This study investigated associations between 90 neurology-related proteins and general fluid cognitive ability in the LBC1936, LBC1921 and INTERVAL. Twenty-two proteins were associated with general fluid cognitive ability in the LBC1936 and in a meta-analysis of LBC1936 and an age-matched INTERVAL sample. Effect sizes, although smaller in INTERVAL-Old, were all in the same direction as in the LBC1936. Another study that measured proteins in two different populations, by using an Olink panel, showed similar differences in the effect sizes of associations with insulin resistance23. Differences in effect sizes may be due to blood from the two cohorts being collected in different tube types (citrate for LBC1936, EDTA for INTERVAL-Old) or differences in the selection bias between the two cohorts. Similar effect sizes to LBC1936 were found for the majority of these 22 proteins in the older LBC1921, indicating that associations do not change between the ages of 73 and 87 years. No replication was identified in INTERVAL-Young, suggesting that age-related changes in protein associations with general cognitive ability may occur. Mediation analysis showed that brain volume mediated the association between ten of the proteins and general fluid cognitive ability. The two proteins that showed the strongest association with total brain, grey matter and normal-appearing white matter volumes (NCAN, BCAN) were not significantly associated with general fluid cognitive ability in the LBC1936, LBC1921 or INTERVAL-Old groups, but were associated in the INTERVAL-Young sample. Similar effect sizes for the associations with cognitive ability were found in LBC1936 and INTERVAL-Young, but these associations were not significant in the smaller LBC1936. The EDA2R protein showed the strongest association with general fluid cognitive ability in the meta-analysis of the LBC1936 and age-matched INTERVAL-Old samples. EDA2R (Ectodysplasin A2 Receptor) is a member of the type III transmembrane protein of the TNFR (tumor necrosis factor receptor) superfamily encoded by EDA2R on chromosome X. This protein is important in hair and tooth development24, and levels of EDA2R have been shown to increase with age in blood25 and lung tissue26. It was also associated with reactive astrogliosis in mice27 and enriched in mouse astrocytes28, indicating that higher levels of this protein may reduce cognitive ability by reducing the number of healthy neurons. Other proteins that were relatively strongly associated with general fluid cognitive ability in the LBC1936 and the meta-analysis of the LBC1936 and INTERVAL-Old sample included sialoadhesin encoded by the SIGLEC1 gene on chromosome 20, a member of the immunoglobulin family29, which may influence cognitive ability through its roles in demyelination and neuroinflammation30; poliovirus receptor encoded by the PVR gene on chromosome 19—viral infections have been previously linked to neurodegeneration31; R-spondin-1 encoded by the RSPO1 gene on chromosome 1 and expressed in the central nervous system during development32; discoidin domain receptor family, member 1 encoded by the DDR1 gene on chromosome 6, which is important in myelination33. The addition of smoking status and antihypertensive drug use as covariates slightly attenuated many of the results.

Interestingly, two chondroitin sulfate proteoglycans (CSPGs) that are common constituents of the extracellular matrix (ECM) and specific to the CNS were strongly associated with brain volume in LBC1936. CSPGs are key members of perineuronal nets (PNNs), which are ECM structures surrounding neurons, important in storage and maintenance of long-term memories34,35,36,37,38,39. Neurocan and brevican are encoded by NCAN (chromosome 19) and BCAN (chromosome 1), respectively, and are expressed in astrocytes and neurons. BCAN is also expressed in oligodendrocytes. These were the only CSPGs on the Olink assay. Neurocan inhibits neuronal adhesion and neurite outgrowth in vitro40. Common genetic variation in NCAN is associated with bipolar disorder41. NCAN is the closest relative of BCAN, and animal knockouts of BCAN and NCAN have a similar phenotype (normal development and memory with deficient hippocampal long-term potentiation)42,43. NCAN peaks in development and declines in the adult brain. In contrast, BCAN is one of the most common CSPGs in the adult brain. It is not yet known what role CSPGs and the PNN may play in age-related cognitive decline; however, our data suggest that NCAN and BCAN are associated with brain volume and may potentially play a neuroprotective role for general fluid cognitive ability in early adulthood. Although expression of NCAN and BCAN is highly specific to the brain, we have shown that levels detected in plasma, in which it is much easier to obtain samples of, also correlate with brain structure. Future studies will be required to confirm these proteins as blood biomarkers of brain structure.

