An UNC5C Allele Predicts Cognitive Decline and Hippocampal Atrophy in Clinically Normal Older Adults

INTRODUCTION

More than half of the heterogeneity in late-life cognitive decline remains unexplained even after accounting for common neuropathologies such as Alzheimer’s disease (AD), vascular brain injury, or Lewy body disease [1]. Understanding the genetic architecture of this variable resilience to neuropathologies could enable personalized prognostication and novel therapeutic target identification.

A recent genome-wide association study (GWAS) showed a relationship between two independent common alleles, UNC5C rs3846455^G and ENC1 rs766629990^A, and worse cognition after accounting for known neuropathologies [2]. UNC5C rs3846455^G is of a particular interest, given other studies that implicated two other independent variants within the gene in cognitive aging: a rare UNC5C mutation (T835M) increases AD dementia risk by increasing hippocampal susceptibility to pathologic insults such as amyloid-β (Aβ) [3], and a common variant, rs28660566^T, is associated with a higher burden of cerebral amyloid angiopathy, an Aβ-related cerebrovascular pathology [4]. UNC5C is abundantly expressed in adult human hippocampus, suggesting the gene’s potential role in hippocampus [5].

However, the association of rs3846455^G with vulnerability to late-life cognitive decline should be further examined in an independent cohort, and the relationship between rs3846455^G and longitudinal hippocampal neurodegeneration in older adults needs to be investigated. This knowledge gap should be addressed, especially in clinically normal older adults for whom neuroprotective treatments are most likely to prevent extensive neurodegeneration [6].

Therefore, we sought to do this in the current study, leveraging the structural and molecular neuroimaging data from the Harvard Aging Brain Study (HABS), a longitudinal cohort study that enrolls clinically normal older adults [7]. We aimed to replicate the previously reported association between rs3846455^G and greater cognitive decline [2], adjusting for baseline Aβ burden measured in vivo. In addition, we hypothesized that rs3846455^G would predict accelerated hippocampal atrophy, which could potentially account for the cognitive decline associated with rs3846455^G.

METHODS

RESULTS

The characteristics of the study participants (n = 174) are summarized in Table 1. Our participants had average 5.7 PACC measurements (range 4 to 7), and average 2.6 HV measurements (n = 87 with two measures, n = 63 with three measures, and n = 24 with four measures). Among these participants, there were 15 rs3846455^G minor allele carriers (14 heterozygotes and one homozygote), and the MAF was 0.05, similar to the European reference population MAF of 0.06 from the 1000 Genomes project [15, 16]. Baseline characteristics were not significantly different between rs3846455^G carriers and non-carriers (Table 1).

The rs3846455^G carrier status was not associated with baseline PACC (p = 0.17; adjusted for age, sex, years of education, and EV1–3), but the SNP was weakly associated with longitudinal decline in PACC, after controlling for the time interaction terms of baseline Aβ, baseline age, sex, years of education, and EV1–3 (β= –0.087/year, 95% CI –0.169 to –0.005, p = 0.039; Table 2 model 2; Fig. 1B). The standardized effect size (estimated effect (“β-value”) divided by standard error) of rs3846455^G carrier status was –2.07, which is about 40% of the standardized effect size of baseline Aβ (–5.42) from the same model. Excluding a participant with rs3846455^GG genotype from Table 2 model 2 did not significantly change the result (β= –0.106/year, 95% CI –0.191 to –0.021, p = 0.015). Unadjusted association between rs3846455^G carrier status and longitudinal PACC also reached statistical significance (β= –0.090/year, 95% CI –0.179 to –0.001, p = 0.048), supporting that statistical over-fitting did not drive our result.

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Fig.1

rs3846455^G carrier status and longitudinal changes in bilateral hippocampal volume and Preclinical Alzheimer Cognitive Composite. A) PACC change over time was predicted from a linear mixed effect model, controlling for interaction of time (years from baseline) with baseline Aβ, baseline age, sex, years of education, and EV1–3. Predicted trajectories of PACC were plotted by UNC5C rs3846455^G minor allele carrier status. B) HV change over time was predicted from a linear mixed effect model, controlling for total intracranial volume and interaction of time (years from baseline) with baseline Aβ, baseline age, sex, and EV1–3. Predicted trajectories of HV were plotted by UNC5C rs3846455^G minor allele carrier status. Aβ, baseline amyloid-β measured by Pittsburgh compound B (PiB) positron emission tomography distributed volume ratio (DVR) from composite cortical regions; EV1–3, first three principal components from the genotype covariance matrix; HV, hippocampal volume; PACC, preclinical Alzheimer cognitive composite.

