Gene expression profiles in microdissected neurons from human hippocampal subregions

Department of Pathology, University of Miami School of Medicine (R-5), 1550 NW, Tenth Avenue, Miami, FL 33136, USA.
Molecular Brain Research (Impact Factor: 2). 09/2004; 127(1-2):105-14. DOI: 10.1016/j.molbrainres.2004.05.017
Source: PubMed


Pyramidal neurons in hippocampal subregions are selectively vulnerable in certain disease states. To investigate, we tested the hypothesis that selective vulnerability in human hippocampus is related to regional differences in neuronal cell death and cell receptor gene expression in CA1 vs. CA3 subregions. We used laser capture microdissection to remove approximately 600 CA1 and 600 CA3 pyramidal neurons each from five fresh-frozen normal post-mortem brains, extracted total RNA and double-amplified mRNA. This was reverse transcribed and labeled for hybridization onto human cDNA array chips containing probes to 10,174 genes and unknown ESTs. RNA from additional microdissections was pooled for replicate hybridizations and quantitative RT-PCR validation. Gene expression differences were few (< 1%). We found 43 enriched genes in CA1 neuronal samples that included peripheral benzodiazipine receptor-associated protein, nicotinic cholinergic receptor, two chemokine receptors (CCR1 and CCR5) and several transcriptional factors. We found 17 enriched genes in the CA3 neuronal samples that included fibroblast growth factor receptor and prostaglandin-endoperoxide synthase 1. We found no differential gene expression for 23 calcium channel proteins; nine transporter proteins; 55 cell death and apoptotic regulator proteins; and an additional 497 cell receptors, including 24 glutamate receptors. Quantitative RT-PCR of four differentially expressed genes confirmed the microarray data. The results confirm the ability to examine gene expression profiles in microdissected neurons from human autopsy brain. They show only minor gene expression differences between two distinct neuronal populations in the hippocampus and suggest that selective hippocampal vulnerability is due to factors other than intrinsic differential expression in glutamate receptors and cell death genes.

