Journal: Nature Communications

Article Title: The MK2/3 cascade regulates AMPAR trafficking and cognitive flexibility

doi: 10.1038/ncomms5701

Figure Lengend Snippet: ( a ) Images of cells expressing actin-EGFP. Note that actin accumulates at spine heads in WT cells, whereas in DKO cells actin accumulates along the process (arrows) and the spines display longer neck and smaller head diameters (arrowheads). ( b ) Graphs showing decreased spine density per 50 μm (WT: 27.6±1.34, 29 processes; DKO: 18.9±0.91; 28 processes), volume (μm 3 ; WT: 0.43±0.01; 796 spines, 17 cells; DKO: 0.39±0.01; 801 spines, 23 cells), head diameter (μm; WT: 0.56±0.01; 796 spines, 17 cells; DKO: 0.48±0.01; 801 spines, 23 cells) but increased neck length (μm; WT: 0.88±0.03; 472 spines, 29 processes; DKO: 1.69±0.06; 464 spines, 28 processes). ( c ) Cumulative distribution of data shown in b . Note that the majority of the spines in DKO cells display longer necks compared with WT (*** P <0.001), smaller spine head diameter (*** P <0.001) and volume (** P <0.01). ( d (i)) WT and DKO cells expressing EGFP or actin-EGFP co-stained with PSD95. Note the co-localization of the long and thin protrusions with PSD95 in DKO cells (arrowheads). (ii) Non-transfected cells co-stained with SV2a and PSD95. (iii) Co-localization analysis of pre- and postsynaptic marker. ( e ) WT and DKO neurons expressing a range of MK2- and MK3-EGFP-tagged constructs. ( f ) WT cells expressing MK2-K79R shows increased spine neck length (EGFP: 0.81±0.03, 412 spines; MK2-WT: 0.89±0.05, 189 spines; MK2-EE: 0.99±0.05, 385 spines; MK2-K79R: 1.73±0.07, 454 spines) and reduced head diameter (EGFP: 0.50±0.01, 629 spines; MK2-WT: 0.51±0.01, 672 spines; MK2-EE: 0.52±0.01, 564 spines; MK2-K79R: 0.44±0.01, 727 spines). Interestingly, spine neck length was reduced to WT levels in DKO cells expressing MK2-WT and MK2-EE, but not in cells expressing MK3-WT (EGFP: 1.67±0.08, 220 spines; MK2-WT: 1.17±0.05, 367 spines; MK2-EE: 0.72±0.03, 257 spines; MK2-K79R: 2.05±0.09, 296 spines; MK3-WT: 1.694±0.05, 700 spines), and head diameter was increased in DKO cells expressing MK2-WT, MK2-EE, MK2-K79R and MK3-WT (EGFP: 0.40±0.01 μm, 468 spines; MK2-WT: 0.50±0.01 μm, 909 spines; MK2-EE: 0.49±0.01 μm, 709 spines; MK2-K79R: 0.45±0.01 μm, 370 spines; MK3-WT: 0.509±0.01μm, 700 spines). An unpaired (Mann–Whitney) T -test ( b ), Kolmogorov–Smirnov ( c ) and one-way analysis of variance ( d ) with appropriate post-hoc test was used. Error bars=±s.e.m., * P <0.05, ** P <0.01, *** P <0.001. Scale bar, 10 μm.

Article Snippet: The blots were incubated overnight at 4 °C with primary antibodies including phospho (p)-p38 MAPK (Thr180/Tyr182; Cell Signaling, 4511), total p38 MAPK (Cell Signaling, 9212), p-MK2 (Thr222/Thr334; Cell Signaling, 3316/3007), p-cofilin (Ser3) (Cell Signaling, 3313), total cofilin1 (Cell Signaling, 3312), GluA1 (Millipore, AB1504), GluA2 (Millipore, MAB397), Arc/Arg3.1 (Synaptic Systems, 156–003), β-Tubulin (Abcam, AB6046) and GAPDH (Abcam, AB8245).