The rs3846455^G carrier status was not associated with baseline HV (p = 0.33; adjusted for age, sex, intracranial volume, and EV1–3). However, rs3846455^G was associated with longitudinal decline in HV (β= –57.1 mm³/year, 95% confidence interval (CI) –103.0 to –11.2, p = 0.015; Table 2 model 3), even after additionally adjusting for baseline Aβ (β= –57.3 mm³/year, 95% CI –102.8 to –11.9, p = 0.014; Table 2 model 4, Fig. 1A). The standardized effect size of rs3846455^G carrier status on longitudinal HV change was –2.49, slightly greater than the standardized effect size of baseline Aβ (–2.01) from the same model. The association between rs3846455^G and longitudinal HV was consistently observed in an unadjusted model without covariates (β= –48.1 mm³/year, 95% CI –95.3 to –0.85, p = 0.046). Notably, this association was no longer statistically significant when we excluded one participant with rs3846455^GG genotype from Table 2 model 4 (β= –42.9 mm³/year, 95% CI –90.1 to 4.3, p = 0.075). We note that in a post-hoc analysis only assuming random intercepts (i.e., not allowing random slopes) in Table 2 model 4, we observed an association between rs3846455^G and longitudinal HV, even when the rs3846455^GG participant was excluded (β= –52.3 mm³/year, 95% CI –95.0 to –9.6, p = 0.017).

Finally, we performed a post-hoc mediation analysis to assess whether the association between rs3846455^G and hippocampal volume loss (Table 2 model 4) mediates the more rapid PACC decline observed with the rs3846455^G allele (Table 2 model 2). In this mediation model with rs3846455^G as the independent variable, HV slope as the mediator variable, and PACC slope as the dependent variable, the effect of rs3846455^G on PACC decline was partially mediated by faster progression of hippocampal atrophy in rs3846455^G carriers (mediated effect on PACC slope = –0.014, 95% CI –0.032 to –6.0×10^–4, p = 0.039; Fig. 2). The direct effect of rs3846455^G on PACC decline not mediated through HV slope was not statistically significant (p = 0.29; Fig. 2).

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Fig.2

Mediation of the association of rs3846455^G with cognitive decline by accelerated hippocampal atrophy. Causal mediation analysis was performed using quasi-Bayesian Monte Carlo method (with 10,000 simulations), having rs3846455^G as an independent (causal) binary variable, HV slope (adjusted for baseline age, sex, and ICV) as a continuous mediator variable, and PACC slope (adjusted for baseline age, sex, and years of education) as the continuous outcome variable. We also adjusted for baseline Aβ and EV1–3 in the mediator and outcome models that we used for the simulation. Mediated (indirect) effect (ACME) and direct effect (ADE) were calculated. Solid arrow indicates a significant mediated effect (p < 0.05), and dotted arrow indicates a direct effect that did not reach statistical significance (p = 0.29). Aβ, amyloid-β measured by Pittsburgh compound B (PiB) positron emission tomography distributed volume ratio (DVR) from composite cortical regions; ACME, average causal mediation effect (the effect of the independent variable on the outcome that is mediated through the mediator); ADE, average direct effect (the effect of the independent variable on the outcome that is independent from the mediator); EV1–3, first three principal components from the genotype covariance matrix; HV, hippocampal volume; ICV, intracranial volume; PACC, Preclinical Alzheimer Cognitive Composite.

DISCUSSION

In this study, leveraging neuroimaging data from HABS, we replicated the previous finding from clinical-pathologic cohorts (the Religious Orders Study (ROS) and the Rush Memory and Aging Project (MAP)) that an UNC5C haplotype captured by rs3846455^G is associated with worse cognitive trajectories in older adults, even after controlling for measurable neuropathology burden [2]. As HABS and ROS-MAP are independent, non-overlapping observational studies of cognitive aging, our study provides an independent validation of a role of the UNC5C rs3846455^G haplotype in interpersonal variability in cognitive decline, and demonstrates the utility of neuroimaging biomarkers in the ante-mortem studies of genetically determined susceptibility to age-related cognitive decline.

UNC5C is a gene with roles in axon guidance during development [17, 18] and apoptosis regulation [19]. UNC5C is a functional dependence receptor that can induce apoptosis in the absence of its ligand (netrin) [19] and has an enriched expression in adult human and rodent hippocampus [3, 5]. Overexpression of UNC5C increased susceptibility of cultured rat hippocampal neurons to various neurotoxic insults [3], suggesting the role of UNC5C in modulating hippocampal susceptibility to neurodegeneration. Moreover, the previously observed association of rs3846455^G with cognitive decline was specific to the episodic memory domain that depends on hippocampus [2], further implicating UNC5C in hippocampal susceptibility to neurodegeneration.

