In this study, we used a psychometrically-optimized composite measure of overall episodic memory performance as a phenotype for GWAS in a sample of MCI and AD patients and controls. Then, through a genome-wide pathway analysis, we identified 27 canonical pathways with enrichment against this composite memory phenotype. These enriched pathways suggest that the genetic architecture of memory impairment in this sample spans both processes classically understood to be involved in normal memory consolidation as well as processes with broader roles in cognition and aging, such as those involving neuronal cell adhesion and inflammation.
Moreover, the results of this pathway enrichment study have valuable implications for the future. First, these results illuminate prime cellular, molecular, and genetic targets for future studies of normal and impaired memory states. Indeed, it should be emphasized that pathway-based approaches analyze genetic data in the context of its operative functional groups; as a result, pathway analysis findings are uniquely and naturally connected to the functional biology underlying complex phenotypes. This insight is vital for future investigations, given that pathway mechanisms are principal sources for developing strategies to diagnose, treat, and prevent complex disorders. It is also important to note that our analysis elucidated pathways with robust enrichment despite using GWA input data that included a relatively modest distribution of SNP-level phenotype associations. This insight affirms the potential of pathway-based analytical approaches to detect significant relationships that are otherwise concealed within single-SNP or single-gene analysis. Finally, the use of genome-wide pathway analysis in this study facilitated the detection of unexpected relationships with memory performance, including pathways not classically related to memory signaling, and subsequently, interesting transcriptional and expression networks. While targeted “candidate pathway” approaches have advantages, these unexpected relationships would not have been easily predicted as candidates for analysis based on prior knowledge.
At a functional level, the enriched pathways identified in this study present interesting biological implications in relation to memory impairment. In a sense, it might be expected that cellular and molecular processes classically understood as mediating memory consolidation would constitute a major part of the genetic architecture of memory impairment. However, the processes underlying memory consolidation are numerous and diverse, and to date it has not been clear which specific pathways are essential objects of the impact of genetic variants. The results of our enrichment analysis suggest prime potential components of this genetic architecture. In particular, we observed significant enrichment of pathways related to neurotransmitter receptor activation and downstream signaling. These pathways and their resultant calcium-mediated signaling are vital in converting short-term memories, which exist as axonal firing patterns, into long-lasting changes in synaptic strength (Sweatt 2011
). As a parallel, it makes sense that a composite long-term potentiation pathway (comprising multiple processes leading to long-lasting increases in synaptic strength) would include genetic determinants related to memory performance. Finally, our results indicate the need for further exploration of long-term depression as a substrate for memory impairment, particularly given its proposed roles in mediating the cognitive effects of acute stress and the elimination of synapses in neurodegenerative diseases (Collingridge et al. 2010
Meanwhile, other pathways enriched with regard to memory impairment in this study have previously been implicated in other roles involving neuronal development and cognition. There is an extensive literature on the roles of neuronal cell adhesion molecules (NCAMs) in susceptibility for schizophrenia, bipolar disorder, and autism-spectrum disorders (Corvin 2010
). Genetic variants of NCAMs have also been associated with CSF biomarkers for AD (Han et al. 2010
), and cell adhesion molecule pathways have exhibited enrichment in a GWA-based pathway analysis of AD susceptibility (Liu et al. 2012
). In addition, expression of NCAMs in cholinergic neurons appears to increase susceptibility to AD-related neurodegeneration (Aisa et al. 2010
), and there is emerging evidence of interactions among NCAMs, the MAPK pathway, and amyloid precursor protein (Chen and Dou 2012
). More broadly, these findings suggest a prominent role for cell adhesion pathways in maintaining the processes of synaptic plasticity that are believed to underlie learning and memory (Ho et al. 2011
Finally, it is interesting that pathways on axon guidance, including those involving functions of ephrins, semaphorins, and rho GTPases, were enriched in this study. Axon guidance pathways are key in forming guided neuronal network connections, and have been previously implicated in early neuronal development and associated genetic conditions (Izzi and Charron 2011
). Together with these enrichment results, the proposed interaction between vascular and neuronal factors related to axon guidance (Arese et al. 2011
) in relation to memory may be an important direction for further studies. In addition, given the complex interactions among brain cells and immune system functions, the immune-related pathways enriched in this study suggest additional candidates for modulation of memory and synaptic plasticity (Yirmiya and Goshen 2011
). It may be particularly fruitful to examine immune mediators of memory dysfunction that exert influences independent from amyloid-related activation of microglia in AD (Emilio 2010
A related perspective – and additional interesting targets for future investigations – can be achieved by examining the set of genes that were highly-represented across the enriched pathways in this study. Prominent groups of gene products represented in this set are particularly important in memory consolidation. For example, integrins, cadherins, and alpha-actinin are known to regulate neuronal cytoskeletal structure to mediate synaptic plasticity; further, these molecules are proposed to signal through MAPK cascades for localized protein synthesis at the specific dendrites being activated to precisely potentiate their synaptic connections (Sweatt 2009
). Another important group of gene products is related to the calcium influx that follows neurotransmitter receptor activation at synapses: this calcium influx leads to activation of a signaling axis involving calmodulin, protein kinases (PKA, PKC-α, CAMKII subtypes, and CAMKIV), and transcription factors (CREB subtypes), among other molecules (Sweatt 2009
). Overall, since the highly-represented genes from our data act in numerous pathways, our results demonstrate the importance of studying their gene variants within a pathway-based framework: in this context, variants of moderate individual effect sizes can nevertheless be identified as exerting strong and wide-ranging effects when juxtaposed with other meaningful variants in the same functional unit (Ramanan et al. 2012
). Extensions of this pathway-based analytic framework will be extremely valuable in identifying localized effects of specific pathways on particular brain regions, particularly given that imaging correlates have been identified for loci with known effects on memory, such as the impact of KIBRA
gene variants on hippocampal activation (Pawlowski and Huentelman 2011
). Notably, innovative voxelwise SNP- (Stein et al. 2010a
; Stein et al. 2010b
) and gene-based (Hibar et al. 2011
) imaging genetics approaches have been successfully employed in studies of AD, as has a novel method for generating multivariate “genetic components” for imaging analysis (Meda et al. 2012
). These strategies will serve as rich foundations for future pathway-based imaging genetics analyses.