Techniques: Expressing, Staining, Transfection, Marker, Construct, MANN-WHITNEY

Journal: Nature Communications

Article Title: The MK2/3 cascade regulates AMPAR trafficking and cognitive flexibility

doi: 10.1038/ncomms5701

Figure Lengend Snippet: ( a ) AMPAR-dependent mEPSC traces from WT, DKO and DKO neurons expressing EGFP-tagged-MK2-WT (rescue) at baseline. ( b ) AMPAR-dependent mEPSC events recorded from WT, DKO and DKO neurons expressing MK2-WT and MK3-WT. ( c ) Cumulative distribution of all mEPSC amplitudes recorded. ( d ) Mean mEPSC amplitude and ( e ) frequency showing that the decrease in amplitude and increase in frequency observed in DKO are reversed by re-insertion of MK2-WT. ( f ) Note that the double peaks in mEPSC amplitude distribution in DKO cells, suggesting multiquantal release, are not seen in WT nor in DKO expressing MK2-WT. Shown are data from WT ( n =8 cells), DKO ( n =10 cells) and DKO overexpressing MK2-WT ( n =6 cells) from three to four independent preparations. Error bars represent ±s.e.m. and * P <0.04; ** P <0.01.

Article Snippet: The blots were incubated overnight at 4 °C with primary antibodies including phospho (p)-p38 MAPK (Thr180/Tyr182; Cell Signaling, 4511), total p38 MAPK (Cell Signaling, 9212), p-MK2 (Thr222/Thr334; Cell Signaling, 3316/3007), p-cofilin (Ser3) (Cell Signaling, 3313), total cofilin1 (Cell Signaling, 3312), GluA1 (Millipore, AB1504), GluA2 (Millipore, MAB397), Arc/Arg3.1 (Synaptic Systems, 156–003), β-Tubulin (Abcam, AB6046) and GAPDH (Abcam, AB8245).

Techniques: Expressing

Journal: Nature Communications

Article Title: The MK2/3 cascade regulates AMPAR trafficking and cognitive flexibility

doi: 10.1038/ncomms5701

Figure Lengend Snippet: ( a ) AMPAR-dependent mEPSC traces from WT, DKO and DKO neurons expressing eGFP-tagged-MK2-WT (rescue) at baseline and 30 min after DHPG exposure. ( b ) Graphs showing average time course of changes in mEPSC amplitude and frequency. Average mEPSC amplitude was 60.6±7.4% of baseline at 45±5 min (blue trace; n =8) in WT, 87.9±6.3% in WT cells pre-incubated with the p38 inhibitor SB 203580 (5 μM; orange trace; n =7) and 95.7±7.2% (red trace; n =10) in DKO. Note that LTD was rescued in DKO expressing MK2-WT 66.5±7.7% (green trace; n =6). Average mEPSC frequency of WT cells was 51.9±8.9 of the baseline at 45±5 min following LTD induction (blue), compared with 88.3±5.8 in WT cells pre-incubated with p38 inhibitor (orange) and to 96.7±7.2 in DKO cells (red). Re-insertion of MK2-WT in DKO cells rescued the frequency (63.2±8.5% of baseline; green) to WT values. ( c ) Example of mEPSC waveforms recorded during baseline (black traces) and after DHPG exposure (coloured traces) for the cell recorded in a . ( d ) Cumulative mEPSC amplitude distributions from WT and DKO cells. ( e ) Graphs of mean mEPSC quantal size for all experimental conditions. Note that the DHPG-dependent reduction in activity observed in wild-type cells (blue bars) is occluded in DKO cells (red bars) but restored in DKO cells expressing MK2-WT protein. ( f ) Graphs showing that the DHPG-dependent reduction in frequency and amplitude observed in WT cells are impaired in DKO cells. ( g ) Representative mEPSC waveforms recorded from DKO; DKO cells expressing MK3-WT at baseline and 30 min after DHPG exposure. ( h ) Graph of mean mEPSC amplitude (DKO: 10.9±3.4 pA and DKO+MK3-WT: 11.7±3.5 pA) and frequency (DKO: 1.85±0.72 Hz and DKO+MK3-WT: 1.71±0.74 Hz) at baseline and ( i ) after DHPG exposure. Data shown are from DKO ( n =14 cells); DKO cells expressing MK3-WT at baseline ( n =6 cells) and after DHPG exposure ( n =4 cells). Error bars=±s.e.m. Recordings were done in the presence of tetrodotoxin (1 μM), picrotoxin (50 μM) and L689–560 (5 μM). Error bars indicate the ±s.e.m. and * P <0.02, ** P <0.01, *** P <0.005.

