Our results yielded four primary findings. First, the earliest impetus for pathogenic processes in APOE4 individuals may come from a variety of abnormalities in signaling cascades and biological processes that involve calcium regulation, mitochondrial function, cell-cycle regulation abnormalities, apoptosis and wnt signaling. Several of these have been at the periphery of discussions about the pathogenesis of AD; in our study, they were found to be central. Second, there may be active protective processes as well as pathological processes. Third, our data suggest that there is a long prodromal period before the onset of clinical AD, given that mean age at death of our sample was 42 years. Finally, we identified lower levels of APOE transcript in BA 1/2/3 compared with BA 21. Thus, even in individuals who carry an APOE4 allele, relatively low amounts of this isoform are present in BA 1/2/3. Speculatively, this may account for the region’s relative invulnerability to histopathology, either because expression levels are not high enough to promote pathogenesis or to interfere with neuroprotective adaptations. In contrast, conformation of the E4 variant including its structural and biophysical features,51,52
in conjunction with relatively high expression levels, may result in increased pathology in susceptible regions.
The MDS clustering also discriminated APOE3 and APOE4 cases, thus indicating that even in this early stage in a putative disease process, separation at the level of individual cases seemed to be present. The heat map data were consistent with this interpretation. Transcripts that are included as significant in these analyses were EGFR, CNTFR, CASP6, ZNF580, GRIA2, CTNNB1, FKBPL, LGALS1 and PSMC5. These transcripts were not only statistically highly significant, but were in key biological or signaling pathways that have relevance to AD pathogenesis. Analyses in IPA were consistent with this view. We found that the transcripts that we validated in RT-qPCR had multiple direct and indirect relationships (‘edges’) among themselves and APP and MAPT processing, strongly suggesting that they affect the disease process.
We identified 22 signaling pathways and biological processes that differed between the APOE3 and APOE4 groups. Several may be of special interest because they have previously been implicated in AD, including oxidative phosphorylation/mitochondrial function, calcium signaling, cell-cycle reentry, neuroactive ligand–receptor interactions, wnt signaling and proteasome functionality. Our study suggests that they are involved in early, presumptively pathogenic features of the disease process. Our results in mitochondrial function were particularly striking. Although mitochondria have become an area of increasing importance in the study of AD and other dementias,53-56
it has generally been assumed that amyloid plaques or toxic amyloid fragments interfere with mitochondrial function.57
Our results suggest that compromises in mitochondrial function may precede plaque formation, in keeping with experiments suggesting that mitochondrial dysfunction may precede amyloid aggregation.56
In our study, the transcripts found to be most different between the APOE3 and APOE4 groups in this pathway were consistently downregulated in temporal lobe in the APOE4 group and included NDUF7. This is consistent with previous microarray work on mitochondrial function in AD brain tissue, that found abnormalities in complexes I, II, III, IV and V.58
Nevertheless, it is likely that later in the disease process amyloid misprocessing amplifies mitochondrial dysfunction.59
Although we did not identify molecules directly associated with amyloid processing, we are not suggesting that the amyloid cascade is not relevant for AD. There is overwhelming evidence for its importance from highly penetrant genetic mutations, transgenic animals, neuropathology and molecular experiments.60
Rather, we think that the data in this study suggest that in APOE4-related AD, the amyloid cascade may be a consequence of earlier abnormalities in multiple neurobiological pathways; that is, it may be a cascade within a cascade.
The cluster analyses of signal intensity profiles in 70 transcripts suggest the possibility that active or dynamic protective responses occur in the BA 1/2/3, as presumptive susceptibility related changes in expression are occurring in BA 21. Nevertheless, it is possible that these changes are obligatory adaptations to E4 and are neither protective nor pathogenic in nature, that is, are neutral. Although we cannot fully rule out the latter hypothesis, we do not believe that is it likely because many of the individual genes and pathways have been implicated in AD. We also appreciate that not every individual with an APOE4 allele will develop AD. Nevertheless, as based on population prevalence figures, we estimate that only about two to three APOE3 individuals (of 28), while five to six APOE4 individuals (of 13) in the sample would have gone on to manifest AD.
The fold-change differences that we observed were often subtle. However, they survived rigorous statistical procedures. Moreover, because our subjects died B30 years on average before any of the clinical manifestations of dementia, we expected that fold-change differences would be rather small, but nevertheless measurable and reproducible.
Several previous human microarray studies with emphasis on APOE or preclinical AD are relevant to our study. Although these studies did not directly examine regional differences, nor use APOE status in younger cases to separate early pathogenic processes from the consequences of pathology, there are nevertheless several points of comparison, including abnormalities in the expression of such individual transcripts as NDUFB7 and GRIA2, transcription-repressor factor upregulation and mitochondrial dysfunction.61-65
Detailed discussion of this human post-mortem study, as well as alternative cellular approaches to vulnerability, can be found in the Supplementary Discussion
More broadly, the long molecular prodrome that we found in APOE4-related AD is consistent with several neurophysiological studies and some neurocognitive studies. Both glucose metabolism reductions and abnormalities in fMRI BOLD signal in temporal lobe structures of APOE4 carriers have been found.66,67
APOE4 may also negatively affect memory function and rate of decline in younger populations.68
To summarize, we found that APOE variants differed in multiple transcripts and multiple biological pathways, including those affecting calcium regulation, cell-cycle reentry and apoptosis, mitochondrial function and transcription factors. This may be consistent with broad notions about why the differences in APOE isoforms can ‘translate up’ to a devastating neurodegenerative disease,69
as several pathways may interact and ultimately promote amyloid aggregation or tau fibrillization.