doi: 10.1016/j.cmet.2015.05.022.
Epub 2015 Jun 25.
HMG-CoA Reductase Inhibitors Bind to PPARα to Upregulate Neurotrophin Expression in the Brain and Improve Memory in Mice
Affiliations
- PMID: 26118928
- PMCID: PMC4526399
- DOI: 10.1016/j.cmet.2015.05.022
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HMG-CoA Reductase Inhibitors Bind to PPARα to Upregulate Neurotrophin Expression in the Brain and Improve Memory in Mice
Cell Metab.
.
doi: 10.1016/j.cmet.2015.05.022.
Epub 2015 Jun 25.
- PMID: 26118928
- PMCID: PMC4526399
- DOI: 10.1016/j.cmet.2015.05.022
Item in Clipboard
Abstract
Neurotrophins are important for neuronal health and function. Here, statins, inhibitors of HMG-CoA reductase and cholesterol lowering drugs, were found to stimulate expression of neurotrophins in brain cells independent of the mevalonate pathway. Time-resolved fluorescence resonance energy transfer (FRET) analyses, computer-derived simulation, site-directed mutagenesis, thermal shift assay, and de novo binding followed by electrospray ionization tandem mass spectrometry (ESI-MS) demonstrates that statins serve as ligands of PPARα and that Leu331 and Tyr 334 residues of PPARα are important for statin binding. Upon binding, statins upregulate neurotrophins via PPARα-mediated transcriptional activation of cAMP-response element binding protein (CREB). Accordingly, simvastatin increases CREB and brain-derived neurotrophic factor (BDNF) in the hippocampus of Ppara null mice receiving full-length lentiviral PPARα, but not L331M/Y334D statin-binding domain-mutated lentiviral PPARα. This study identifies statins as ligands of PPARα, describes neurotrophic function of statins via the PPARα-CREB pathway, and analyzes the importance of PPARα in the therapeutic success of simvastatin in an animal model of Alzheimer's disease.
Copyright © 2015 Elsevier Inc. All rights reserved.
Figures
Figure 1. Anti-inflammatory, but Not Neurotrophic, Activity…
Figure 1. Anti-inflammatory, but Not Neurotrophic, Activity of Simvastatin Depends on Mevalonate Metabolites and Isoprenylation…
(A and B) Mouse astrocytes pre-treated with different doses of farnesyl pyrophosphate (FPP) and mevalonate for 1 hr were stimulated with 10 μM simvastatin for another 5 hr under serum-free condition followed by mRNA analysis of neurotrophins by semi-quantitative RT-PCR (A) and real-time PCR (B). Results represent three independent analyses. ap < 0.001 versus control-BDNF; bp < 0.001 versus control-NT3. (C) Astrocytes pre-treated with mevalonate, farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP) in the presence of 10 μM simvastatin for 6 hr were stimulated with IL-1β (20 ng/ml) for another 12 hr followed by analysis of iNOS by western blot. (D) Bands were scanned and presented as relative to control. ap < 0.001 versus control; bp < 0.001 versus IL-1β; cp < 0.001 versus IL-1β + Simva. (E) Astrocytes were transfected with pcDNA3 (empty vector) and dominant-negative mutants of p21ras (ΔRas) and p21rac (ΔRac). After a 24-hr transfection, cells were incubated with serum-free media for 6 hr followed by analysis of mRNA expression of BDNF and NT-3 by real-time PCR. (F) Similarly, 24 hr after transfection, cells were stimulated with IL-1β (20 ng/ml) for 6 hr followed by monitoring the expression of iNOS mRNA by real-time PCR. (G) After transfection with control and p21rassiRNA, cells were incubated with serum-free media for 6 hr followed by monitoring Bdnf and Nt3 mRNAs by real-time PCR. (H) Similarly, after transfection, cells were stimulated with IL-1β for 12 hr followed by analysis of iNOS protein by western blot. (I) Bands were scanned and presented as relative to control. ap < 0.001 versus control; bp < 0.001 versus control-siRNA-IL-1β. (J) Astrocytes were incubated with farnesyl phosphotransferase inhibitor (FPTi) and geranylgeranyl phosphotransferase inhibitor (GGTi) for 6hr followed by analysis of Bdnf mRNA by realtime PCR. (K) Astrocytes pretreated with FPTi and GGTi for 1 hr were stimulated with IL-1β followed by monitoring iNOS protein after a 12-hr incubation. (L) Bands were scanned and presented as relative to control. ap < 0.001 versus control; bp < 0.001 versus IL-1β. Results represent three independent analyses. See also Figure S1.