Article Snippet: The blots were incubated overnight at 4 °C with primary antibodies including phospho (p)-p38 MAPK (Thr180/Tyr182; Cell Signaling, 4511), total p38 MAPK (Cell Signaling, 9212), p-MK2 (Thr222/Thr334; Cell Signaling, 3316/3007), p-cofilin (Ser3) (Cell Signaling, 3313), total cofilin1 (Cell Signaling, 3312), GluA1 (Millipore, AB1504), GluA2 (Millipore, MAB397), Arc/Arg3.1 (Synaptic Systems, 156–003), β-Tubulin (Abcam, AB6046) and GAPDH (Abcam, AB8245).

Techniques: Expressing, Incubation, Activity Assay

Journal: Nature Communications

Article Title: The MK2/3 cascade regulates AMPAR trafficking and cognitive flexibility

doi: 10.1038/ncomms5701

Figure Lengend Snippet: ( a – f ) Representative blots of non-stimulated (C), DHPG-incubated (100 μM, 10 min; D), pre-incubated with p38 inhibitor SB 203580 (5 μM) alone (SB) or pre-incubated with DHPG application (SB+D) cultures from WT and MK2/3 DKO hippocampus. ( a ) DHPG causes a significant increase in p-p38 at (Thr180/Tyr182) and ( b ) a significant reduction in p-cofilin1 in WT cells, an effect that is blocked in DKO cells. Note that no change in total cofilin1 ( c ) or mGluR5 ( d ) expression is detected between genotypes. ( e ) Blot showing a significant increase in phosphorylation of MK2 (Thr334) after DHPG-exposure in WT cells, an effect blocked by pre-incubation with SB. ( f ) Blot showing that the DHPG-dependent reduction in levels of p-cofilin1 (Ser3) is blocked by pre-incubation of p38 inhibitor SB 203580 (5 μM) in WT hippocampal cultures. Incubation of SB alone does not promote any significant changes in cofilin1 phosphorylation. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin were used as loading controls. Western blot band densitometry analyses were obtained from a minimum of three different primary hippocampal preparations from the WT and DKO mice. T -tests, one-way analysis of variance and the appropriate post-hoc test were conducted accordingly for each data set. Error bars indicate ±s.e.m. and * P <0.05, ** P <0.01.

Article Snippet: The blots were incubated overnight at 4 °C with primary antibodies including phospho (p)-p38 MAPK (Thr180/Tyr182; Cell Signaling, 4511), total p38 MAPK (Cell Signaling, 9212), p-MK2 (Thr222/Thr334; Cell Signaling, 3316/3007), p-cofilin (Ser3) (Cell Signaling, 3313), total cofilin1 (Cell Signaling, 3312), GluA1 (Millipore, AB1504), GluA2 (Millipore, MAB397), Arc/Arg3.1 (Synaptic Systems, 156–003), β-Tubulin (Abcam, AB6046) and GAPDH (Abcam, AB8245).

