite these limitations, strengths of the study are notable. Our study provided novel insight by combining metabolomics with genomics to help understand the pathogenesis of AD. The use of two sample MR approach also enabled us to use the very large AD case–control data, giving sufficient power to detect even small effects. And there is no overlapping among exposure and outcome datasets, as is unachievable in many MR studies that may bias effect estimates. Stronger evidence of causal relationships is of great importance because the AD underlying pathophysiological mechanisms are unclear. If these circulating metabolites levels truly reduce AD risk, it would be promising markers for early detection and potential avenues for effective therapeutic intervention in AD.