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Microarray analyses of laser-captured hippocampus reveal distinct gray and white matter signatures associated with incipient Alzheimer's disease.

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 such studies have focused on the disease's critical early stages. In a prior microarray study we correlated gene expression in hippocampus with degree of Alzheimer's disease and found close associations between upregulation of apparent glial transcription factor/epigenetic/tumor suppressor genes and incipient AD. The results suggested a new model in which AD pathology spreads along myelinated axons (Blalock et al., 2004). However, the microarray analyses were performed on RNA extracted from frozen hand-dissected hippocampal CA1 tissue blocks containing both gray and white matter, limiting the confidence with which transcriptional changes in gray matter could be distinguished from those in white matter. Here, we used laser capture microdissection (LCM) to exclude major white matter tracts while selectively collecting CA1 hippocampal gray matter from formalin-fixed, paraffin-embedded (FFPE) hippocampal sections of the same subjects assessed in our prior study. Microarray analyses of this gray matter-enriched tissue revealed many transcriptional changes similar to those seen in our past study and in studies by others, particularly for downregulated neuron-related genes. Additionally, the present analyses identified several previously undetected pathway alterations, including downregulation of molecules that stabilize ryanodine receptor Ca2+ release and upregulation of vasculature development. Conversely, we found a striking paucity of the upregulated changes in the putative glial and growth-related genes that had been strongly overrepresented in the prior mixed-tissue study. We conclude that FFPE tissue can be a reliable resource for microarray studies of brain tissue, that upregulation of growth-related epigenetic/transcription factors during incipient AD is predominantly localized in and around white matter (supporting our prior findings and model), and that novel alterations in vascular and ryanodine receptor-related pathways in gray matter are closely associated with incipient AD.

SUBMITTER: Blalock EM

PROVIDER: S-EPMC3163806 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Microarray analyses of laser-captured hippocampus reveal distinct gray and white matter signatures associated with incipient Alzheimer's disease.

Blalock Eric M EM Buechel Heather M HM Popovic Jelena J Geddes James W JW Landfield Philip W PW

Journal of chemical neuroanatomy 20110702 2

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 such studies have focused on the disease's critical early stages. In a prior microarray study we correlated gene expression in hippocampus with degree of Alzheimer's disease and found close associations between upregulation of apparent glial transcription factor/epigeneti ...[more]

PMID: 21756998

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Project description: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.

Project description:It is well established that brain structures and cognitive functions change across the life span. A long-standing hypothesis called "age differentiation" additionally posits that the relations between cognitive functions also change with age. To date, however, evidence for age-related differentiation is mixed, and no study has examined differentiation of the relationship between brain and cognition. Here we use multigroup structural equation models (SEMs) and SEM trees to study differences within and between brain and cognition across the adult life span (18-88 years) in a large (N > 646, closely matched across sexes), population-derived sample of healthy human adults from the Cambridge Centre for Ageing and Neuroscience (www.cam-can.org). After factor analyses of gray matter volume (from T1- and T2-weighted MRI) and white matter organization (fractional anisotropy from diffusion-weighted MRI), we found evidence for the differentiation of gray and white matter, such that the covariance between brain factors decreased with age. However, we found no evidence for age differentiation among fluid intelligence, language, and memory, suggesting a relatively stable covariance pattern among cognitive factors. Finally, we observed a specific pattern of age differentiation between brain and cognitive factors, such that a white matter factor, which loaded most strongly on the hippocampal cingulum, became less correlated with memory performance in later life. These patterns are compatible with the reorganization of cognitive functions in the face of neural decline, and/or with the emergence of specific subpopulations in old age.SIGNIFICANCE STATEMENT The theory of age differentiation posits age-related changes in the relationships among cognitive domains, either weakening (differentiation) or strengthening (dedifferentiation), but evidence for this hypothesis is mixed. Using age-varying covariance models in a large cross-sectional adult life span sample, we found age-related reductions in the covariance among both brain measures (neural differentiation), but no covariance change among cognitive factors of fluid intelligence, language, and memory. We also observed evidence of uncoupling (differentiation) between a white matter factor and cognitive factors in older age, most strongly for memory. Together, our findings support age-related differentiation as a complex, multifaceted pattern that differs for brain and cognition, and discuss several mechanisms that might explain the changing relationship between brain and cognition.

Project description:Huntington disease (HD) is a fatal progressive neurodegenerative disorder for which only symptomatic treatment is available. A better understanding of the pathology, and identification of biomarkers will facilitate the development of disease-modifying treatments. HD is potentially a good model of a neurodegenerative disease for development of biomarkers because it is an autosomal-dominant disease with complete penetrance, caused by a single gene mutation, in which the neurodegenerative process can be assessed many years before onset of signs and symptoms of manifest disease. Previous MRI studies have detected abnormalities in gray and white matter starting in premanifest stages. However, the understanding of how these abnormalities are related, both in time and space, is still incomplete. In this study, we combined deep gray matter shape diffeomorphometry and white matter DTI analysis in order to provide a better mapping of pathology in the deep gray matter and subcortical white matter in premanifest HD. We used 296 MRI scans from the PREDICT-HD database. Atrophy in the deep gray matter, thalamus, hippocampus, and nucleus accumbens was analyzed by surface based morphometry, and while white matter abnormalities were analyzed in (i) regions of interest surrounding these structures, using (ii) tractography-based analysis, and using (iii) whole brain atlas-based analysis. We detected atrophy in the deep gray matter, particularly in putamen, from early premanifest stages. The atrophy was greater both in extent and effect size in cases with longer exposure to the effects of the CAG expansion mutation (as assessed by greater CAP-scores), and preceded detectible abnormalities in the white matter. Near the predicted onset of manifest HD, the MD increase was widespread, with highest indices in the deep and posterior white matter. This type of in-vivo macroscopic mapping of HD brain abnormalities can potentially indicate when and where therapeutics could be targeted to delay the onset or slow the disease progression.

Dataset Information

Microarray analyses of laser-captured hippocampus reveal distinct gray and white matter signatures associated with incipient Alzheimer's disease.

Publications

Microarray analyses of laser-captured hippocampus reveal distinct gray and white matter signatures associated with incipient Alzheimer's disease.

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