Techniques: Incubation, Expressing, Phospho-proteomics, Western Blot

Journal: Nature Communications

Article Title: The MK2/3 cascade regulates AMPAR trafficking and cognitive flexibility

doi: 10.1038/ncomms5701

Figure Lengend Snippet: ( a , b ) A schematic representation of the putative mechanism by which the p38-MK2/3 cascade regulates cofilin1 activity and GluA1 trafficking. ( a ) At non-stimulated dendritic spines of WT neurons levels of p38, MK2 and cofilin1 activity are low, and the balance between G- and F-actin is skewed towards F-actin; resulting in low levels of GluA1/2 endocytosis. By comparison, in spines of non-stimulated DKO neurons, where the p38-MK2 signalling cascade is blocked, there is a long lasting increase in basal cofilin1 activity levels, resulting in increased G-actin and abnormal spine morphology. Interestingly, the spines of MK2/3 DKO neurons, which display decreased head diameters and longer neck lengths, also have deficits in glutamatergic synaptic transmission. ( b ) Activation of mGluRs in WT spines induces an increase in p38 and MK2 activity, which correlates to an increase in cofilin1 activity and a shift in balance between G- and F-actin towards G-actin and increased endocytosis of GluA1 receptors. In DKO mice, the block of p38-MK2/3 cascade results in reduced cofilin1 activity and GluA1 endocytosis combined with deficits in mGluR-LTD.

Article Snippet: The blots were incubated overnight at 4 °C with primary antibodies including phospho (p)-p38 MAPK (Thr180/Tyr182; Cell Signaling, 4511), total p38 MAPK (Cell Signaling, 9212), p-MK2 (Thr222/Thr334; Cell Signaling, 3316/3007), p-cofilin (Ser3) (Cell Signaling, 3313), total cofilin1 (Cell Signaling, 3312), GluA1 (Millipore, AB1504), GluA2 (Millipore, MAB397), Arc/Arg3.1 (Synaptic Systems, 156–003), β-Tubulin (Abcam, AB6046) and GAPDH (Abcam, AB8245).

Techniques: Activity Assay, Comparison, Transmission Assay, Activation Assay, Blocking Assay

Journal: Current Issues in Molecular Biology

Article Title: Mitigation of 3.5 GHz Electromagnetic Field-Induced BV2 Microglial Cytotoxicity by Polydeoxyribonucleotide

doi: 10.3390/cimb47060386

Figure Lengend Snippet: Effect of SP600125, SB203580, or PD98059 on 3.5 GHz EMF-induced growth suppression of BV2 cells and phosphorylation of JNK-1/2, p38 MAPK (MK2), or ERK-1/2 in BV2 cells. ( A ) BV2 cells were exposed to 3.5 GHz EMFs without or with SP600125 (25 μM), a JNK-1/2 inhibitor, SB203580 (25 μM), a p38 MAPK inhibitor, or PD98059 (50 μM), an ERK-1/2 inhibitor for 2 h. The number of surviving cells was analyzed using cell count analysis. Data represent the mean ± SE of three independent experiments. * p < 0.01 compared with the values of the control (no 3.5 GHz EMF exposure). # p < 0.01 compared with the values of 3.5 GHz EMF exposure (no drug), as determined by one-way ANOVA followed by Sidak’s post hoc test. ( B ) A representative image of morphological changes in the conditioned cells in ( A ). ( C ) BV2 cells were exposed to 3.5 GHz EMFs without or with SP600125 (25 μM), SB203580 (25 μM), or PD98059 (50 μM) for 2 h. Whole-cell lysates from the conditioned cells were prepared and analyzed using Western blotting with antibodies.

Article Snippet: The primary antibodies, including eukaryotic initiation factor-2α (eIF-2α) (cat. no. 9722), phosphorylated (p)-extracellular signal-regulated protein kinase-1/2 (p-ERK-1/2) (T202/Y204) (cat. no. 9101), ERK-1/2 (cat. no. 9102), p-JNK-1/2 (T183/Y185) (cat. no. 9251), JNK-1/2 (cat. no. 9252), and p-p38 MAPK (T180/Y182) (cat. no. 9211) p38 MAPK (cat. no. 9212), p-MK2 (T334) (cat. no. 3007), and T-MK2 (cat. no. 3042), were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA).

Techniques: Phospho-proteomics, Cell Counting, Control, Western Blot