Journal: Aging (Albany NY)

Article Title: Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways

doi: 10.18632/aging.103551

Figure Lengend Snippet: T-006 stimulates MEF2D/PGC1α/Nrf2 signal pathway through regulation of the Akt/GSK3β pathway in PD animal models. ( A ) Representative Western blots and densitometric analysis the expression of CDK5, p-MEF2D, MEF2D and PGC1α. ( B , E ) Representative Western blots and densitometric analysis of the expression of p-Akt and p-GSK3β. ( C , F ) Representative Western blots and densitometric analysis of the expression of Nrf2, HO-1 and TFAM. ( D ) Representative Western blots and densitometric analysis the expression of MEF2D and PGC1α. Data are expressed as mean±SEM (n=3 per group). # P<0.05, ## P<0.01, ### P<0.001 vs. sham group, and * P<0.05, ** P<0.01 and *** P<0.001vs. MPTP or 6-OHDA group.

Article Snippet: After blocking with a blocking buffer, the polyvinyldifluoride membranes were co-incubated using primary antibodies overnight at 4 o C respectively against TH (Cell Signaling Technology), CDK5, phospho-Ser444-MEF2D (p-MEF2D, Merck Millipore, Germany), MEF2D (Merck Millipore, Germany), PGC1α, Nrf2, HO-1, TFAM, phospho-Ser473-Akt (p-Akt), total-Akt (Akt), p-GSK3β, total-GSK3β (GSK3β), phospho-Ser133-CREB (p-CREB, Cell Signaling Technology), CREB (Cell Signaling Technology), mature BDNF (abcam, Britain), synaptophysin (SYP, Cell Signaling Technology) and β-actin.

Techniques: Western Blot, Expressing

Journal: Aging (Albany NY)

Article Title: Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways

doi: 10.18632/aging.103551

Figure Lengend Snippet: T-006 prevents the loss of SN DA neuron loss by activating MEF2D/PGC1α/GSK3β signal pathway. Representative images of middle brain sections co-stained with antibodies against ( A , D ) TH (green) and p-GSK3β (red); ( B , E ) TH (red) and MEF2D (green); ( C , F ) TH (green) and PGC1α (red). DAPI (blue) indicates nucleus. ( G , H ) Quantitative analysis of immunofluorescence intensity in TH-positive cells. Data are expressed as mean±SEM (n=3 to 4 per group). # P<0.05, ### P<0.001 vs. sham group, and * P<0.05, ** P<0.01 vs. MPP + or 6-OHDA group.

Article Snippet: After blocking with a blocking buffer, the polyvinyldifluoride membranes were co-incubated using primary antibodies overnight at 4 o C respectively against TH (Cell Signaling Technology), CDK5, phospho-Ser444-MEF2D (p-MEF2D, Merck Millipore, Germany), MEF2D (Merck Millipore, Germany), PGC1α, Nrf2, HO-1, TFAM, phospho-Ser473-Akt (p-Akt), total-Akt (Akt), p-GSK3β, total-GSK3β (GSK3β), phospho-Ser133-CREB (p-CREB, Cell Signaling Technology), CREB (Cell Signaling Technology), mature BDNF (abcam, Britain), synaptophysin (SYP, Cell Signaling Technology) and β-actin.

Techniques: Staining, Immunofluorescence

Journal: Aging (Albany NY)

Article Title: Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways

doi: 10.18632/aging.103551

Figure Lengend Snippet: T-006 activates the MEF2/PGC1α/Nrf2 pathway through regulation of the Akt-GSK3β pathway. A53T and corrected DA neurons treated with T-006 and the positive drug at the indicated concentration for 24 hours. For MPP + -treatment assay, CGNs were pretreated with or without LY294002 (1 μM), Akt-iv (1 μM), or LiCl (10 μM) for 2 h, incubated with or without T-006 for 2 h, and finally exposed to MPP + . Cell viability was examined using an MTT assay. Luciferase reporter gene assays respectively included MEF2 ( A ), PGC1α ( B ), PGC1α-ΔMEF2 ( C ) and ARE ( D ). ( E ) Representative images of neurons co-stained with antibody against Nrf2 (green). DAPI (blue) indicates nucleus. ( F ) Luciferase reporter gene assays of MEF2 with MPP + induction. ( G – K ) respectively represent the fold changes of CDK5, MEF2D, PGC1α, Nrf1 and Nrf2 at mRNA level. ( L ) Effect of MEF2D reduction on MPP + -induced neurotoxicity in CGNs. ( M ) Effects of Akt pathway inhibitor LY294002 and GSK3β inhibitor on MEF2 transcriptional activity. ( N ) Effects of Akt pathway inhibitors LY294002 and Akt-iv, and GSK3β inhibitor on MPP + -induced neurotoxicity in CGNs. Data above are all from three independent experiments, expressed as mean±SEM. # P<0.05, ## P<0.01 and ### P<0.001 vs. control (Ctrl) group, and * P<0.05, ** P<0.01, *** P<0.001 vs. MPP + group.

