Downregulation of CREB expression in Alzheimer's brain and in Aβ-treated rat hippocampal neurons
© Pugazhenthi et al; licensee BioMed Central Ltd. 2011
Received: 31 May 2011
Accepted: 19 August 2011
Published: 19 August 2011
Oxidative stress plays an important role in neuronal dysfunction and neuron loss in Alzheimer's brain. Previous studies have reported downregulation of CREB-mediated transcription by oxidative stress and Aβ. The promoter for CREB itself contains cyclic AMP response elements. Therefore, we examined the expression of CREB in the hippocampal neurons of Tg2576 mice, AD post-mortem brain and in cultured rat hippocampal neurons exposed to Aβ aggregates.
Laser Capture Microdissection of hippocampal neurons from Tg2576 mouse brain revealed decreases in the mRNA levels of CREB and its target, BDNF. Immunohistochemical analysis of Tg2576 mouse brain showed decreases in CREB levels in hippocampus and cortex. Markers of oxidative stress were detected in transgenic mouse brain and decreased CREB staining was observed in regions showing abundance of astrocytes. There was also an inverse correlation between SDS-extracted Aβ and CREB protein levels in Alzheimer's post-mortem hippocampal samples. The levels of CREB-regulated BDNF and BIRC3, a caspase inhibitor, decreased and the active cleaved form of caspase-9, a marker for the intrinsic pathway of apoptosis, was elevated in these samples. Exposure of rat primary hippocampal neurons to Aβ fibrils decreased CREB promoter activity. Decrease in CREB mRNA levels in Aβ-treated neurons was reversed by the antioxidant, N-acetyl cysteine. Overexpression of CREB by adenoviral transduction led to significant protection against Aβ-induced neuronal apoptosis.
Our findings suggest that chronic downregulation of CREB-mediated transcription results in decrease of CREB content in the hippocampal neurons of AD brain which may contribute to exacerbation of disease progression.
KeywordsAlzheimer's disease CREB Oxidative stress Apoptosis Tg2576 mice Laser capture microdissection
Cyclic AMP response element binding protein (CREB) is a constitutively expressed nuclear transcription factor that regulates the expression of genes involved in neuronal survival and function [1–3]. CREB is essential for the formation and retention of memory in several species [4, 5]. CREB-mediated gene expression is increased in the hippocampus during LTP . Spatial learning deficits in rats are observed after intra-hippocampal infusion of CREB antisense oligos . CREB is also an important nuclear target that couples neurotrophin-mediated signaling to neuronal survival . CREB undergoes phosphorylation at serine 133 in response to multiple signaling pathways [9, 10]. The phosphorylated form of CREB binds to the coactivators, CREB binding protein (CBP) and p300 resulting in the facilitation of target gene expression . We have reported that IGF-I induces CREB-regulated expression of the anti-apoptotic gene, bcl-2 through multiple signaling pathways [12, 13].
Previous studies have reported that CREB-mediated gene expression is impaired in Alzheimer's brain. The active form of p90RSK, a critical CREB kinase, is decreased in a transgenic rat AD model . Gong et al  reported a decrease in the levels of phosphorylated CREB in hippocampal neurons of PS1/APP double mutant transgenic mice. Treatment of these mice with rolipram, a phosphodiesterase inhibitor that increases CREB phosphorylation results in a significant improvement in cognitive function. In AD post-mortem brain, there is a decrease in the levels of CREB-regulated BDNF . In cultured neurons, Aβ interferes with CREB activation by cAMP and BDNF [17–19]. However, paradoxical CREB activation in conditions associated with AD is also reported [20, 21]. We demonstrated that lipid peroxidation products increase the levels of active phosphorylated form of CREB without a parallel increase in CREB-mediated gene expression . We also reported oxidative stress-induced downregulation of CREB-dependent bcl-2 expression in cultured rat hippocampal neurons . In the same study, we showed that hydrogen peroxide decreases CREB protein levels in neurons which were normalized by preincubation with the antioxidant, N acetyl cysteine. The objective of the present study was to determine the expression of CREB in three different models of AD, namely the Tg2576 mouse which expresses human APP with the Swedish mutation, AD post-mortem brain and cultured rat hippocampal neurons exposed to Aβ.
