Fig 1: Astrocyte-specific expression of dnEGFR blocked injury-induced ErbB activation in reactive astrocytes. (a) Representative images of TRE-YFP expression in astrocytes of Mlc1-tTA mice at 1-month old. AAV-TRE-YFP was stereotaxically injected into indicated brain regions. Fixed brains collected 1 day later were sectioned and immunostained for Acsbg1 or GFAP to label astrocytes in the cerebral cortex or hippocampal hilus, respectively. White arrows, double-positive cells. (b) Efficient inhibition on activity of each ErbB receptor by dominant-negative mutant dnEGFR. Complementary DNA sequence of dnEGFR was amplified from genomic DNA of TRE-dnEGFR mice by PCR and cloned into pcDNA3.1-His/myc vector. pcDNA3.1-dnEGFR-myc was transfected into HEK293 cells by polyethylenimine (PEI) together with one of ErbB1–4 plasmids or an empty vector. Cells were lysed 24 h later and processed into WB with indicated antibodies. Any ErbB receptor when overexpressed in HEK293 cells would autophosphorylate itself independent of ligand stimulation. Shown are representative WB results, demonstrating the inhibition of dnEGFR on phosphorylation of each ErbB receptor. (c) Quantitative analyses of experiments in (b). ***P<0.001; **P<0.01; n=3 for each ErbB receptor, paired t-test. (d) Schematic illustration of the Tet-off system in Mlc1-tTA;TRE-dnEGFR (Mlc1-dnEGFR) mice. (e, f) Active ErbB3 (pErbB3), but not total ErbB3 levels, was suppressed in the reactive astrocytes of Mlc1-dnEGFR cortex. Cortical slices from indicated mice 3 days post injury were immunostained with antibodies against pErbB3 (e) or total ErbB3 (f) together with GFAP. White arrows, double positive cells. Arrowheads, cells positive for GFAP alone.
Fig 2: ErbB overactivation in Plp-ErbB2V664E mice induced inflammatory demyelination. Unless otherwise indicated, quantitative data were presented in the graphs as mean ± SEM and analyzed by unpaired t test. A, Dox treatment setting for indicated mice and littermate controls. B, Real-time RT-PCR results of ErbB2 expression at indicated day post-Dox withdrawal (dpd). Statistical information for 1 dpd, t(4) = 6.72, p = 0.0025; for 4 dpd, t(4) = 3.432, p = 0.026. C, Walking speed and percentage of foot slips of Plp-ErbB2V664E mice and littermate controls at P35 with 14 dpd in the grid walking test. n = 4 mice for each group. For steps per second, t(6) = 3.773, p = 0.0093; for foot slip percentage, t(6) = 12.31, p < 0.0001. D, Western blotting of indicated proteins in white matter isolated from Plp-ErbB2V664E (PB) mice in comparison with that from littermate control mice (Ctrl). Activation status of each ErbB receptor or downstream signaling protein was examined by Western blotting with the specific antibody against its phosphorylated form. E, Quantitative data of Western blotting results. For EGFR, t(4) = 27.64, p < 0.0001; for ErbB3, t(4) = 19.98, p < 0.0001; for ErbB4, t(4) = 10.06, p = 0.00055; for Akt, t(4) = 8.096, p = 0.0013; for Erk, t(4) = 4.353, p = 0.012. F, LFB staining and MBP immunostaining results of coronal sections through the genu of the corpus callosum (CC) in Plp-ErbB2V664E and control mice. Black arrows indicate the lower staining intensity of myelin stained in the CC. Statistical information for quantitative data of LFB intensity: the middle part at 9 dpd, t(6) = 6.345, p = 0.00072; the lateral part at 9 dpd, t(6) = 3.914, p = 0.0078; the middle part at 14 dpd, t(6) = 9.89, p < 0.0001; the lateral part at 14 dpd, t(6) = 23.07, p < 0.0001. Statistical information for MBP intensity: CC, t(4) = 4.884, p = 0.0081; cortex (CX), t(4) = 2.834, p = 0.047. Statistical information for MBP distribution: CC, t(4) = 14.53, p = 0.00013; CX, t(4) = 20.51, p < 0.0001. G, Western blotting results of MBP and ErbB2 in multiple brain regions (CX; CE, cerebellum; SC, spinal cord; ME, medulla) isolated from Plp-ErbB2V664E (PB) and littermate control mice (Ctrl) at 14 dpd. GAPDH served as a loading control. Statistical information for CE, t(4) = 6.35, p = 0.0032; for CX, t(4) = 9.243, p = 0.00076; for SC, t(4) = 4.118, p = 0.0146; for ME, t(4) = 3.634, p = 0.022. H, Representative EM images showed that myelin sheath ruptured and broke down in Plp-ErbB2V664E mice at 14 dpd (red arrow). Note the associated nuclei (red asterisk) showed no chromatin condensation and nucleation. I, EM images of the CC, optic nerve (ON), and prefrontal CX (PFC) from Plp-ErbB2V664E and littermate controls at 14 dpd. Quantitative data were shown for g-ratio analysis of myelinated axons detected by EM. Averaged g-ratio for each mouse were plotted as insets, presented as mean ± SEM, and analyzed by unpaired t test. For CC, t(4) = 3.412, p = 0.027; for ON, t(4) = 