Apoptosis (2010) 15:1461–1469 DOI 10.1007/s10495-010-0541-5
LXR-a genomics programmes neuronal death observed in Alzheimer’s disease Ashvinder Raina • Deepak Kaul
Published online: 7 October 2010 Ó Springer Science+Business Media, LLC 2010
Abstract Keeping in view the fact that the most pathognomonic feature of Alzheimer’s disease is the abnormal processing of neuronal cell membrane amyloid precursor protein accompanied by significantly elevated human serum and CSF levels of 24-hydroxycholesterol recognised widely as the specific endogenous ligand of Liver X receptor (LXR-a), the present study was addressed to explore the epigenomic-pathway (if any) that connects LXR-a activation with the genes recognised to be involved in the regulation of aberrant Abeta production leading to the generation of toxic and inflammatory mediators responsible for neuronal death. The results of such a study revealed that LXR-a activation by its specific endogenous or exogenous ligands within neuroblastoma cells resulted in the over-expression of PAR-4 gene accompanied by suppression of AATF gene through its inherent capacity to regulate genes coding for SREBP and NF-jB. Overexpression of PAR-4 gene was accompanied by aberrant Abeta production followed by ROS generation and subsequent death of neuroblastoma cells used in the present study as a cellular model for neurons. Further based upon these results, it was proposed that Abeta-induced heme oxygenase-1 can ensure cholesterol-oxidation to provide endogenous ligands for the sustained activation of neuronal LXR-a dependent epigenomic-pathway leading to neuronal death observed in Alzheimer’s disease. Keywords LXR-a activation PAR-4 AATF Abeta production Toxic mediators Neuronal death
A. Raina D. Kaul (&) Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India e-mail: [email protected]
Introduction There exists a general recognition of the fact that a central event in the development of Alzheimer’s disease (AD) is the abnormal processing of neuronal cell membrane amyloid precursor protein followed by deposition of b-amyloid protein in the form of amyloid plaques responsible for the activation of microglia leading to the production of toxic and inflammatory mediators like hydrogen peroxide, nitric oxide and cytokines [1, 2]. Mounting evidence suggests that PAR-4 (Prostate apoptosis response-4) is an endogenous regulator of b-secretase-dependent cleavage of the Alzheimer amyloid precursor protein  and this phenomenon is inhibited by AATF (Apoptosis antagonizing transcription factor) by interacting directly with PAR-4 . Further, b-amyloid-protein-induced microglial proliferation has been shown to be mediated through NADPH oxidase-dependent of hydrogen peroxide [5, 6]. Keeping in view the fact that most of the circulating 24S-hydroxy cholesterol in the human body originates in the brain ; recent study revealed that serum concentration of 24S-OHcholesterol was significantly higher in Alzheimer and vascular demented patients than the depressed patients and healthy controls . It is interesting to note that 24S-OHcholesterol is a natural ligand of LXR-a protein that belongs to the superfamily of ligand-dependent transcription factors and is known to play critical role in the lipid metabolism and inflammation [9, 10]. Consequently, the present study was addressed to explore three specific issues: (a) whether or not LXR-a has the inherent capacity to regulate the genes coding for AATF and PAR-4?; (b) If yes, does LXR-a activation result in the generation of b-amyloid leading to ROS production followed by neuronal apoptosis?; (c) Do ROS scavengers inhibit this LXR-a induced neuronal-apoptotic pathway?
Materials and methods
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also confirmed by real time PCR using 19 SYBER GreenERTM qPCR super mix universal (Stratagene).
Cellular model employed and reagents LXR-a gene knock-down and PAR-4 gene expression For the present study human neuroblastoma cell line (IMR32) and THP-1 cell line were procured from National Centre for Cell Science (NCCS), Pune and were subsequently maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) at 37°C. Cells were counted in haemocytometer chamber and viability of the cells was measured by Trypan blue dye exclusion. Revert AidTM first strand cDNA synthesis kit was obtained from Fermentas; LXR-siRNA and siRNA Transfection reagent were obtained from Santa Cruz; TOPO reporter plasmid and b-Gal assay kit were obtained from Invitrogen; siPORTTM XP-1 transfection reagent from Applied Biosystems and Withaferin A (WA) was obtained from Merck BioSciences.