In addition, the network analyses in this study reinforce the notion that key genes related to memory impairment function coordinately. We found that a preponderance of the most highly-represented genes in our enriched pathways were constituents of a transcriptional regulation network driven by the SP1
transcription factor (). The SP1
transcription factor has known binding regions in the promoters of genes related to beta-amyloid precursor protein (Yu et al. 2010
; Rossner et al. 2006
), tau protein (Santpere et al. 2006
), and APOE
. In particular, SP1
has been proposed as a regulator of APOE
promoter activity in relation to two promoter polymorphisms with significant association to AD (Maloney et al. 2010
). Given that networks of common regulation represent prime targets for identifying common functions, further investigation of the transcriptional network that we have identified may elucidate the as-yet-unknown mechanistic connections among APOE
and other susceptibility loci, AD pathogenesis, and MCI- and AD-related memory impairment.
Finally, expression analysis using the Allen Human Brain Atlas revealed additional functional relationships among key genes. Since strong co-expression of a set of 10 key genes in the brain may indicate common modes of function, further study of this and other similar sets may be of great value. In addition, the co-expression of highly-represented genes from the enriched pathways in this study with known AD susceptibility genes suggests the possibility of significant crosstalk between AD pathogenesis and basic memory processes. While the data in the Allen Human Brain Atlas has several limitations, including a small number of subjects and the inclusion of only postmortem brain tissue from neuropsychologically- and neuropathologically-normal subjects, at present it is the only available resource which integrates multi-modal brain imaging data with whole-brain genome-wide expression data. As such, this and other functional annotation resources will be vital for identifying mechanistic connections between AD pathogenesis and memory impairment, including future efforts to quantitatively assess the significance of overlap between memory pathways and AD pathways. There are some notable limitations to the current study. First, a pathway analysis is only as good as the functional information underlying its pathway definitions. Importantly, some intragenic SNPs may not affect the function or expression of their assigned gene, while other SNPs may functionally impact distant genes or even multiple genes (Kapranov et al. 2007
; Portin 2009
). As functional annotation of the genome becomes more extensive, the power of pathway analyses will heighten. For this study, we used a collection of canonical pathways curated through expert review. While these pathway annotations are expected to have high accuracy, differences across pathway databases can lead to divergent enrichment analysis results (Elbers et al. 2009
). For example, similarly-named pathways can have vastly different gene constituents, while distinctly-named pathways can nevertheless include significant gene overlap. As a result, an early discussion of pathway analysis methods recommended the use of multiple databases for each analysis (Cantor et al. 2010
). While we have followed this recommendation for this analysis, future studies may benefit from formally assessing the relationships in biological coverage among the diverse pathways tested. In addition, at this time there is no gold standard for pathway-based study design. Indeed, different enrichment algorithms and different parameters, such as those guiding SNP-to-gene mapping, can impact analytical results (Gui et al. 2011
). As such, pathway enrichment results benefit from further study using independent replication data sets and using alternative enrichment strategies. While differences across annotation resources, data sets, and analytical strategies may impact SNP- or gene-level statistics (Luo et al. 2010
), legitimately-associated pathways will likely exhibit significant enrichment or strongly-trending signal across a healthy percentage of studies. Finally, while it is beyond the scope of this study, future efforts will benefit from examining key memory-implicated genes and gene sets for epistatic (gene-gene) interactions with each other and with APOE
In addition, there are several caveats about the clinical setting for this study. First, the ADNI cohort represents a clinical trial population and is not a sample of the general population. As a result, the extent to which the present findings can be extended to account for episodic memory impairment in the general population remains to be determined. In addition, it is probable that the memory deficits in this study’s MCI and AD participants are at least partially driven by AD-related pathology. While using APOE
ε4 allele status as a covariate in these analyses likely attenuated this effect, a better understanding of the pathways underlying normal memory and other pathologies than AD may be achieved through studies of normal cohorts and other memory-impaired populations without AD-related pathology. In particular, further exploration of the relationships among APOE
genotype status, amyloid-β load and pathology, and cognition in normal adults (Kantarci et al. 2012
; Buchman and Bennett 2012
) may be especially fruitful. Additionally, meta-analytic approaches to achieve larger study sample sizes may reveal greater SNP-level phenotype associations which could impact the pathway enrichment results. Finally, while this study used a composite episodic memory score optimized on the basis of modern psychometric theory, similar pathway-based studies using other quantitative memory phenotypes may provide different sensitivity and specificity to fine-grained memory deficits and would potentially serve as a validation for the discoveries of pathways enriched against the phenotype used in this study.
Nevertheless, the present results provide several new insights into key functional pathways associated with memory deficits in older adults with MCI or AD and controls. Importantly, these results highlight numerous candidates for further explorations of the SNPs, genes, and gene sets underlying normal memory processes and memory impairment. Overall, these findings encourage further use of pathway-based genetic analyses of quantitative memory phenotypes as statistically-powerful vehicles for discovery and as bridges to underlying biological mechanisms.