PCA indicated that the levels of the individual proteins were not independent, with 30% of the variance explained by the first PC. The first three PCs derived from the 90 proteins were highly congruent between the four cohorts, providing cross-sample validation of the stability of the proteins’ correlational structure. The first PC was associated with general fluid cognitive ability in the LBC1936, LBC1921, and a meta-analysis of LBC1936 and INTERVAL-Old samples. This association was not mediated by brain variables in the LBC1936, suggesting that the influence on general fluid cognitive ability was independent of the micro- and macrostructural brain variables measured at the global level. Proteins that loaded highly on protein–PC1 included RGM domain family member B (RGMB) that is involved in patterning of the developing nervous system44, and Ephrin-A4 (EFNA4) and Ephrin type-B receptor 6 (EPHB6), both of which are members of the ephrin family that is implicated in the development of the nervous system45. Our data suggest that these proteins may also be important in the ageing nervous system. These findings can serve to sharpen downstream mechanistic and molecular work on the role of specific proteins in processes involved in CNS ageing. Protein–PC3 (like BCAN and NCAN that load highly on protein–PC3) was not associated with general fluid cognitive ability, but was associated with total brain, grey matter and normal-appearing white matter volumes in the LBC1936, suggesting that although it is related to brain volume, it does not do so in a way that affects general fluid cognitive ability. A review looking at how components of PNNs, including BCAN and NCAN, control plasticity, and on their role in memory in normal ageing, concluded that interventions that target PNNs may allow the brain to function well, despite pathology36. Therefore, components of the PNN may protect against changes in brain volume.

The fairly common pattern of protein–cortical associations in the cingulate, temporal and frontal lobes is of interest, as these are among the regions implicated in higher cognitive function46,47,48,49. The five proteins in these analyses showed a moderate level of correlation, but despite this the same vascular risk-type covariates (smoking and hypertension) lead to slightly different levels of attenuation. As was shown in the analyses looking at protein levels and general cognitive ability the least attenuation was identified for PVR. The fact that these vascular risk factors attenuated the associations might indicate the differential relevance of these specific blood biomarkers in the well-established associations between vascular risk and brain structure50.

The strengths of this study include the fact that protein levels, cognitive ability and structural brain variables were measured in the same individuals at about the same time in ~600 members of the LBC1936. Participants in the LBC1936 have a narrow age range and are an ancestrally homogeneous population, which reduces the variability compared with other cohorts. The age-matched INTERVAL cohort for replication of associations with general fluid cognitive ability and the ability to investigate these associations in both an older (LBC1921) and younger (INTERVAL-Young) cohort were further strengths of this study, giving a total sample size larger than most other studies of this type. A key strength of the INTERVAL sample is that they are all healthy blood donors, which minimises confounding by disease status. The Olink Neurology panel was particularly well suited to this study as all proteins were chosen because of a prior link to neurology-related diseases, traits or processes and because it has high sensitivity and specificity20.

The limitations of the study included the fact that the proteins were measured in blood rather than brain tissue. However, as blood samples are relatively easy to obtain, proteins in the blood that are associated with cognitive function and brain structure are more likely to be useful as future biomarkers. Also, a panel of pre-selected neurology-related proteins was used, rather than bespoke assays for proteins that we specifically hypothesised to be associated with cognitive ability and brain structure. One other potential limitation of our investigation is the use of non-fasting plasma samples. However, a recent study concluded that timing of food intake only had a modest effect on the levels of the Olink neurology-related biomarkers used in this study51. The use of citrate blood collection tubes for the LBCs and EDTA blood collection tubes for INTERVAL is potentially a limitation. However, the fact that the within-protein correlational structure was consistent across cohorts, suggests that it was not a significant confound. Another limitation was the lack of a replication cohort that included brain MRI variables. A further limitation is that we investigated cognitive measures at the global level. Potentially counterintuitive findings (such as the protein–PC3 associations with brain volumes but not general fluid cognitive ability) are plausible where specific cognitive abilities are affected. A further potential limitation is the use of different cognitive tests in the LBC1936, the LBC1921 and the INTERVAL sample. Although research has shown that general factors created from different cognitive batteries are highly consistent52,53, and specifically in LBC1936 two general cognitive function phenotypes calculated from two non-overlapping batteries of cognitive tests had a correlation of r = 0.799, a more ideal study would have administered the same cognitive tests to each cohort and extracted a general factor from the combined cohorts.

In conclusion, we have identified several proteins associated with general fluid cognitive ability and brain volume that should be replicated in an independent study before being considered as reliable and possibly useful biomarkers of cognitive ability in later life. Integrating information about these proteins with information about established biomarkers for dementia, such as amyloid β42 and neurofilament light, may help to identify biological pathways to potentially target therapeutically for age-related cognitive decline.