Genomic expression: effect of Withaferin A or 24S-OH-cholesterol In order to explore the transcriptional expression of genes coding for PAR-4, AATF and LXR-a within neuroblastoma cells (IMR-32), the cells were processed for RNA isolation using standard method  and the integrity of RNA was verified by electrophoretic size separation in 1% ethidium bromide-stained agarose gel. Subsequently cDNA was synthesized from RNA using Revert AidTM first strand synthesis kit (Fermentas). The transcriptional expression of these genes was then studied using gene specific primers and standard RT-PCR method. The relative expression of each gene was measured by using b-actin gene as an invariant control. Reaction products for PAR-4, AATF and LXR-a genes were resolved on 2% ethidium bromidestained agarose gels followed by densitometric scanning of each band on the gels. Intensity ratio of target mRNA to b-actin mRNA was plotted. Keeping in view our recent study indicating that the dietary compound Withaferin A has the inherent capacity to act as a specific exogenous ligand for LXR-a protein , the neuroblastoma cells were seeded at an initial density of 5 9 104 cells per well in RPMI-1640 medium containing 10% FCS at 37°C in 5% CO2, and were then exposed to medium enriched with different concentrations of Withaferin A (0–10 lM) or 24S-OH-cholesterol (0–10 lM) for 48 h at 37°C and subsequently these cells were processed to study the effect of Withaferin A and 24S-OH-cholesterol on the transcriptional expression of genes coding for PAR-4, AATF and LXR-a by employing RT-PCR method mentioned above. The validation of gene expression of PAR-4, AATF and LXR-a in response to 10 lM Withaferin A or 10 lM 24S-OH-cholesterol were
To study the LXR-a dependent regulation of genes coding for AATF and PAR-4, neuroblastoma cells maintained in vitro culture were used as a cellular model to knock-down the LXR-a gene. These cells were seeded at a density of 6 9 104 cells per well in RPMI-1640 medium containing 10% FCS at 37°C in 5% CO2 atmosphere. After synchronisation, these cells were transfected with LXR-a siRNA as well as scrambled control siRNA (Santa Cruz Biotechnology, USA) using transfection reagent from the same company. In order to determine the efficiency of siRNA transfection flourescein-A conjugated control scrambled sequence was employed. The efficiency was found to be around 70–80%. After 72 h of transfection, the cells were harvested and processed for RNA and protein isolation by standard methods and subsequently the expression of genes coding for LXR-a, PAR-4 and AATF was analyzed by RTPCR. The LXR-a silencing was also checked at the protein level through immuno-detection. For this transfected cells were harvested in Laemmli sample buffer (10% 2-mercaptoethanol, 6% SDS, 20% glycerol, 0.2 mg/ml bromophenol blue). After lysis, the proteins from the above cellular extract were separated by SDS-PAGE on 12.5% polyacrylamide gels. Proteins were then transferred electrophoretically onto nitrocellulose membranes. Non-specific sites were blocked by incubating membranes in 2% BSA. Membranes were then probed with primary antibody against LXR-a and b-actin (Santa Cruz Biotechnology, USA) followed by alkaline phosphatase-conjugated secondary IgG antibody. Alkaline phosphatase activity was visualised on adding BCIP/NBT substrate. Bands visualised on addition of substrate were analysed using SCION IMAGE analysis software. LXR-a activation and PAR-4 promoter activity Bio-informatic analysis of PAR-4 gene promoter revealed putative regulatory sites corresponding to transcription factors SREBP and NF-jB. Since LXR-a is known to down regulate NF-jB gene and up regulate SREBP gene at the transcriptional level, the regulatory sequences (corresponding to transcription factors SREBP and NF-jB) present in PAR-4 gene promoter were incorporated in the TOPO reporter plasmid (Invitrogen) which was subsequently transfected in cultured human neuroblastoma cells (maintained at a density of 6 9 104) by making use of siPORTTM XP-1 transfection reagent (Applied Biosystems). After 6 h of transfection, the cells were exposed to medium with no additions or Withaferin A (10 lM) for
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48 h at 37°C. After this incubation period the b-galactosidase activity was measured by b-Gal assay kit (Invitrogen) according to manufacturers instructions.
