APOE4 Allele Disrupts Resting State fMRI Connectivity in the Absence of Amyloid Plaques or Decreased CSF Aβ42

Discussion

In the current study, participants carrying an APOE4 allele had clear-cut abnormalities in precuneus resting-state functional connectivity in the absence of any cognitive impairment, and in the absence of fibrillar cerebral Aβ deposits detectable by PET PIB imaging. This result suggests that genetic influences altered functional connectivity within the DMN before the onset of clinical disease. We have previously shown an APOE4 dose-dependent increase in MCBP values, with an odds ratio of 5.04 (95% CI, 2.53–10.2) for likelihood of high MCBP in individuals with an APOE4 allele (Morris et al., 2010). Effects of the APOE4 allele have been demonstrated in amyloid imaging studies in individuals with moderate AD (Drzezga et al., 2009) and in cognitively normal individuals (Reiman et al., 2009; Small et al., 2009; Morris et al., 2010). In addition, in AD, the APOE4 allele has been shown to have greater impairment on memory retention, whereas noncarriers were more impaired on tests of working memory, executive function, and language (Wolk et al., 2010). We observed, in the absence of fibrillar Aβ plaque deposition, regional alterations in fcMRI in many of the same brain areas that show disrupted connectivity in PIB+ individuals and in frank AD (Sheline et al., 2010). Interestingly, most of the regions identified with effects of APOE4, in particular, bilateral hippocampus and left parahippocampus, had decreased connectivity with the precuneus. This result stands in contrast to two small resting-state studies of APOE4 effects on DMN finding increased connectivity (Filippini et al., 2009; Fleisher et al., 2009). There was no effect of regression analysis on results using CSF Aβ₄₂ as a covariate. Further, in post hoc analyses of the subset of 70 participants screened to exclude those with abnormally low CSF Aβ₄₂ levels, we found similar patterns of abnormal functional connectivity, although there were less regions that survived, perhaps due to a reduced sample size (n = 70 vs 100) or to an effect of CSF Aβ₄₂ levels. These results suggest that mechanisms resulting in increased neuronal toxicity might already be in place before lowered Aβ₄₂ CSF levels.

Altered functional connectivity between the posterior and anterior portions of the default-mode network has been described in AD (Greicius and Menon, 2004) and in preclinical Aβ plaque deposition (Hedden et al., 2009; Sheline et al., 2010). We found these same regional differences in precuneus connectivity in the current study as seen in AD, with the exception of increased anticorrelation with dorsal AC and left superior temporal cortex/frontal-parietal operculum. In addition to differences predicted from a priori regions in AD, we also found additional regions with alterations in precuneus connectivity in APOE4 allele carriers in whole-brain exploratory analyses, indicating that altered resting-state functional organization already had occurred in normal aging in the presence of APOE4 genotype but in the absence of deposition of Aβ plaques. However, interpretive caution is warranted since differences were observed in exploratory analyses and require replication in an independent sample. One interesting discrepancy was observed between the increased connectivity with MPFC in the a priori analysis versus decreased connectivity with MPFC in whole-brain analysis. We note, however, that the MPFC region from whole-brain analysis was considerably more caudal (z = −12) than the a priori region (z = 18), and as such has different medial network connections (Carmichael and Price, 1996). The significance of these regions of altered connectivity remains to be determined but, as discussed below, may reflect activity-dependent APOE4 effects on Aβ amyloid deposition and on neuronal stability. We note that most of the alterations involved decreased connectivity with the precuneus and most of these additional regions were also in the DMN, highlighting the importance of early damage to the DMN as a potential manifestation of preclinical AD.

Regions in the DMN (Raichle et al., 2001) exhibit structural and functional connectivity that converge on the precuneus and posterior cingulate (Hagmann et al., 2008). During attention-demanding tasks, there are decreases in activity, correlated with task difficulty and successful memory encoding (Lustig et al., 2003; Daselaar et al., 2004). Patients with mild AD (Lustig et al., 2003), mild cognitive impairment (Petrella et al., 2007), and preclinical Aβ amyloid deposition (Sperling et al., 2009), however, have demonstrated a paradoxical reversal in this pattern, with increased task-related fMRI activity in areas of Aβ deposition, implicating amyloid deposition in dysfunctional brain networks supporting memory formation. Further, we have shown that the regions with earliest Aβ deposition lie within the DMN and also have the highest resting metabolic activity (Buckner et al., 2005). Since DMN regions are known to have the highest resting-state metabolic activity, it may be that those APOE4-mediated mechanisms that depend on metabolic activity, such as decreased transport and damage to astroglia, occur at a higher rate within the DMN regions, and that these effects predate deposition of Aβ plaques.