Figure 2. PPARα Is Involved in Neurotrophic,…
Figure 2. PPARα Is Involved in Neurotrophic, but Not Anti-inflammatory, Properties of Statins
(A–H) Astrocytes…
(A–H) Astrocytes isolated from WT and Ppara null (α KO) mice were treated with simvastatin for 6 hr followed by stimulation with IL-1β. After 6 hr, the mRNA expression of iNOS was monitored by RT-PCR (A) and real-time PCR (B). Results are mean ± SD of three independent experiments. After 24 hr, the protein level of iNOS was monitored by western blot (C) and immunofluorescence (D). Microglia (E and F) and astrocytes (G and H) isolated from WT and α KO mice were treated with simvastatin. After 6 hr, cells were analyzed for mRNA encoding BDNF and NT-3 by RT-PCR (E and G) and real-time PCR (F and H). ap < 0.001 versus control-BDNF; bp < 0.001 versus control-NT-3. (I–K) WT and α KO primary astrocytes were incubated with 10 μM simvastatin (J) and pravastatin (K) under serum-free conditions. After 24 hr, the BDNF level was analyzed by double-label immunofluorescence. Results represent three independent experiments. See also Figures S2 and S3.
Figure 3. A High Throughput Analysis to…
Figure 3. A High Throughput Analysis to Study the Interaction between PPARα and Statin
(A–D)…
(A–D) PPARα-LBD affinity-purified nuclear fraction extracted from control (A) and simvastatin-treated (B) primary astrocytes was analyzed by electrospray ionization tandem mass spectrometry. Counts versus retention time shows the peak of simvastatin at 4.56 min in simvastatin-treated, but not control, sample. The magnified view of counts versus mass-to-charge ratio clearly shows the peak of simvastatin in the simvastatin-treated (D), but not control (C), sample. (E) To identify stain group of drugs as ligand of PPARα, time-resolved fluorescence energy transfer (TR-FRET) technology was adopted. Successful binding of statin with PPARα would transfer fluorescence energy from Terbium (Tb)-tagged anti-GST antibody to Fluorescein (FL)-tagged PGC-1α co-activator. The optimum level of emitted fluorescence was measured at 400 μ-second integration time and 40 μ-second delay time set in Molecular Device Analyst instrument. (F–H) Fluorescein emission was recorded with increasing doses of (F) simvastatin, (G) pravastatin, and (H) mevastatin. (I) TR-FRET was also performed for atorvastatin and rosuvastatin and maximum emitted fluorescence was compared among different statins. Results represent three independent analysis.
Figure 4. Proteomic Analysis of Interaction between…
Figure 4. Proteomic Analysis of Interaction between PPARα and Simvastatin
(A) Ribbon representations of the…
(A) Ribbon representations of the docked complex of PPARα (3VI8.pdb, X-ray, green) and simvastatin show the distribution of amino acids within a 5.00-Å distance of simvastatin. (B) Magnified view of the docking site shows the interaction between simvastatin and Leu331 and Tyr334. (C and D) Thermal shift assay of (C) full-length (GFP-FLPpara) and (D) statin-binding domain mutated (GFP-ΔsbdPpara) PPARα was performed. (E) De novo binding assay of simvastatin where mouse astrocytes transduced with FLPpara and ΔsbdPpara constructs were treated with simvastatin for 2 hr, nuclear extracts were isolated, passed through GFP column for affinity-purification, and then analyzed by ESI-MS. Representative counts versus acquisition time ratio of simvastatin analyzed in the nuclear extracts of GFP-transduced (i and ii), GFP-FLPpara-transduced (iii and iv), and GFP-ΔsbdPpara-transduced (v and vi) astroglial cells. (F) PPRE-driven luciferase activity in FLPpara and ΔsbdPpara-transduced Ppara null astrocytes after treatment with simvastatin (top), pravastatin (middle), and mevastatin (bottom). Results are mean ± SD of three independent experiments. ap <0.001 versus control; bp < 0.001 versus FLPpara.(G and H) Docked poses of gemfibrozil (G) and WY14673 (H) in PPARα ligand binding core. (I and J) PPRE-driven luciferase activity in FLPpara and ΔsbdPpara-transduced Ppara null astrocytes after treatment with WY14643 (I) and gemfibrozil (J). Results are mean ± SD of three independent experiments.