Article Snippet: After blocking with a blocking buffer, the polyvinyldifluoride membranes were co-incubated using primary antibodies overnight at 4 o C respectively against TH (Cell Signaling Technology), CDK5, phospho-Ser444-MEF2D (p-MEF2D, Merck Millipore, Germany), MEF2D (Merck Millipore, Germany), PGC1α, Nrf2, HO-1, TFAM, phospho-Ser473-Akt (p-Akt), total-Akt (Akt), p-GSK3β, total-GSK3β (GSK3β), phospho-Ser133-CREB (p-CREB, Cell Signaling Technology), CREB (Cell Signaling Technology), mature BDNF (abcam, Britain), synaptophysin (SYP, Cell Signaling Technology) and β-actin.

Techniques: Concentration Assay, Incubation, MTT Assay, Luciferase, Staining, Activity Assay, Control

Journal: Journal of Neuroscience

Article Title: Suppression of a MEF2-KLF6 Survival Pathway by PKA Signaling Promotes Apoptosis in Embryonic Hippocampal Neurons

doi: 10.1523/jneurosci.3609-11.2012

Figure Lengend Snippet: Figure2. ExogenousexpressionofactivatedPKAsuppressestheprosurvivalroleofMEF2in hippocampal neurons. A, Primary E18 hippocampal neuronal cells (7DIV) were transiently transfectedwithpGL3–4XMEF2-Lucreportergene,pCMV--galactosidasetonormalizetrans- fection efficiencies, and increasing amounts of the catalytic subunit of PKA (pFC-PKA 250 ng-1 g). B, Primary hippocampal neuronal cells were cotransfected either with empty vector or pcDNA3-MEF2D,incombinationwithorwithoutpFC-PKA(asindicated).MEF2-mediatedtran- scriptional activity was determined by pGL3–4XMEF2-Luc reporter gene and pCMV-- galactosidase assays. C, Primary hippocampal neuronal cells were cotransfected either with emptyvectororpFC-PKA(asindicated).Thirty-sixhoursaftertransfection,primaryhippocam- palneuronalcellswerestainedwithannexinV-FITCandPI(annexinV-FITCapoptosisdetection kit,Sigma).Necrosisandapoptosisweredeterminedbyflowcytometryanalysis.(Thepercent- age of apoptotic cells labeled with annexin V-FITC appeared in the lower right quadrant of the density plot, shown in the bottom right corner of each panel).

Article Snippet: Primary monoclonal antibodies - -tubulin III (T8660), and -MEF2D (610775), and GFP(B-2) (s.c.-9996) were purchased from Sigma, BD Biosciences, and Santa Cruz Biotechnology, respectively.

Techniques: Plasmid Preparation, Activity Assay, Labeling

Journal: Journal of Neuroscience

Article Title: Suppression of a MEF2-KLF6 Survival Pathway by PKA Signaling Promotes Apoptosis in Embryonic Hippocampal Neurons

doi: 10.1523/jneurosci.3609-11.2012

Figure Lengend Snippet: Figure 3. MEF2D-S121/190A confers resistance to PKA in hippocampal neurons. A, Schematic of mouse MEF2D indicating PKA phospho-acceptor sites. B, Primary hippocampal neuronal cells (7DIV) were transiently transfected with empty vector or mutated forms of MEF2D S121/190A (neutralizing) and S121/190D (phospho-mimetic) with or without PKA. Thirty-six hours after transfection, cells were stained with annexin V-FITC and PI (annexin V-FITC apoptosis detection kit, Sigma). Neuronal apoptosis was determined by flowcytometry analysis (FACS analyzer). C, Primaryhippocampalneuronsweretransientlycotransfectedat7DIVwithpGL3–4XMEF2-LucreportergeneandpCMV--galactosidasetonormalizetransfectionefficienciesandwithemptyvector or double mutated forms of MEF2D S121/190A and S121/190D, in combination with or without pFC-PKA (as indicated). MEF2-mediated transcriptional activity was determined by Luciferase and pCMV--Galassays.D,Primaryhippocampalneuronalcellsweretransientlycotransfectedwith5XGAL4-Luciferasereportervector,GAL-DBDorGAL4-MEF2D(87-507),withandwithoutpFC-PKA and -galactosidase to normalize transfection efficiencies. (Data are the mean SEM; n 3). E, Primary hippocampal neuronal cells (7DIV) were transiently transfected with empty vector or mutated forms of MEF2D S121/190A (neutralizing) and S121/190D with or without PKA. Cells were harvested and lysates were prepared 36 h later. Equal amounts of total protein were separated by 10% SDS-PAGE followed by immunoblot analysis using MEF2D monoclonal antibody (1:1000) to detect the expression levels of transfected constructs. Actin (polyclonal, 1:2000) was used as a loading control.

