CCAAT-Enhancer Binding Protein-β Expression and Elevation in Alzheimer’s Disease and Microglial Cell Cultures

Breitkopf T (2014) CCAAT-Enhancer Binding Protein-b Expression and Elevation in Alzheimer's Disease and Microglial Cell Cultures. PLoS ONE 9(1): e86617. doi:10.1371/journal.pone.0086617 CCAAT-Enhancer Binding Protein-b Expression and Elevation in Alzheimer's Disease and Microglial Cell Cultures Ron Strohmeyer 0 Jadd Shelton 0 Christopher Lougheed 0 Trisia Breitkopf 0 Hongwei Gao, Harvard Medical School, United States of America 0 Department of Biology, Northwest Nazarene University , Nampa, Idaho , United States of America CCAAT-enhancer binding proteins are transcription factors that help to regulate a wide range of inflammatory mediators, as well as several key elements of energy metabolism. Because C/EBPs are expressed by rodent astrocytes and microglia, and because they are induced by pro-inflammatory cytokines that are chronically upregulated in the Alzheimer's disease (AD) cortex, we have investigated whether C/EBPs are expressed and upregulated in the AD cortex. Here, we demonstrate for the first time that C/EBPb can be detected by Western blots in AD and nondemented elderly (ND) cortex, and that it is significantly increased in AD cortical samples. In situ, C/EBPb localizes immunohistochemically to microglia. In microglia cultured from rapid autopsies of elderly patient's brains and in the BV-2 murine microglia cell line, we have shown that C/ EBPb can be upregulated by C/EBP-inducing cytokines or lipopolysaccharide and exhibits nuclear translocation possibly indicating functional activity. Given the known co-regulatory role of C/EBPs in pivotal inflammatory mechanisms, many of which are present in AD, we propose that upregulation of C/EBPs in the AD brain could be an important orchestrator of pathogenic changes. - Funding: INBRE Program, NIH Grant Nos. P20 RR016454 (National Center for Research Resources) and P20 GM103408 (National Institute of General Medical Sciences), http://inbre.uidaho.edu/. MJ Murdock College Research Program for Life Sciences #2005247:JVZ:2/23/2006 http://www.murdock-trust.org/grants/ college-research-lifesciences.php. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Competing Interests: The authors have declared that no competing interests exist. Chronic, micro-localized inflammation, consisting primarily of innate and acute phase mechanisms, is widely regarded as a potentially important pathogenic element in Alzheimers disease (AD). Nonetheless, despite meticulous cataloging of inflammatory components that are increased in the AD brain (reviewed in [1]), the molecular mechanisms that might underlie or orchestrate such processes are still the objects of intense investigation. Transcription factors represent a primary point for regulation of gene and subsequent protein expression, and they typically act on sets of genes within multiple pathways. As such, they are in a position to broadly organize cellular responses, including pathogenic responses. CCAAT-enhancer binding proteins (C/EBPs) consist of a family of six transcription factor isoforms that include C/EBPa, C/EBPb, C/EBPd, C/EBPe, C/EBPc, and C/EBPf (reviewed in [27]). C/EBPs function as pleiotropic transactivators of a wide array of genes involved in energy metabolism, cell differentiation, and inflammation, most often in concert with other transcription factors [6]. For example, C/EBP family members C/ EBPb and C/EBPd can be important co-regulators with nuclear factor-kB (NF-kB) in a host of inflammatory responses [812], although this role appears to have been largely overlooked in AD studies of NF-kB-related mechanisms. Because the protein products of many of the inflammatory genes of which the C/ EBPs have been shown to regulate in the periphery [5,6,13] are also known to be altered in AD [1], our goal is determining which C/EBP isoforms are expressed in human brain and which are upregulated in pathologically-vulnerable regions of the AD brain. In a previous study, we reported for the first time that C/EBPd is present in the human brain and is upregulated in the AD cortex, with predominant localization to astrocytes [14]. Here, we show for the first time that the isoform C/EBPb is also present in the human brain with increased expression in AD brain microglia by immunohistochemistry and western blot and that C/EBPb is also expressed and upregulated in the AD brain and in microglial cultures. Further, C/EBP upregulation and nuclear localization in response to lipopolysaccharide (LPS) and pro-inflammatory cytokines is also shown in primary human elderly microglia cell cultures and BV-2 murine microglia cell line cultures. These results are consistent with recent findings showing a key role for C/EBPb in regulating microglial activation in pro-inflammatory environments within the brain [1520]. Materials and Methods Ethics Statement Northwest Nazarene University is committed to the highest standard of integrity in research. All human research activities are governed by the principles outlined in Title 45 Code of Federal Regulations Part 46. The Universitys Code of Conduct for the Responsible Practice of Research sets out the obligations on all University researchers, staff, and students to be aware of the ethical framework governing research at the University and to comply with institutional and regulatory requirements. All human tissues and cells used in this study meet the criteria for NIH exemption 4 for human research as determined by the Banner Sun Health Research Institute Brain Bank and per approval by the Northwest Nazarene University Human Research Review Committee and by the University of Idaho and the Idaho INBRE grant IRB approval. 2.1 Brain Autopsy Specimens All brain tissues for both glial cell cultures (2.2) and immunohistochemistry (2.3) were kindly provided by the Banner Sun Health Research Institute Brain Bank. (http://www. bannerhealth.com/Research/Research+Institutes/ Banner+Sun+Health+Research+Institute/Research/ Research+Programs/Brain+and+Body+Donation/ Tissue+request.htm). Neocortex and limbic cortex samples were dissected from routine autopsies of AD and ND patients. Postmortem intervals averaged less than 3 hours, and did not exceed 4 hours. Complete neuropathologic evaluations were performed in all cases. AD and ND groups were well-matched for age, gender, and postmortem interval, and did not differ significantly with respect to these variables (data not shown) [21]. 2.2 Elderly Microglia Cultures The method for developing isolated microglia from rapid autopsies of AD and ND patients, including a relatively complete characterization of these cells, has been previously published [22 26]. Microglia cultures have been characterized with respect to their morphology, immunoreactivity, repertoire of inflammationrelated secretory activity, and chemotactic and phagocytic responsiveness to amyloid b peptide (Ab) [23,24,26,27]. They are non-immunoreactive for multiple neu (...truncated)