ABSTRACT: Alzheimer's disease (AD) is a devastating neurodegenerative disorder that threatens to reach epidemic proportions as our population ages. Although much research has examined molecular pathways associated with AD, relatively few studies have focused on critical early stages. Our prior microarray study correlated gene expression in human hippocampus with AD markers. Results suggested a new model of early-stage AD in which pathology spreads along myelinated axons, orchestrated by upregulated transcription and epigenetic factors related to growth and tumor suppression (Blalock et al., 2004). However, the microarray analyses were performed on RNA from fresh frozen hippocampal tissue blocks containing both gray and white matter, potentially obscuring region-specific changes. In the present study, we used laser capture microdissection to exclude major white matter tracts and selectively collect CA1 hippocampal gray matter from formalin-fixed, paraffin-embedded (FFPE) hippoc ampal sections of the same subjects assessed in our prior study. Microarray analyses of this gray matter-enriched tissue revealed many correlations similar to those seen in our prior study, particularly for neuron-related genes. Nonetheless, in the laser-captured tissue, we found a striking paucity of the AD-associated epigenetic and transcription factor genes that had been strongly overrepresented in the prior tissue block study. In addition, we identified novel pathway alterations that may have considerable mechanistic implications, including downregulation of genes stabilizing ryanodine receptor Ca2+ release and upregulation of vascular development genes. We conclude that FFPE tissue can be a reliable resource for microarray studies, that upregulation of growth-related epigenetic/ transcription factors with incipient AD is predominantly localized to white matter, further supporting our prior findings and model, and that alterations in vascular and ryanodine receptor-relat ed pathways in gray matter are closely associated with incipient AD. RNA was extracted using RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Ambion) according to manufacturer’s instructions (3h incubation at 55 C followed by glass fiber filtration). This system has recently been shown to outperform other FFPE methods/ kits regarding yield of amplifiable RNA (Okello et al., 2010). Quality assessment of extracted material was performed with the Paradise Reagent Quality Assessment Kit (Molecular Devices), as well as via NanoDrop (Thermoscientific). All samples yielded sufficient genetic material (>50 ng) for subsequent reactions. 50 ng of extracted purified nucleic acid underwent RNA amplification using WT-Ovation FFPE System (NuGen) followed by FL-Ovation cDNA Biotin Module V2 (NuGen) for labeling and microarray (Affymetrix HGU133 v2) hybridization. All 30 microarrays (one per specimen) performed within acceptable limits (Scaling factor: 32.6 +/- 3.7; RawQ: 1.28 +/- 0.01; GapDH 3’:5’: 1.48 +/- 0.08; % present 35.4 +/- 1.5) and were not significantly different across treatment (p < 0.5 for all measures, 1-ANOVA). In general, these results indicate a smaller signal in laser captured FFPE samples than in prior fresh frozen samples (Scaling factor: 5.9 +/- 0.6; RawQ 2.7 +/- 0.04; GapDH 3’:5’: 3.65 +/- 0.55; % present: 44.6 +/- 1.1) with an increased scaling factor decreased RawQ and reduced % present all indicating reduced signal volume, while the smaller GapDH ratio suggests more degraded material- consistent with other reports of the dynamics of small FFPE sample results. Further, the % present call, while lower than found in fresh frozen tissue, is much greater than would be expected by chance (5%).This suggests that the extracted genetic material still contains a large amount of valid data. Probe sets were annotated, and transcriptional profiles were generated, using the MAS5 algorithm and annotation data sets (Affymetrix Expression Console v. 1.1; HGU133 annotation October, 2003) in order to facilitate comparison with prior work. Results were filtered for presence, redundancy, and annotation status and analyzed by Pearson’s test for correlation with each subject’s Mini-Mental Status Exam (MMSE) score and Neurofibrillary Tangle (NFT) counts. The false discovery rate (FDR) (Hochberg and Benjamini, 1990) was used to estimate the error of multiple testing’s contribution to False Positives and the DAVID suite of bioinformatic tools was used to identify transcriptional pathways using the ‘table cluster’ option.