Laser capture microdissection (LCM) reveals decrease of CREB mRNA in hippocampal neurons of Tg2576 mouse brain
Decrease in CREB protein levels of Alzheimer's mouse hippocampal neurons
Markers of oxidative stress in Tg2576 mouse brain
Inverse correlation between Aβ deposition and CREB content
Decreased expression of CREB-regulated proteins and markers of oxidative stress in AD brain
Aβ-induced decrease in CREB expression in cultured rat hippocampal neurons
CREB-mediated protection of rat hippocampal neurons from Aβ-induced apoptosis
CREB is a constitutively expressed nuclear transcription factor needed for cognition and neuronal survival. Its regulation is primarily through phosphorylation at serine 133 by several signaling kinases and thus responds to a variety of extracellular signals [10, 13, 25, 26]. CREB-regulated gene expression has been shown to be downregulated in AD brain. In this study, we show that the expression of CREB itself is decreased in Tg2576 mouse hippocampal neurons, AD post-mortem hippocampal samples and in Aβ-treated rat hippocampal neurons. Our findings suggest that persistent downregulation of CREB-regulated gene expression could lead to decrease in the levels of CREB itself which could exacerbate neurodegeneration in AD.
Tg2576 mice overexpressing human APP with the Swedish mutation are characterized by Aβ deposition and cognitive dysfunction [27, 28]. Previous studies have suggested that markers of CREB function are downregulated in the hippocampal neurons of this mouse model [14, 15, 29]. We demonstrate in the present study by laser capture microdissection (LCM) that the levels of CREB mRNA are significantly decreased in the hippocampal neurons of Tg2576 mouse brain (Figure 1). The expression of BDNF, a target gene of CREB was also found to be reduced in LCM-captured neurons. Further examination of CREB protein by immunohistochemical analysis showed age-dependent decreases in hippocampal neurons, more significantly in the transgenic mouse brain (Figure 2). Markers of oxidative stress including HNE and MDA were also elevated. We observed decreases in CREB levels especially in the regions where astrocytes were abundantly present (Figure 3E). Astrocytosis is known to play a role in neurodegeneration through release of cytokines [30, 31]. We have previously reported in cultured MIN6 cells, a mouse pancreatic cell line that a combination of proinflammatory cytokines, IL-1β, TNF-α and IFN-γ decreases CREB expression following chronic exposure .
Oxidative stress is known to play an important role in the neurodegenerative process of Alzheimer's brain. Accumulation of free radicals is an important feature of aging, which is also a risk factor for AD. Markers of oxidative stress are found in aged rats, especially in those with impaired spatial learning . Hensley et al. demonstrated that Aβ aggregation leads to generation of oxidative stress in vitro [34, 35]. In addition, several other agents, including iron, aluminum, and advanced glycosylation end products, induce oxidative stress in AD [36–38]. Lipid peroxidation and DNA oxidation products accumulate in AD brains as a result of oxidative stress [36, 39–41]. We observed increased protein-bound HNE and protein oxidation in AD post-mortem samples as shown by Oxyblot analysis (Figure 6). SDS-extractable Aβ accumulation showed inverse correlation with CREB levels in AD post-mortem brain (Figure 4E). These findings suggest that Aβ-generated oxidative stress could play a role in downregulation of CREB expression. To determine the direct effects of Aβ on CREB expression we examined the activity of a luciferase reporter gene driven by the CREB promoter in cultured rat hippocampal neurons. CREB promoter activity was downregulated by Aβ fibrils but not by Aβ oligomers (Figure 7B). The oligomers did decrease the activity of CREB-dependent BDNF promoter activity (Figure 7A). Furthermore, Aβ fibril-induced decrease in CREB mRNA levels was prevented by preincubation of neurons with the antioxidant, NAC (Figure 7D). Thus our findings with cultured hippocampal neurons provide direct evidence for the downregulation of CREB expression by oxidative stress. Although Aβ oligomers did not play a role in the downregulation of CREB expression in this study, their role in AD pathology is well characterized.