LXR-a activation and Abeta generation Neuroblastoma cells were exposed to Withaferin A (0–10 lM) or 10 lM 24S-OH-cholesterol for 48 h at 37°C in order to explore whether or not LXR-a activation by Withaferin A or 24S-OH-cholesterol can generate extracellular Abeta. At the end of the incubation period, the extracellular medium was processed for protein isolation. The proteins were separated on 15% SDS-PAGE which were subsequently transferred to nitrocellulose paper and Abeta detection was done by using specific antibody against Abeta (Ab-42) (Sigma). Densitometric analysis of each band was performed by SCION IMAGE Analysis software in order to determine the relative level of Abeta with respect to total protein in the medium.
LXR-a activation, ROS production and cellular apoptosis
Effect of Withaferin A or 24S-OH-cholesterol on LXR-a knockdown cellular model In order to confirm that the ROS production and cellular death in neuroblastoma cells in response to Withaferin A or 24S-OH-cholesterol is mediated through LXR-a, we checked the extent of ROS production and cell death in LXR-a knockdown cellular model of neuroblastoma in response to 10 lM Withaferin A or 10 lM 24S-OHcholesterol. Cellular response of neuroblastoma cells after treatment with Withaferin A and Ab neutralising antibody In order to check that the observed cell death and ROS production are mediated through Ab, the neuroblastoma cells were exposed to 10 lM Withaferin A and 1 lg of Ab neutralising antibody (Sigma) for 48 h and subsequently the cells were processed to analyze the extent of ROS production and cell death.
Results LXR-a, AATF and PAR-4: Molecular link
In order to explore whether or not LXR-a activation within neuroblastoma cells (regarded as a model for neurons) generated Abeta stimulate THP-1 cells (regarded as a model for microglial cells) to produce reactive oxygen species (ROS) which in turn induces the apoptosis of neuroblastoma cells, two types of experiments were designed. In one set of experiments neuroblastoma cells were co-cultured with THP-1 cells and exposed to Withaferin A (0–10 lM) for 48 h at 37°C in 5% CO2 atmosphere. At the end of this incubation period the ROS produced by these cells was analyzed by flow cytometry by using dichlorofluorescein diacetate dye (DCF-DA; Sigma). In another set of experiments the neuroblastoma in culture were exposed to either H2O2 (0.5 mM) or Withaferin A (10 lM) or H2O2 ? Withaferin A or H2O2 ? Withaferin A ? ROS scavengers such as DMSO (0.5%) as chelator of OH- radicals or 1, 10-phenanthroline (150 lM) as chelator of iron or TEMPOL (1.2 mM) as chelator of superoxide for 48 h at 37°C in 5% CO2 atmosphere. At the end of the incubation period, the cells were harvested and subsequently subjected to flow cytometric analysis using dichlorofluorescein diacetate dye for the detection of ROS production and Annexin V-propidium iodide staining (Sigma) for the detection of cellular apoptosis. Keeping in view that 24S-OH-cholesterol is a natural ligand for LXR-a, we also checked the extent of ROS production and cell death in neuroblastoma cells in response to 10 lM 24S-OH-cholesterol.