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Available from: Jorge E Torres-Muñoz
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    • "Modules were characterized using the following strategy: first, modules were annotated using EASE (as described above); second, modules were further annotated by measuring their overlap with modules from previous WGCNA studies of human and mouse brain [14,25]; third, cell type annotations were confirmed by measuring the overlap between our modules and experimentally derived lists of cell type-specific genes using the function userListEnrichment [22]; fourth, modules were annotated for region and disease specificity by measuring their overlap with lists of differentially expressed genes from the six studies discussed in the text [3,4,20,29-31]; and finally, module eigengenes were associated with all phenotypic traits available in this study (region, disease, age, and so on) in order to gain insight into the role each module might play in AD pathophysiology. To test for significant overlap between gene lists from our study and those from previous lists, the hypergeometric distribution (Fisher's exact test) was used. "
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    ABSTRACT: Background Transcriptional studies suggest Alzheimer's disease (AD) involves dysfunction of many cellular pathways, including synaptic transmission, cytoskeletal dynamics, energetics, and apoptosis. Despite known progression of AD pathologies, it is unclear how such striking regional vulnerability occurs, or which genes play causative roles in disease progression. Methods To address these issues, we performed a large-scale transcriptional analysis in the CA1 and relatively less vulnerable CA3 brain regions of individuals with advanced AD and nondemented controls. In our study, we assessed differential gene expression across region and disease status, compared our results to previous studies of similar design, and performed an unbiased co-expression analysis using weighted gene co-expression network analysis (WGCNA). Several disease genes were identified and validated using qRT-PCR. Results We find disease signatures consistent with several previous microarray studies, then extend these results to show a relationship between disease status and brain region. Specifically, genes showing decreased expression with AD progression tend to show enrichment in CA3 (and vice versa), suggesting transcription levels may reflect a region's vulnerability to disease. Additionally, we find several candidate vulnerability (ABCA1, MT1H, PDK4, RHOBTB3) and protection (FAM13A1, LINGO2, UNC13C) genes based on expression patterns. Finally, we use a systems-biology approach based on WGCNA to uncover disease-relevant expression patterns for major cell types, including pathways consistent with a key role for early microglial activation in AD. Conclusions These results paint a picture of AD as a multifaceted disease involving slight transcriptional changes in many genes between regions, coupled with a systemic immune response, gliosis, and neurodegeneration. Despite this complexity, we find that a consistent picture of gene expression in AD is emerging.
    Full-text · Article · May 2013 · Genome Medicine
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    • "These results led us to evaluate the specific role of CCR5 receptor, a receptor for CCL5/RANTES, in this model. CCR5 is constitutively expressed in astrocytes, microglia and neurons [18-20]. Its role in the healthy brain is still poorly understood, but CCR5 seems to regulate leukocyte maturation and trafficking in models of brain disease [21]. "
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    ABSTRACT: Background The neuroinflammatory response aimed at clearance of herpes simplex virus-1 (HSV-1) plays a key role in the pathogenesis of neuroaxonal damage in herpetic encephalitis. Leukocytes activated in an adaptive immune response access brain tissue by passing through the blood–brain barrier. The chemokine CCL5/RANTES is involved in recruitment of these cells to the brain acting via the receptors CCR1, CCR3 and mainly CCR5. Here, we evaluated the role of CCR5 on traffic of leukocytes in the brain microvasculature, cellular and cytokines profile in a severe form of herpetic encephalitis. Results Wild type and mice lacking CCR5 (CCR5-/-) were inoculated intracerebrally with 104 PFU of neurotropic HSV-1. We evaluated the traffic of leukocytes in the brain microvasculature using intravital microscopy and the profile of cytokines by Enzyme-Linked Immunosorbent Assay at 1 day post infection. Flow cytometry and histopathological analyses were also carried out in brain tissue. Absence of CCR5 leads to lower viral load and an increased leukocyte adhesion in brain microvasculature, predominantly of neutrophils (CD11+ Ly6G+ cells). Moreover, there was a significant increase in the levels of MIP-1/CCL2, RANTES/CCL5, KC/CXCL1 and MIG/CXCL9 in the brain of infected CCR5-/- mice. Conclusions These results suggest that the absence of CCR5 may boost the immune response with a high neutrophil recruitment which most likely helps in viral clearance. Nonetheless, the elevated immune response may be detrimental to the host.
    Full-text · Article · Feb 2013 · BMC Neuroscience
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    • "Molecular differences between the three hippocampal fields consist of differential gene and protein expression. Namely, different anatomical fields of the hippocampus have been shown to have distinct transcription and protein expression patterns (Datson et al., 2001; Zhao et al., 2001; Bonaventure et al., 2002; Gozal et al., 2002; Lein et al., 2004; Torres-Munoz et al., 2004; Greene et al., 2009). These anatomical, connectivity and molecular properties that characterize each region are altered in aging. "
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    ABSTRACT: Aging is associated with cognitive decline in both humans and animals and of all brain regions, the hippocampus appears to be particularly vulnerable to senescence. Age-related spatial learning deficits result from alterations in hippocampal connectivity and plasticity. These changes are differentially expressed in each of the hippocampal fields known as cornu ammonis 1 (CA1), cornu ammonis 3 (CA3), and the dentate gyrus. Each sub-region displays varying degrees of susceptibility to aging. For example, the CA1 region is particularly susceptible in Alzheimer's disease while the CA3 region shows vulnerability to stress and glucocorticoids. Further, in animals, aging is the main factor associated with the decline in adult neurogenesis in the dentate gyrus. This review discusses the relationship between region-specific hippocampal connectivity, morphology, and gene expression alterations and the cognitive deficits associated with senescence. In particular, data are reviewed that illustrate how the molecular changes observed in the CA1, CA3, and dentate regions are associated with age-related learning deficits. This topic is of importance because increased understanding of how gene expression patterns reflect individual differences in cognitive performance is critical to the process of identifying new and clinically useful biomarkers for cognitive aging.
    Preview · Article · Oct 2010 · Frontiers in Aging Neuroscience
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