In this context, our hypothesis-driven characterization of a candidate polymorphism shows that the UNC5C rs3846455^G haplotype is associated with accelerated hippocampal atrophy in older adults who were clinically normal at baseline. This association partially mediated the association of rs3846455^G with accelerate cognitive decline, in a post-hoc mediation modeling. This finding is line with previous experimental and clinical-pathologic studies of UNC5C, and expands our understanding of the rs3846455^G haplotype. However, our study has a modest sample size, and the association between rs3846455^G carrier status and accelerated hippocampal atrophy was no longer statistically significant after excluding the single participant that is homozygous for the risk allele (rs3846455^GG genotype) (p = 0.075). Therefore, the previously unreported association between rs3846455^G and accelerated hippocampal atrophy requires further validation by replication studies with larger sample size.

UNC5C rs3846455^G haplotype was not associated with baseline Aβ, hippocampal volume, or cognition in our study. This is consistent with the previous clinical-pathologic study with close to 1,000 participants that did not find a significant relationship of rs3846455^G with any of the measured post-mortem neuropathologies or baseline cognition [2], as well as a large GWAS meta-analysis on a cross-sectional mid-adulthood hippocampal volume in more than 26,000 participants [20]. Thus, the clinical implication of the haplotype captured by rs3846455^G is likely specific to the older adults at risk of cognitive decline.

The molecular mechanism by which the UNC5C haplotype captured by rs3846455^G increases the susceptibility to cognitive decline in older adults remains unclear. As cellular overexpression of wild-type UNC5C increases apoptosis in cultured hippocampal neurons [3], it is plausible to hypothesize that functional variants increasing UNC5C expression in hippocampus could lead to accelerated hippocampal neurodegeneration. UNC5C rs3846455^G is an intronic SNP within an enhancer region in human hippocampus, per the ChromHMM core 15-chromatin state prediction [21] based on the Roadmap Epigenomics Project [22]. This functional prediction suggests that the SNP might alter UNC5C gene expression in the hippocampus, but rs3846455 was not significantly associated with hippocampal UNC5C mRNA expression in the Genotype-Tissue Expression database (n = 111) [22]. Given the SNP’s relatively low MAF (0.06), larger number of hippocampal samples from older adults will be required to examine the expression quantitative trait locus association.

Our study has several important limitations. First and foremost, we had a very limited sample size for a genetic association study (n = 177), and the study sample included only fifteen rs3846455^G carriers. Therefore, our results should be interpreted with caution, and the newly found association between rs3846455^G and accelerated hippocampal atrophy as well as the subsequent mediation analysis requires further validation. Also, due to our limited sample size, we could not assess interactive association of Aβ and rs3846455^G, a potential genetic risk factor of hippocampal neurodegeneration, despite multiple previous reports that Aβ and neurodegeneration have synergistic predictive value for cognitive decline among clinically normal older adults [23 –25]. Nonetheless, our study is an independent replication of the previously reported link between rs3846455^G and cognitive decline [2], and the link between rs3846455^G and hippocampal neurodegeneration was found through a hypothesis-driven candidate polymorphism analysis based on previous reports [2, 3]. Thus, our results are likely capturing a true effect rather than spurious associations. Second, our analyses were restricted to participants from European ancestry, and the results cannot be easily extrapolated to people of non-European ancestry. Third, HABS participants are highly educated and in good physical and cognitive health at the time of enrollment. As such, they are not entirely representative of the general population of older adults. Still, MAF of rs3846455^G in our participants was not significantly different from the reference panel [15, 16], indicating a genetically representative sampling. Fourth, there were only 14% of APOE ɛ4 carriers among the rs3846455^G carriers, while 31% of the rs3846455^C homozygotes were APOE ɛ4 carriers. Although this imbalance did not reach statistical significance, it might have led to a slight underestimation of the effect of the rs3846455^G allele on hippocampal atrophy and cognitive decline. Finally, the mediation analysis should be interpreted cautiously, given that this was a simplified post-hoc model assuming a linear relationship among rs3846455^G carrier status, HV change, and PACC change.

In conclusion, leveraging longitudinal data with approximately five-year average follow-up, we validated the previously reported association of the UNC5C rs3846455^G haplotype and faster cognitive decline in older adults. Moreover, our results suggest that accelerated hippocampal atrophy might partially mediate the association between 3846455^G and faster cognitive decline in clinically normal older adults. This study also demonstrates the utility of modestly sized, but deeply phenotyped cohorts in investigating the genetics of complex endophenotypes such as cognitive decline not explained by neuropathology [26]. Nonetheless, neuroimaging studies with larger numbers of participants including individuals of non-European descent are required to further validate our results and understand the role of the UNC5C rs3846455^G haplotype in more diverse populations. Further investigations of the functional mechanism of the UNC5C rs3846455^G haplotype may open additional therapeutic avenues for neuroprotection in older adults at high risk of cognitive decline.