Using neuroblastoma (IMR-32) cell line as the cellular model for Neuron, we were able to demonstrate that this cell line has the ability to express all the three genes coding for AATF, PAR-4 and LXR-a (Fig. 1). Keeping in view the
Fig. 1 Transcriptional expression of genes coding for AATF, PAR-4 and LXR-a in neuroblastoma (IMR-32) cells. Relative expression of these genes with each bar representing mean ± SD of experiments done in triplicate. Both genes coding for PAR-4 and LXR-a shows significantly low expression (p \ 0.05) with respect to that of AATF gene
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Fig. 2 Dose dependent expression of AATF, Par4 and LXR-a genes within neuroblastoma cells exposed to various concentrations of Withaferin A and 24S-OHcholesterol. Each point in the graph represents mean of experiments done in triplicate
fact that 22-OH-cholesterol, 24-OH-cholesterol and dietary compound Withaferin A have the capacity to act as specific ligands for LXR-a protein , we studied the effect of Withaferin A as well as 24S-OH-cholesterol on the transcriptional expression of genes coding for PAR-4, LXR-a and AATF within neuroblastoma (IMR-32) cells maintained in vitro culture. The results of this study revealed that Withaferin A and 24S-OH-cholesterol had not only the ability to down-regulate AATF gene expression but also up-regulate the expression of genes coding for LXR-a and PAR-4 in a dose-dependent fashion (Fig. 2a, b). This fact was also confirmed by the real time PCR which showed the similar results (Fig. 3). In order to further verify the LXR-a dependent regulation of genes coding for AATF and PAR-4, we made use of neuroblastoma cells in vitro culture to knock-down the LXR-a gene. The results of this study revealed that LXR-a gene knock-down cells exhibited significantly higher expression of AATF gene as well as lower expression of PAR-4 gene as compared to the corresponding control cells (Fig. 4a, b). Further, dissection of PAR-4 promoter using bioinformatics –tools revealed that the promoter sequence of this gene contains binding sites of transcription factors especially SREBP and NF-jB. The existence of response elements (having binding affinity for SREBP and NF-jB) within PAR-4 promoter established the molecular basis as to how LXR-a could regulate PAR-4 gene transcription (Figs. 2–4) because of the fact that LXR-a upon activation with its specific ligands is known to up-regulate SREBP gene expression as well as down-regulate NF-jB gene expression . To verify this phenomenon, we made use of reporter plasmid (having PAR-4 promoter sequence containing SREBP and NF-jB response
Fig. 3 Expression of AATF, Par4 and LXR-a with real time PCR after exposure of neuroblastoma (IMR-32) to (a) 10 lM Withaferin A and (b) 10 lM 24S-OH-cholesterol. Each bar represents mean ± SD of experiments done in triplicate. ** represents p value \ 0.05
elements) in order to study PAR-4 promoter activity within neuroblastoma (IMR-32) cells exposed to either no stimulus or Withaferin A or 24S-OH-cholesterol. The results of this study revealed that both agonists of LXR-a protein were able to significantly increase PAR-4 promoter activity within these cells (Fig. 5).
LXR-a activation, b-amyloid generation and apoptosis In order to explore whether or not increased expression of PAR-4 induced by LXR-a activation leads to generation of
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Fig. 4 Expression of LXR-a protein after 72 h of LXR-a siRNA transfection (a) and genes coding for AATF and PAR-4 in LXR-a gene knock down neuroblastoma cells (b) as compared to that found in the control cells. Each bar represents mean ± SD of experiments done in triplicate. ** represents p value \ 0.05
LXR-a activation, ROS production and apoptosis
Fig. 5 PAR-4 gene promoter dependent reporter activity in neuroblastoma cells exposed to Withaferin A and 24S-OH-cholesterol. Each bar represents mean ± SD of the experiments done in triplicate. ** represents significance value p \ 0.05 with respect to control transfected cells
b-amyloid (Ab-42) within neuroblastoma (IMR-32), these cells in vitro culture were exposed to medium enriched Withaferin A. The results of this study revealed that b-amyloid was generated within these cells in a dosedependent fashion by Withaferin A (Fig. 6a) leading to significant increase in their apoptosis (Fig. 6b, c). We also found the increased production of b-amyloid as well as increased cell death and ROS production in response to 10 lM 24S-OH-cholesterol (Fig. 7a–c).