3.083, p = 0.037; for PFC, t(4) = 7.11, p = 0.0021. The red arrow indicates the axon with myelin breakdown. J, The densities of myelinated axons, as well as the percentages of axons with myelin breakdown, examined by EM in different brain regions of Plp-ErbB2V664E (PB) and littermate control mice (Ctrl) at 14 dpd were quantified. Statistical information for myelinated-axon density, in CC, t(4) = 2.683, p = 0.046; in ON, t(4) = 1.818, p = 0.143. For the percentage of axons with myelin breakdown, in CC, t(4) = 29.32, p < 0.0001; in ON, t(4) = 6.108, p = 0.0036; in PFC, t(4) = 8.125, p = 0.0012. K, Axons were reduced in the subcortical white matter (white arrows) of Plp-ErbB2V664E mice at 14 dpd. Sagittal sections of Plp-ErbB2V664E and littermate control mice were immunostained by monoclonal antibody TuJ1. Subcortical staining intensities were measured and plotted as mean ± SEM and analyzed by unpaired t test. t(4) = 6.019, p = 0.0009. L, M, Astrocytes (GFAP+) and microglia (Iba1+) examined in the subcortical white matter of indicated mice by immunostaining. Cell densities in the CC were quantified, and data were presented as mean ± SEM and analyzed by unpaired t test. In L, t(10) = 4.753, p = 0.0008. In M, t(4) = 36.4, p < 0.0001. N, Real-time RT-PCR results of GFAP and IL-1ß transcripts in the CC and ON; *p < 0.05, **p < 0.01, ***p < 0.001, PB versus Ctrl; #p < 0.05, CC versus ON. See also Extended Data Figures 1-1, 1-2.
Fig 3: ErbB overactivation caused noninflammatory hypomyelination in Sox10-ErbB2V664E mice. Unless otherwise indicated, quantitative data were presented as mean ± SEM and analyzed by unpaired t test. A, Dox treatment setting for indicated mice and littermate controls. B, Real-time RT-PCR results of ErbB2 expression at indicated day with Dox treatment (dwd). Statistical information for 1 dwd, t(10) = 4.081, p = 0.0022; for 4 dwd, t(4) = 6.37, p = 0.0031. C, Walking speed and percentage of foot slips of Sox10-ErbB2V664E mice and littermate controls at 9 dwd in the grid walking test. n = 4 mice for each group. Statistical information for steps per second, t(6) = 3.504, p = 0.0128; for foot slip percentage, t(6) = 4.429, p = 0.0044. D, Western blotting of indicated proteins in white matter tissues isolated from Sox10-ErbB2V664E (SB) mice in comparison with that from littermate control mice (Ctrl). Activation status of each ErbB receptor or downstream signaling protein was examined by Western blotting with the specific antibody against its phosphorylated form. E, F, Quantitative data of Western blotting results. In E, statistical information for EGFR, t(4) = 0.1983, p = 0.852; for ErbB3, t(4) = 28.34, p < 0.0001; for ErbB4, t(4) = 9.181, p = 0.00078; for Akt, t(4) = 3.380, p = 0.028; for Erk, t(4) = 4.899, p = 0.008. In F, for MBP, t(4) = 48.82, p < 0.0001. G, LFB staining and MBP immunostaining results of coronal sections through the genu of the corpus callosum (CC) in Sox10-ErbB2V664E (SB) and control mice (Ctrl). Black arrows indicate the lower staining intensity of myelin stained in the CC. Statistical information for quantitative data of LFB intensity: the middle part, t(6) = 15.17, p < 0.0001; the lateral part, t(6) = 10.23, p < 0.0001. Statistical information for MBP intensity: CC, t(8) = 5.14, p = 0.0009; CX, t(4) = 4.091, p = 0.015. Statistical information for MBP distribution: CC, t(8) = 4.622, p = 0.0017; CX, t(4) = 0.997, p = 0.375. H, EM images of the CC, optic nerve (ON), and prefrontal cortex (PFC) from Sox10-ErbB2V664E and littermate controls at 9 dwd. Quantitative data were shown for g-ratio analysis of myelinated axons detected by EM. Averaged g-ratio for each mouse were plotted as insets, presented as mean ± SEM, and analyzed by unpaired t test. For CC, t(4) = 3.295, p = 0.0301; for ON, t(4) = 3.775, p = 0.0195; for PFC, t(4) = 1.196, p = 0.298. I, The densities of myelinated axons examined by EM in different brain regions of Sox10-ErbB2V664E (SB) and littermate control mice (Ctrl) at 9 dwd were quantified. Statistical information for CC, t(4) = 0.2773, p = 0.795; for ON, t(4) = 0.1455, p = 0.891. J, K, Astrocytes (GFAP+) and microglia (Iba1+) examined in the subcortical white matter of indicated mice by immunostaining. Cell densities in the CC were quantified, and data were presented as mean ± SEM and analyzed by unpaired t test. In J, t(4) = 0.0501, p = 0.962. In K, t(4) = 1.637, p = 0.178. L, Real-time RT-PCR results of IL-1β and TNFα transcripts in the CC. Data were presented as mean ± SEM and statistical analysis by unpaired t test revealed no differences. See also Extended Data Figure 4-1 as well as Extended Data Figures 1-1, 1-2.
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