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: Oncogene

Article Title: Divergent functions for eIF4E and S6 kinase by Sonic hedgehog mitogenic signaling in the developing cerebellum

doi: 10.1038/onc.2010.564

Figure Lengend Snippet: Shh-treated proliferating CGNPs show increased levels of translation initiation components and suppressed S6 kinase activity. (A) Western blot analysis of protein isolated from vehicle- or Shh-treated CGNP primary cultures. Shh treatment stimulates mTOR activity as demonstrated by eIF4G and 4EBP2 hyperphosphorylation but blocks S6 kinase activity measured by rpS6 phosphorylation. Levels of the mTOR effector eIF4E increase in Shh-treated CGNPs. (B) Quantification of western blots by densitometry. Phosphorylated protein levels were compared to total protein levels after normalization to β-Tubulin n=3, (**p<0.002, ***p<0.0005). (C to I) Immunofluorescence analysis of vehicle- and Shh-treated CGNPs plated on coverslips. eIF4E increases in Shh-treated CGNPs (D compared to C) while phospho-S235/236-rpS6 accumulates in vehicle-treated CGNPs (E compared to F). Total rpS6 levels do not change in proliferating or differentiated cells (G, H). Furthermore phospho-S235/236-rpS6 is expressed in vehicle-treated CGNPs marked by the differentiation marker, MEF2D (I arrowheads), while eIF4E protein colocalizes with PCNA positive cells (J arrowheads) in Shh-treated CGNPs. (K) Quantification of immunostaining in C-H, n=3, (***p<0.0001, **p<0.001). (L) Percentage of cells expressing eIF4E only, PCNA only or both eIF4E and PCNA in Shh-treated CGNPs. (M) Percentage of cells expressing MEF2D only, phospho-rps6 only or both MEF2D and phospho-rpS6 in vehicle-treated CGNPs.

Article Snippet: Primary antibodies included eIF4E, phospho-S6 (235/236), GFAP, NeuN (Cell Signaling), MEF2D, p27 (BD-PharMingen), BrdU (Becton Dickinson), and PCNA (Calbiochem).

Techniques: Activity Assay, Western Blot, Isolation, Phospho-proteomics, Immunofluorescence, Marker, Immunostaining, Expressing

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