Laser-captured microglia in the Alzheimer's and Parkinson's brain reveal unique regional expression profiles and suggest a potential role for hepatitis B in the Alzheimer's brain

Abstract

Expression array data from dozens of laboratories, including our own, show significant changes in expression of many genes in Alzheimer's disease (AD) patients compared with normal controls. These data typically rely on brain homogenates, and information about transcripts specific to microglia and other central nervous system (CNS) cell types, which far outnumber microglia-specific transcripts, is lost. We therefore used single-cell laser capture methods to assess the full range of microglia-specific expression changes that occur in different brain regions (substantia nigra and hippocampus CA1) and disease states (AD, Parkinson's disease, and normal controls). Two novel pathways, neuronal repair and viral processing were identified. Based on KEGG analysis (Kyoto Encyclopedia of Genes and Genomes, a collection of biological pathways), one of the most significant viruses was hepatitis B virus (HBV) (false discovery rate < 0.00000001). Immunohistochemical analysis using HBV-core antibody in HBV-positive control, amnestic mild cognitive impairment, and HBV-positive AD cases show increased HBV immunoreactivity as disease pathology increases. These results are the first, to our knowledge, to show regional differences in human microglia. In addition, these data reveal new functions for microglia and suggest a novel risk factor for AD.

Keywords: Alzheimer's disease; Laser capture; Microglia; Parkinson's disease; RNA sequencing; Synapse; Virus.

Figure 1:

Microglia exhibit different expression profiles in different brain regions of the non-diseased human elderly brain. Heat maps of the significant (p<.05) log2 fold changes in non-demented elderly control microglia within two brain regions, CA1 of the hippocampus and substantia nigra (SN), demonstrate unique expression profiles within regions. Heuristic cluster analysis of cluster 1 vs. cluster 2, or NC CA1 vs NC SN reveal three significant (p<.05, FDR<.02), upregulated biological processes in NC CA1 microglia: nervous system development, regulation of neuronal synaptic plasticity, and neurogenesis. Further analysis in the same subjects, cluster 3 vs. cluster 4, shows highly significant (p<.05, FDR<.000000000001) log2 fold changes in genes associated with mitochondrial function.

Figure 2:

Microglia exhibit different expression profiles in CA1 of the diseased human brain. Heat maps are provided that represent the significant (p<. 05) log2 fold changes in laser capture microglia from AD and PD cases compared to NC patients. A,B) Heuristic heat map illustrations of differentially expressed (log2 fold changes) in genes in AD CA1 vs. NC CA1 (A) and PD CA1 vs. NC CA1 (B). C) Pathway Analysis (2D-single cluster analysis) using Cytoscape network visualizer. The pie charts depict molecular interaction network and biological pathways in AD CA1 (C) and PD CA1 (D). C) 367 significant (p<.01-Bonferroni step down correction) genes were used in the pathway analysis for AD/NC comparisons. The top three pathways in AD microglia were thyroid hormone synthesis, icosanoid metabolic process, and regulation of viral process. D) 409 significant (p<.01-Bonferroni step down correction) genes were used in the pathway analysis for PD/NC comparisons. The top three pathways in PD microglia were aldosterone synthesis and secretion, positive regulation of protein complex assembly, and focal adhesion assembly.

Figure 3:

Microglia exhibit different expression profiles in different regions of the diseased human brain. Heat maps are shown that illustrate the significant (p<. 05) log2 fold changes in a heuristic hierarchical clustering output from laser capture microglia from AD and PD brain. A) A heat map, within group comparison (e.g. AD CA1 vs. AD SN) reveals 100 differentially expressed genes in laser captured microglial cells. Analysis of the log2 fold changes between cluster 1 and cluster 2 identifies three significant (p<.05, FDR<.002) processes: synaptic transmission, cell-cell signaling, and metal ion transport. Comparison of cluster 3 and cluster 4 shows no significant biological pathway. B) 313 significantly altered, log2 fold changes were found in PD CA1 vs. PD SN. Cluster analysis—cluster 1 versus cluster 3—shows three significant (p<.05, FDR<.00000001) biological processes: behavior, regulation of transport, and, like AD, synaptic transmission. No significant biological pathways were identified for cluster 3 and cluster 4.

Figure 4:

Percent of viral associated genes in laser capture microglia. AD and PD microglia were both compared to age-matched NC subjects. Signficant alterations in viral-associated transcripts were some 5-fold more common in AD CA1 than in PD CA1, AD SN, or PD SN.

Figure 5:

Representative photomicrographs of HBV core immunoreactivity in hippocampus CA1 in a Braak I NC subject (A), Braak IV MCI subject (B), and Braak V AD subject (C).