Several reports have shown that caspase activation, a marker of apoptosis, is observed in the neurons in AD brain (reviewed in ). Caspase activation can also play a role in AD pathology independent of causing neuronal death. For example, caspase-cleaved tau undergoes conformational changes that facilitate filament formation, and Aβ localizes to cleaved tau . Although significant loss of neurons is observed in human AD brain, marked neuronal apoptosis is not observed in mouse models of AD. We observed modulation of several proteins in the apoptosis pathway in AD post-mortem brain (Figure 5). The levels of anti-apoptotic Bcl-2 and caspase inhibitors, BIRC3 and BIRC4 decreased and proapoptotic Bax increased in AD brain leading to activation of caspase-9, a marker for the intrinsic pathway of apoptosis. CREB plays an important role in increasing the levels of proteins that maintain mitochondrial membrane integrity and prevent the release of cytochrome c, an activator of caspase-9. Oxidative stress is known to induce neuronal apoptosis by a dual mechanism of downregulation of anti-apoptotic genes and upregulation of proapoptotic genes .
We have previously reported that IGF-I induces CREB-mediated bcl-2 expression in PC12 cells through multiple signaling pathways [12, 13]. We also reported that oxidative stress downregulates bcl-2 expression in rat hippocampal neurons . In the present study, we observed that Aβ-induced neuronal apoptosis is exaggerated when dominant negative mutant forms of CREB (KCREB and MCREB) were overexpressed along with GFP by plasmid transfection (Figure 8A). KCREB is mutated at the DNA binding region of CREB. This mutant will heterodimerize with endogenous CREB and sequester it away from target promoters. MCREB is mutated at the critical phosphorylation site (S133A). MCREB will bind to CRE but cannot bind to the coactivators. We also tested the effects of modulation of CREB function by adenoviral transduction on Aβ toxicity (Figure 8E). Apoptosis by the intrinsic pathway was induced by Aβ as shown by the activation of caspase-9 as observed in AD post-mortem brain (Figure 5). Expression of wild type CREB decreased neuronal death induced by Aβ significantly. There was also exacerbation of Aβ-induced apoptosis when the neurons were transduced with adenoviral KCREB or MCREB. Thus chronic CREB downregulation caused by oxidative stress could be an important cause of loss of neurons in AD brain.
The primary events occurring in the pathogenesis of AD include the deposition of Aβ-containing senile plaques and the formation of neurofibrillary tangles. This triggers a slow neurodegenerative process involving multiple insults to neurons, including free radical generation and inflammatory responses. During this slow progressive phase, the cellular defense mechanism fails, due to oxidative stress-induced changes in transcriptional events including downregulation of CREB expression. In the treatment of AD, downstream interventions cannot be discounted as it is essential to repair neuronal injury while attempting to help clear Aβ accumulation. Small molecule enhancers of CREB-mediated gene expression are being considered as potential therapeutic agents in the treatment of AD .
Cell culture media and supplies were purchased from Gemini BioProducts (Woodland, CA, USA) and Invitrogen-Life Technologies (Carlsbad, CA). Antibodies to CREB, phosphorylated form of CREB, synaptophysin, BIRC3, BIRC4, Bax, active caspase-9, GFAP, and β actin were from Cell signaling (Beverly, MA). The antibodies to HNE and MDA and caspase-9 assay kit were obtained from Millipore (Billerica MA). The cDNA encoding a chimeric protein for GFP and MnSOD was provided by Dr. Sonia Flores (University of Colorado, Aurora, CO). IgG linked to Cy3 or FITC were obtained from Jackson ImmunoResearch (West Grove, PA, USA). All other fine chemicals were from Sigma (St. Louis, MO). We obtained 24 post-mortem samples (12 each of control and AD) from the AD Brain bank at University of Colorado. The mean age was 75 y for AD and 73 y for controls. The postmortem delay (PMD) was 3-12 h (mean 7 h). This is comparable to PMD reported in other studies [46–49]. The groups of subjects were characterized as AD or normal controls using the criteria from the Consortium to Establish a Registry for AD .