The effect of APOE4 appears to operate through both Aβ metabolism effects and non-Aβ effects. Clear evidence supports Aβ metabolism effects as a major mechanism for APOE genotype influence on AD. Histopathological studies demonstrated a positive correlation between APOE4 dose and Aβ plaque density (Rebeck et al., 1993) and increased neuritic plaques (Tiraboschi et al., 2004). Further, cognitively normal subjects had an APOE4 dose-dependent increase in fibrillar Aβ plaque burden, detected by amyloid imaging (Reiman et al., 2009). Studies have also found that APOE4 was more effective than APOE3 at increasing Aβ40 aggregation (Wisniewski et al., 1994). In addition to effects on fibrillogenesis, there is evidence that APOE4 alters both brain transport and metabolism of Aβ. Aβ in complex with APOE2 and APOE3 is cleared more rapidly from the brain than Aβ complexed to APOE4 (Deane et al., 2008).

Additional evidence suggests that APOE4 also plays a role in non-Aβ-mediated mechanisms (Zhong and Weisgraber, 2009), resulting in decreased neuronal stability and plasticity. Among critical differences are a lower stability of the APOE4 isoform with greater propensity to form toxic aggregates (Hatters et al., 2006); lower E4 affinity for small lipoproteins, resulting in less effective transport of lipids required for neuronal synaptogenesis, maintenance, and repair (Dong et al., 1994); and increased cleavage of APOE4 by neuronal protease, forming fragments that escape the secretory pathway and lead to neuronal death (Harris et al., 2003). An isoform-specific effect of APOE on neurite outgrowth has been reported and, although inconclusive regarding the effects of APOE on neuronal processes (Kim et al., 2009), it is possible that astrocytes contribute more directly to the association of APOE4 and AD than had previously been thought. An interesting phenomenon involves a switch in APOE4 expression. Normally APOE is only expressed in astrocytes and other glial cells. However, under stressful conditions, neurons also express APOE (Xu et al., 2006). Further, APOE4 produced within neurons is subject to cleavage by neuronal protease, resulting in toxic fragments. These toxic fragments have been observed in AD patients and in transgenic mice expressing APOE4 in neurons (Zhong et al., 2009). An additional pathological mechanism involves APOE4 alteration of the ability of astrocytes to facilitate neuronal function: Over most of a lifetime, although compromised, astrocyte function is sufficient in non-stressed conditions (Zhong et al., 2009). However, in the presence of later-life stressors such as ischemia, oxidative stress, and Aβ toxicity, astrocyte support can become less effective. Thus, APOE4 can contribute to AD pathology by acting through both Aβ-dependent and Aβ-independent pathways.

Although many APOE4 changes in our study occurred in the same areas that are affected in AD and these effects could be interpreted as very early manifestations of AD, our exploratory whole-brain analyses revealed other regions with altered functional connectivity and may represent APOE4 effects that are independent of AD pathology. For example, APOE4 genotype could be associated with a genetically altered functional connectivity, some portion of which is unassociated with AD. Alternatively, these changes could have pathological significance and be associated with toxic APOE4 allele effects independent of AD-related toxicity. The current finding that APOE4 allele carriers had alterations in precuneus resting-state functional connectivity with other important DMN regions highlights the importance of integrity of the DMN (Raichle et al., 2001). Indeed, a role for an APOE4 effect mediated through metabolic demands is suggested by the distribution of preclinical fibrillar Aβ falling within DMN regions (Shulman et al., 1997; Raichle et al., 2001; Buckner et al., 2005), which have very high resting metabolic demands. Evidence from mouse models (Cirrito et al., 2005) suggests neuronal activity-dependent increase in production of Aβ. In APOE4 allele carriers, who have chronic deficient astrocyte support, the vulnerability of DMN regions to neuronal damage could be triggered in the face of additional burden, such as ischemia or oxidative stress. This could help to explain the greater risk and earlier age of onset of dementia in APOE4 carriers. Longitudinal studies will be important to determine the time course of any alterations in resting-state functional connectivity, both those that appear independent of fibrillogenesis and, particularly, those that appear to progress to neuronal dysfunction and damage, to help plan the optimal timing for potential interventional studies in individuals at risk for symptomatic AD.