Using neuroblastoma and THP-1 cells as the cellular model to represent neuronal and microglial cells, the experiments were conducted to explore whether or not b-amyloid generated through LXR-a activation results in ROS production responsible for the observed apoptosis of these cells (Fig. 6). Exposure of neuroblastoma cells co-cultured with THP-1 cells to Withaferin A resulted in dose-dependent increased production of ROS (Fig. 8a) whereas exposure of THP-1 cells to conditioned medium from Withaferin A treated neuroblastoma cells resulted in the increase in ROS production by THP-1 cells in a dose-dependent fashion (Fig. 8b). From these experiments we concluded that LXR-a activation within neuroblastoma cells generated b-amyloid which, in turn, activated THP-1 cells to secrete hydrogen peroxide responsible for accelerated apoptosis of these neuroblastoma cells. In order to further verify this phenomenon, the neuroblastoma cells were exposed in vitro to medium enriched with Withaferin A or H2O2 or Withaferin A ? H2O2 or Withaferin A ? H2O2 ? ROS scavengers (such as TEMPOL; DMSO; 1,10-phenanthroline) and subsequently ROS production and extent of apoptosis was studied in these cells. The results of this study revealed that maximum ROS production paralleled by apoptosis was observed in neuroblastoma cells exposed to Withaferin A ? H2O2 and this phenomenon was significantly reduced in presence of ROS scavengers employed in the study (Fig. 9a, b). Effect of Withaferin A or 24S-OH-cholesterol on LXR-a knockdown cellular model Exposure of LXR-a knockdown neuroblastoma cells with 10 lM Withaferin A or 10 lM 24S-OH-cholesterol
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Fig. 6 Amount of b-amyloid formation (Ab-42) (a) accompanied by extent of apoptosis (b, c) in neuroblastoma cells exposed to various concentrations of Withaferin A. Each point represents mean ± SD of experiments done in triplicate
Fig. 7 Expression of b amyloid and subsequent ROS production accompanied by apoptosis within neuroblastoma cells (IMR-32) exposed with 10 lM 24S-OH-cholesterol. Each bar represents mean ± SD of experiments done in triplicate. ** represents significance value p \ 0.05
revealed a significant decrease in the cell death and ROS production (Fig. 10a, b). The results reported here revealed that the observed cellular response (cell death
and ROS production) are mediated through LXR-a in response to its agonist Withaferin A and 24S-OHcholesterol.
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(Fig. 11a, b), which supports the fact that the reported cellular responses (cell death and ROS production) are mediated through Ab after activation with LXR-a agonist Withaferin A.
Fig. 8 Extent of ROS production within co-cultured neuroblastoma and THP-1 cells exposed to various concentrations of Withaferin A (a) and THP-1 cells exposed to conditioned medium from Withaferin A treated neuroblastoma cells (b). Each point represents mean ± SD of experiments done in triplicate
Cellular response of neuroblastoma cells after treatment with Withaferin A and Ab neutralising antibody Exposure of neuroblastoma cells to Withaferin A along with Ab neutralising antibody, resulted in significant decrease in apoptosis as well as ROS production
Much evidence exists to support the view that the aberrant production and aggregation of amyloid b-peptide (Abeta) is the pathognomonic feature responsible for cerebral degeneration with selective neuronal death observed in Alzheimer’s disease . The molecular mechanism for this aberrant production of Abeta remains obscure despite the fact that interaction of AATF with a leucine zipper protein PAR-4 was shown to be critical for b-secretase dependent cleavage of Alzheimer amyloid precursor protein [3, 4]. Keeping in view that 24S-hydroxycholesterol is mainly a production of brain cholesterol metabolism, the recent findings indicating elevated plasma and CSF levels of 24Shydroxycholesterol in patients with Alzheimer’s disease [8, 12] added a new dimension to the complex mechanism responsible for aberrant production of Abeta leading to selective neuronal death. In order to resolve this complex mechanism, our imagination was caught by the findings