Laser capture microdissection of mouse hippocampal neurons
All animal procedures were performed with the approval of the Subcommittees on Research Safety and Institutional Animal Care and Use at the Denver Department of Veterans Affairs Medical Center, Eastern Colorado Health Care System (ACORP# 08006M). Tg2576 mice and nontransgenic controls were anesthetized and perfused with cold RNase-free phosphate buffered saline. The brain was removed and embedded in Tissue-TeK OCT in a plastic mold and frozen. Frozen brain sections (10 μM) were cut on a cryostat and collected on Superfrost slides (Fischer Scientific, Pittsburgh, PA). DEPC-treated water was used in all procedures. All solutions except wash buffers contained 0.5 U/μl RNase inhibitor. Brain sections (10 μM) were stained using HistoGene™ Stain, (Arcturus) to identify the neurons. LCM was performed using AutoPix LCM system from Arcturus Engineering (Mountain View, CA). The neurons in CA1, CA3 and dentate gyrus regions were selectively captured from each section onto Capsure LCM macro caps. Total RNA was isolated from LCM samples using a PicoPure RNA isolation kit (Arcturus). Because the RNA yield is low, additional amplification steps were used following the RiboAMP RNA amplification kit instructions from Arcturus. Real time RT-PCR analysis using Taqman probes was performed for mouse neurofilament heavy chain (mNFHc) to determine the neuronal nature of the samples and human APP to differentiate the RNA samples isolated from nontransgenic and APP transgenic mice. In addition, the mRNA levels of CREB and BDNF were compared in amplified RNA samples from nontransgenic and transgenic mice. The PCR reactions in 96 wells were monitored in real time in an ABI Prism 7700 sequence detector (Perkin Elmer Corp./Applied Biosystems).
Immunohistochemical analysis of Tg2576 mouse brain
The tissue sections were de-paraffinized and hydrated through serial dilutions of ethanol and then in distilled water. The hydrated sections were steamed in 10 mM sodium citrate buffer (pH 6.0) for 30 min for antigen retrieval. They were treated with 1% hydrogen peroxide for 10 min and blocked with 5% horse serum for 1 h at RT. Subsequently, depending on whether the antibody was from mouse or rabbit, we used a 'mouse on mouse' or rabbit ABC elite kit from Vector laboratories (Burlingame, CA), respectively. The chromogens were Novared and/or DAB. Counterstaining was with methyl green. For double immunostaining, color was developed with the first primary antibody followed by incubation with the second antibody and color development.
Western blot analysis
Proteins were resolved by SDS-PAGE electrophoresis, transferred to polyvinylidene difluoride membranes and blocked with 5% non-fat dry milk for 1 h at RT. The membranes were exposed to primary antibodies at a dilution of 1:1000 overnight at 4°C. After washing with 5% non-fat milk, the membranes were incubated in the presence of appropriate secondary antibodies linked to alkaline phosphatase for 1 h at RT. After treating with alkaline washing buffer (10 mM Tris-HCl (pH 9.5), 10 mM NaCl and 1 mM MgCl2), signals were developed with CDP-Star reagent (New England Biolabs, Beverly, MA) and exposed to X-ray film. The intensity of bands were measured by using Fluor-S MultiImager and Quantity One software (Bio-Rad, Hercules, CA) and corrected for the levels of β actin. Oxyblot analysis: Oxidative modification of proteins by free radicals was determined for AD-post mortem samples along with age-matched controls using a kit from Millipore (Billerica, MA). The protein samples (20 μg) in 5 μl were mixed with 5 μl of 12% SDS for a final concentration of 6% and derivatized with 10 μl of 1X DNPH solution. The samples were incubated at RT for 15 min, neutralized and electrophoresed. Following transfer, the blots were immunoprobed with anti-DNP antibody and the signals were developed with CDP-Star.
Aβ assay and preparation of Aβ aggregates
After extracting the soluble fractions from post-mortem samples with Tris-buffered saline (pH 7.6) containing 1% Triton X-100 and protease inhibitors, the tissue pellets were extracted with 2% SDS containing protease inhibitors. The extracts were appropriately diluted and Aβ 1-42 levels were determined by sandwich ELISA. For capture, the Ban-50 (1-42) antibody was used and the BC-05 antibody was used for detection. To determine Aβ-induced toxicity in cultured neurons, Aβ aggregates were prepared by the following procedure: Aβ (1-42) peptide purchased from Peptide 2.0 (Chantilly, VA) was first monomerized by resuspending in hexafluoroisopropanol followed by incubation at RT for 1 h. After evaporation of solvent under vacuum, Aβ is dissolved in DMSO and stored at -20°C. Aβ oligomer stock (100 μM) was prepared by incubation in F-12 (without phenol red) culture medium at 4°C for 24 h. Aβ fibril stock (100 μM) was prepared by incubation in HCl (10 mM) at 37°C for 24 h.
