Fig 1: Differentiation of OPC-derived GABAergic neurons. (A,B) Representative mCherry + cells that were recorded from in Tol2-control-transfected (A) and Tol2-Dlx2-transfected (B) OPCs at 14 dpt expressing Gad1-GFP (green) and mCherry (red). Scale bar, 30 µm. (C,D) Membrane voltage traces in response to current injection (− 50 to + 250 pA, 500 ms duration, 10 pA increments). Red indicates response to highest current injection. Recorded cells are categorized into four types, based on the electrical response to voltage clamp recordings (C), and listed in detail in table (D). (E) Co-localization of GAD67 (red) and VGAT (blue) at 14 dpt in Dlx2-transfected OPCs. Right panels show higher resolution images of the boxed region in left panel. Arrowheads indicate area where GAD67 and VGAT double positive puncta wrap around MAP2 + (green) dendrites. Scale bars, 20 µm (low resolution image) and 10 µm (higher resolution images). (F) Co-localization of inhibitory post-synaptic protein gephyrin (red) and pre-synaptic inhibitory protein VGAT (blue) at 21 dpt in Dlx2-transfected OPCs. Right panels show higher resolution images of the boxed region in left panel. Arrowheads indicate synapsetic puncta with colocalized VGAT and gephyrin. Scale bars, 20 µm (low resolution image) and 10 µm (higher resolution images). (G) Expression of inhibitory neuron subtype-specific genes at 14 dpt for control- or Dlx2-transfected OPCs analyzed by qPCR. Values are normalized to the average of control-transfected cells for each transcript. The p-values for each transcript are as follows: Sst 0.0200, Satb1 0.0247, Erbb4 0.0305. Asterisk denotes p-values < 0.05. n = 4, Student’s t-test, unpaired.
Fig 2: Dlx2 misexpression converts OPCs into immature GABAergic neurons. (A) Dlx2-transfected OPCs immunolabeled 2 days post transfection (dpt) for mCherry (red) and Olig2 (blue). Scale bars, 20 µm. (B) Control-transfected (top) and Dlx2-transfected (bottom) OPCs immunolabeled at 2 dpt for Tuj1 (green), mCherry (red) and NG2 (blue). Arrowheads in the top control-transfected panels indicate mCherry + cells that are NG2 + but Tuj1-negative. Arrows in the bottom Dlx2-transfected panels indicate mCherry + cells that express both Tuj1 and NG2. Scale bar, 50 µm. (C) Bar graph showing the proportion of transfected cells that became Tuj1 + neurons in control- and Dlx2-transfected OPCs. Means ± standard deviations, n = 3, ** p = 0.0014. Student’s t-test, unpaired. (D) Representative examples of neuronal density in control- and Dlx2-transfected OPCs at 14 dpt, immunolabeled for MAP2 (green) and GAD67 (red). Scale bar, 50 µm. (E) Density of GAD67 + MAP2 + cells in control- and Dlx2-transfected cultures. Means ± standard deviations, n = 4, ** p = 0.0073, Student’s t-test, unpaired.
Fig 3: Neurite outgrowth is accelerated in OGD/R-treated PC12 cells following the knockdown of AK139328 expression. Immunofluorescence staining was used to analyze (A) MAP-2 and (B) GAP-43 expression in PC12 cells transfected with shRNA-AK139328 or shRNA. Original magnification, ×200. (C) Western blotting was performed to analyze the protein expression levels of MAP-2 and GAP-43 in OGD/R-treated PC12 cells transfected with shRNA-AK139328 or shRNA. Data are expressed as the mean ± SD. ***P<0.001 vs. control; ###P<0.001 vs. OGD/R + shRNA. OGD/R, oxygen glucose deprivation/reoxygenation; shRNA, short hairpin RNA; MAP-2, microtubule associated protein-2; GAP-43, growth associated protein-43.
Fig 4: Effect of oestradiol (E2) on neuritogenesis of male and female hippocampal cultures.(a,b) Proportion of neurons in the three stages of differentiation in male and female cultures at 2 DIV treated 24 h with E2. (c) Representative examples of male and female neurons treated with E2 and immunostained for MAP 2 (green), TAU protein and DAPI (blue) at 2 DIV. (d) Number of primary neurites, length of the axon and complexity of the dendritic arbour (number of intersections of dendrites with the circles in the Sholl analysis). Data are the mean ± SEM of 3 hippocampal cultures. ***p < 0.001, **p < 0.01 and *p < 0.05 vs control male values.
Fig 5: Reduced tau microtubule binding and microtubule stability are associated with synapse loss in primary RGCs upon glucolipotoxicity in a GSK3β-dependent manner. (a) Representative images of triple immunostaining for RGC-characteristic marker Thy1 (red), neuronal markers TUJ1 (green) and Map2 (blue). Scale bar, 100 μm. Primary RGCs were then exposed to conditioned medium (HG + PA) for 24 h, in the absence or presence of TWS119. (b) Representative images of subcellular expression of pT231-Tau (green) and Thy1 (red) by double immunofluorescence. Scale bar, 20 μm. (c) Representative synaptophysin (green; scale bar, 20 μm) immunostaining in RGCs. Areas boxed in are shown at higher magnification in the lower panels. (d) Western blotting for synaptophysin from whole cell lysates (Total) or synaptosome fractions (Syn). Intensities were quantified and normalized against the level of GAPDH and expressed as percentages of protein abundance under stimulation relative to control. (e) mRNAs of synaptophysin in total lysates or synaptosomes were quantified by Q-PCR. (f) Microtubule sedimentation assay. Western blotting for Tau 5 and β-tubulin in the supernatant (SN) and the microtubule pellet (pellet). Relative intensities of each protein in its respective fraction were quantified and normalized against the sum of the intensity value of that protein (total, including both supernatant and pellet fractions). MT, microtubule. (g) Western blotting for Ac-tubulin in whole cell lysates. Intensities were normalized against the level of GAPDH. (h) Representative images of double immunofluorescence for Ac-tubulin (green) and Tyr-tubulin (red). Scale bar, 40 μm. Data are means ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 vs control; #P < 0.05 and ##P < 0.01 vs HG + PA
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