Article Snippet: Primary monoclonal antibodies - -tubulin III (T8660), and -MEF2D (610775), and GFP(B-2) (s.c.-9996) were purchased from Sigma, BD Biosciences, and Santa Cruz Biotechnology, respectively.

Techniques: Transfection, Plasmid Preparation, Staining, Activity Assay, Luciferase, SDS Page, Western Blot, Expressing, Construct, Control

Journal: Journal of Neuroscience

Article Title: Suppression of a MEF2-KLF6 Survival Pathway by PKA Signaling Promotes Apoptosis in Embryonic Hippocampal Neurons

doi: 10.1523/jneurosci.3609-11.2012

Figure Lengend Snippet: Figure 5. Cellular localization of MEF2D and KLF6 in hippocampal neurons. A, Primary hippocampal neuronal cells were fixed with4%paraformaldehyde.DoubleimmunofluorescenceanalysiswasperformedusingprimaryantibodiestoMEF2D(green)and aneuronalmarkertubulinIII(red).DAPI(4,6-diamidino-2-phenylindole)wasusedtoidentifynuclei(blue).Themergedpicture demonstrateslocalizationofMEF2D(nuclear)and-tubulinIIIpositivecells(red).B,PrimaryhippocampalneuronsexpressKLF6. HippocampalneuronalcellswerefixedanddoubleimmunofluorescenceanalysiswasperformedwithprimaryantibodiestoKLF6 showninredandto-tubulinIIIshowningreen.ThemergedpicturedemonstrateslocalizationofKLF6(nuclear)and-tubulin III positive cells (green), counterstained with DAPI (blue). C, Double immunofluorescence labeling demonstrating KLF6 (red) and MEF2D (green) in primary hippocampal neurons. The merged picture indicates MEF2D-positive cells are mostly positive for KLF6 and both proteins are predominantly nuclear.

Article Snippet: Primary monoclonal antibodies - -tubulin III (T8660), and -MEF2D (610775), and GFP(B-2) (s.c.-9996) were purchased from Sigma, BD Biosciences, and Santa Cruz Biotechnology, respectively.

Techniques: Immunofluorescence, Labeling

Journal: Journal of Neuroscience

Article Title: Suppression of a MEF2-KLF6 Survival Pathway by PKA Signaling Promotes Apoptosis in Embryonic Hippocampal Neurons

doi: 10.1523/jneurosci.3609-11.2012

Figure Lengend Snippet: Figure 10. PKA induces physical association of HDAC4 with MEF2D in hippocampal neurons. A, Primary hippocampal neurons were transiently transfected with indicated expression plasmids. Cell extracts were immunoprecipitated with anti-MEF2D fol- lowed by immunoblotting with anti-HDAC4. Whole-cell lysates were immunoblots with indicated antibodies. HDAC4 interacts withMEF2DinthepresenceofPKAandthisassociationislostwithHDAC4L175AwithorwithoutPKA.B,ForHDAC4genesilencing inprimaryhippocampalneurons,threeindependentsiRNAsweretransfectedalongwithacontrolscRNA.Cellswereharvestedand lysateswereprepared48hlater.ProteinlevelofHDAC4wasanalyzedbyimmunoblotusingHDAC4polyclonalantibody(1:1000). Actin(polyclonal,1:2000)wasusedasaloadingcontrol.C,Primaryhippocampalneuronsweretransfectedwithtwoindependent siRNAsandacontrolscRNA.48haftertransfection,cellswerestainedwithannexinV-FITCandPIusingannexinV-FITCapoptosis detection kit, as described in Material and Methods. Neuronal apoptosis was measured using flow cytometry (FACS analyzer).

Article Snippet: Primary monoclonal antibodies - -tubulin III (T8660), and -MEF2D (610775), and GFP(B-2) (s.c.-9996) were purchased from Sigma, BD Biosciences, and Santa Cruz Biotechnology, respectively.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Flow Cytometry