Culture of rat hippocampal neurons and transfection
Hippocampal neurons were isolated from E18 rat embryos obtained from timed-pregnant Sprague-Dawley rats as previously described . The astrocyte population was less than 5%. The neurons were cultured in serum-free Eagle's medium containing 2% antioxidant-free B27 supplement (Life Technologies). The experiments were carried out in these neurons after one week in culture . Total RNA was isolated from treated neurons using an isolation kit (Versagene RNA; Fisher Scientific, Pittsburgh, PA). Transient transfection of rat hippocampal neurons cultured in 12 well dishes to about 70% confluence was performed using LipofectAMINE 2000 reagent (Invitrogen-Life Technologies) according to the procedure described previously . BDNF promoter and CREB promoter linked to a luciferase reporter gene were provided by Anne West (Harvard Medical School, Boston) and Dr. Dwight Klemm (University of Colorado) respectively. A constitutively active renilla luciferase (pRL-TK-luc) was included to correct for transfection efficiency and nonspecific actions of oxidative stress. After 6 h of transfection, the neurons were exposed to Aβ oligomers or fibrils for 18 h. The cells were processed for the assay of luciferases using dual luciferase assay kit (Promega). The transfection efficiency for rat hippocampal neurons using LipofectAMINE 2000 reagent was ~30%.
Rat hippocampal neurons were cultured on glass cover slips (Carolina Biological Supply, Burlington, NC) prepared according to the methods of Goslin and Banker . Treated neurons were fixed in 4% paraformaldehyde for 30 min at RT and washed with PBS. The cells were permeabilized in PBS containing 0.2% Triton X-100 and 5% BSA for 90 min at RT, followed by incubation with the primary antibody at 4°C overnight. Double immunostaining was performed using monoclonal and polyclonal antibodies for two targets. After washing with PBS, appropriate secondary antibodies linked to Cy3 and FITC were added along with DAPI (2 μg/ml; nuclear staining) for 90 min at RT. The cells were then washed in PBS, mounted on slides and examined by digital deconvolution microscopy.
Measurement of intracellular oxidative stress
Intracellular ROS (reactive oxygen species) generation was measured in neurons exposed to Aβ aggregates using an oxidation-sensitive dye, 5- (and 6-)chloromethyl-2',-7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). When the cells are loaded with this dye, its acetate groups are cleaved by cellular nonspecific esterases, trapping the dye in the cell. Although this "leuco" form of the dye is colorless, the oxidized dye fluoresces at 535 nm when excited with 485 nm light. The cells were loaded with the dye and the fluorescence was measured using the CytoFluor multiwell plate reader.
Cultured rat hippocampal neurons transduced with adenoviruses followed by exposure to Aβ fibrils were processed for the assay of caspase-9 using a kit from Millipore (Billerica, MA). Supernatant of the cell lysates were incubated in the presence of substrate, p-nitroanilide-labeled LEHD. The released chromophore was read at 405 nm using a microplate reader.
Data presented are Mean ± SE. Statistical evaluation was performed by one-way ANOVA with Dunnett's multiple comparison test.
- AD :
- AMP :
- BDNF :
Brain-derived neurotrophic factor
- CBP :
CREB binding protein
- CREB :
Cyclic AMP response element binding protein
- GFP :
Green fluorescent protein
- HNE :
- IGF-I :
Insulin-like growth factor-I
- LCM :
Laser capture microdissection
- MDA :
- NAC :
- RT-PCR :
Reverse transcription-polymerase chain reaction
This work was carried out with the use of resources and facilities at Denver VA Medical Center. This study was supported by Merit Review grant NEUD-004-07F from the Veterans Administration (S.P.).
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