Figure 5. The Role of PPARα, PPARβ,…
Figure 5. The Role of PPARα, PPARβ, and PPARγ in Transcriptional Regulation of CREB
(A–E)…
(A–E) Mouse astrocytes were transfected with PPRE luciferase and after a 24-hr transfection, cells were treated with mevastatin, pravastatin, simvastatin (A), atorvastatin, and rosuvastatin (B) for 4 hr, followed by the luciferase assay. Activation of WT CREB promoter by pravastatin, simvastatin (C), atorvastatin, and rosuvastatin (D) in astrocytes isolated from WT and Ppara null (αKO) mice. Activation of WT and mutated CREB promoter by mevastatin, pravastatin, and simvastatin in mouse astrocytes (E). Results are means ± SD of three independent experiments. ap < 0.001 versus control. (F–H) The ChIP assay for PPARα (F), PPARβ (G) and PPARγ (H) in pravastatin-treated WT and αKO astrocytes. (I–K) The ChIP assay for PPARα (I), PPARβ (J), and PPARγ (K) in simvastatin-treated WT and αKO astrocytes. Results represent three separate analyses. See also Figures S3 and S5.
Figure 6. Lentiviral Manipulation of FL Ppara …
Figure 6. Lentiviral Manipulation of FL Ppara and Δsbd Ppara in the Adult Brain Hippocampus…
(A) Astrocytes isolated from Ppara null (αKO) mice were transduced with GFP-FLPpara and GFP-ΔsbdPpara to analyze the transduction efficiency of our plasmids. (B) Expression of CREB and BDNF were checked by immunoblot analysis. (C) Bands were scanned and presented as relative to control. Results are mean ± SD of three independent experiments. ap < 0.001 versus lenti-FLPpara control-CREB; bp < 0.001 versus lenti-FLPpara control-BDNF. (D) BDNF expression was confirmed by ELISA from the respective supernatants. ap < 0.001 versus lenti-vector control; bp < 0.05 versus lenti-FLPpara control. (E) Both of these constructs were injected bilaterally in the hippocampus of 6- to 8-week-old male C57BL/6 mice with coordinates of 2.54 mm AP axis, 1.30 mm ML (CA2 layer) and 1.80 mm ML (SGlayer of DG) axis, and 2.4 mm DV axis. Each animal received four injections with two injections perhemisphere. After 3 weeks, the distribution of lentivirus was detected by microscopic analysis of GFP. Injection sites were magnified and shown inside panels. (F) After 2 weeks of simvastatin feeding, the expression of BDNF in GFAP-immunoreactive astrocytes were analyzed (green, GFP-PPARα; red, GFAP; blue, BDNF). (G) CREB and BDNF expression were analyzed by immunoblot in the hippocampal extracts of GFP, GFP-FLPpara, and GFP-ΔsbdPpara animals. (H) Bands were scanned and presented as relative to control. ap < 0.05 versus simvastatin; bp < 0.01 versus lenti-FLPpara-simvastatin. (I) Expression of BDNF was confirmed by ELISA. ap < 0.01 versus simvastatin; bp < 0.01 versus lenti-FLPpara-simvastatin. (J) Performances of statin-fed FLPpara and ΔsbdPpara animals (n = 5 per group) in Barnes maze were compared with statin-fed αKO mice. Target hole placed in first quadrant (Q1) is shown as red. (K–M) Total time spent in all four quadrants (Q1–Q4), (L) latency and (M) errors were calculated. ap < 0.05 (= 0.041) versus statin-fed Ppara null and bp < 0.05 (= 0.037) versus statin-fed FLPpara. *p < 0.01 versus statin-fed αKO and **p < 0.05 (= 0.045) versus statin-fed ΔsbdPpara (0.0015) animals.
Figure 7. Simvastatin Increases BDNF in the…
Figure 7. Simvastatin Increases BDNF in the Brain of FAD5X Mice via PPARα
(A–E) Genotyping…
(A–E) Genotyping of FAD5X/Ppara null (5X/αKO) mice. Five-month-old mice were fed simvastatin for 2 weeks followed by immunofluorescence analysis of astroglial BDNF (GFAP, red; BDNF, green) in the cortices of (B) wild-type (WT), (C) αKO, (D) FAD5X, and (E) FAD5X/αKO animals. (F and G) Immunoblot (F) and densitometric (G) analyses of CREB and BDNF were performed in the hippocampal extracts. (H) ELISA of BDNF in the hippocampal extracts. (I and J) Number of errors (I) and latency (J) in Barnes maze for statin unfed and fed WT, αKO, FAD5X, and FAD5X/αKO. (K–N) All groups of mice were also monitored for body weight (K), stereotypy (L), total distance (M), and horizontal activity (N). Eight mice (n = 8) were used in each group. *p < 0.05 versus statin-unfed wild-type control and **p < 0.05 versus statin-unfed FAD5X mice.
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