Fig. 9 The extent of ROS production (a) accompanied by apoptosis (b) in neuroblastoma cells exposed to either H2O2 or Withaferin A (WA) alone or WA ? H2O2 or WA ? H2O2 ? DMSO or WA ? H2O2 ? 1,10-phenanthroline (PA) or WA ? H2O2 ? TEMPOL. Each bar represents mean ± SD of experiments done in triplicate. ** represents significance value p \ 0.05
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Fig. 10 Extent of ROS production and apoptosis in LXR-a knock down neuroblastoma cells (IMR-32) after exposed with 10 lM Withaferin A and 10 lM 24SOH-cholesterol. Each bar represents mean ± SD of experiments done in triplicate. ** represents significance value p \ 0.05
Fig. 11 Extent of ROS production (a) and apoptosis (b) in neuroblastoma cells (IMR-32) after exposed with 10 lM Withaferin A and Ab neutralizing antibody. Each bar represents mean ± SD of experiments done in triplicate. ** represents significance value p \ 0.05
that revealed: (a) Dietary compound had the capacity to induce PAR-4 gene expression responsible for apoptosis in Prostate Cancer Cells  (b) Withaferin A could regulate LXR-a activity in similar manner to that of its endogenous regulators such as 24S-or 22R-hydroxycholesterol . It is in this context that the results reported here regarding establishment of molecular relationship between LXR-a activation (through exogenous or endogenous regulators), the regulation of its effecter genes (such as PAR-4 and AATF) and aberrant-production of Abeta assume importance. The results reported here also revealed as to how increased PAR-4 gene expression upon LXR-a activation results in increased ROS production leading to increased apoptosis of neuroblastoma cells (Fig. 2-7). Keeping in view the findings indicating that Abeta-induced microglial release of H2O2 (produced from NADPH-oxidase) has been recognized to be responsible for inflammatory
neurodegeneration caused by Abeta, the results reported here show that LXR-a activation within: (a) neuroblastoma co-cultured with THP-1 cells results in significant increase in ROS production (Fig. 8); (b) neuroblastoma cells exposed to medium enriched with H2O2 results in significant increase in ROS production by these cells followed by appreciably high apoptosis and this phenomenon could be inhibited significantly in the presence of ROS scavengers (Fig. 9). Further, Abeta-induced cholesterol oxidation  has been shown to require divalent copper or iron and is accompanied by production of H2O2 . This phenomenon is in conformity with our results indicating that LXR-a induced ROS production by neuroblastoma and their subsequent apoptosis is inhibited to significant extent in presence of iron-chelator or Ab neutralising antibody as well as when the LXR-a gene is knocked-down in these cells (Figs. 9–11). Based upon these results, the sequence
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Fig. 12 Proposed LXR-a dependent signalling pathway responsible for the generation of b-amyloid within neurons leading to their death
of events that connect LXR-a activation with genes responsible for aberrant generation of Abeta followed by selective neuronal apoptosis, have been depicted in Fig. 12 and the salient features are as follows: LXR-a activation by its specific endogenous or exogenous agonists results in the over-expression of PAR-4 gene through upregulation of SREBP coupled with down-regulation of NF-jB as well as suppression of AATF gene transcription through NF-jB down-regulation. The overexpressed PAR-4 gene product can initiate neuronal death through: (a) activation of b-secretase dependent production of Abeta that has the inherent capacity to activate microglial NADPH oxidase to enable these cells to secret H2O2 [3, 5, 16, 17]; (b) suppression of apoptosis repressor gene Bcl-2 ; (c) Abeta-induced heme oxygenase-1 ensures oxidation of cholesterol  to provide endogenous ligands for sustained activation of neuronal LXR-a protein-dependent pathway (Fig. 12) involving aberrant Abeta production followed by ROS production and subsequent neuronal death. Hence molecules that inhibit heme oxygenase-1 may be of therapeutic importance in the treatment of Alzheimer’s disease.
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