(A) MBP immunolabeling of sciatic nerve sections showing delayed loss of myelin in –null nerves compared with controls, 3 d after transection of nerves of 5-d-old mice

Bar, 10 μm. (B) Quantification of the delay in myelin disappearance by quantitative image analysis of MBP-immunolabeled sections (comparable to those shown in A) 2, 3, and 5 d after injury (expressed as percentage of MPB area in uncut P5 nerve). In every case, the difference between c-Jun–null and control nerves is significant (P < 0.01). (C) Electron micrographs showing –null and control nerves from 5-d-old mice, intact and 3 d after injury as indicated. Note preservation of rounded or partially collapsed myelin sheaths in –null nerves. Bar, 4 μm. (D) Counts of myelin sheaths (rounded or collapsed) in c–null and control nerves 3 and 5 d after injury (3 d, P < 0.05; 5 d, P < 0.01). Error bars show standard deviation of the mean.<p><b>Copyright information:</b></p><p>Taken from "c-Jun is a negative regulator of myelination"</p><p></p><p>The Journal of Cell Biology 2008;181(4):625-637.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386103.</p><p></p

(A and B) Cells cotransfected with empty GFP vector (to visualize transfected cells) and an empty control vector (EV; A) or a vector (B)

Both cultures were then treated with 1 mM db-cAMP for 2 d to induce Krox-20 and were immunolabeled for Krox-20. In A, arrows point to induced Krox-20 in nuclei of GFP-positive control cells (yellow nuclei of Krox-20–positive GFP-positive cells). In B, no Krox-20 is induced (arrows) in cells containing . Arrowheads in both panels indicate untransfected cells that have been induced to express Krox-20 by db-cAMP as controls for induction. (C) Activation of JNK inhibits induction of Krox-20. Western blot of cells infected with adenovirus expressing control LacZ or virus expressing activated MKK7 to activate JNK is shown. Note that the Krox-20 and periaxin induced by 2 d of exposure to 1 mM db-cAMP in LacZ control cells is inhibited by MKK7 expression. Note also that MKK7 elevates c-Jun in the presence of db-cAMP. (D–G) In –null cells, loss of Krox-20 expression is significantly delayed. Double immunolabeling of control cells (D and E) and –null cells (F and G) for Krox-20 (red) and periaxin (green) after 2 d in culture in DM containing 20 ng/ml NRG-1 is shown. Note that Krox-20 has disappeared from the control cells, whereas many –null cells still have Krox-20–positive nuclei (G, arrows). Note that –null Krox-20–positive cells are also periaxin positive (F, arrows), whereas control cells have lost periaxin expression (D). Bars, 15 μm.<p><b>Copyright information:</b></p><p>Taken from "c-Jun is a negative regulator of myelination"</p><p></p><p>The Journal of Cell Biology 2008;181(4):625-637.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386103.</p><p></p

(A) Western blot showing that c-Jun is absent from cells infected with CRE-expressing adenovirus

The blot also compares periaxin in control (Con) and –null cells (CRE) infected with GFP control adenovirus (GFP) or a Krox-20/GFP virus (K20). Note high periaxin levels in Krox-20–infected –null cells (CRE). (B–E) control ( con) and –null mouse Schwann cells 2 d after infection with Krox-20/GFP adenovirus. Note that Krox-20 induces much higher levels of P protein in –null cells (D and E) than in control cells (B and C). The reason why P levels in the Krox-20–expressing control cells appear low in this picture (C) compared with other comparable experiments (e.g., ) is that exposure had to be reduced (equally for C and E) to avoid overexposure in E. (F and G) P protein expression in control cells P ( con) and c–null mouse Schwann cells after 3 d of exposure to db-cAMP/NRG-1. Note that cAMP/NRG-1 induces substantially higher P levels in cells without c-Jun. Bars, 15 μm.<p><b>Copyright information:</b></p><p>Taken from "c-Jun is a negative regulator of myelination"</p><p></p><p>The Journal of Cell Biology 2008;181(4):625-637.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386103.</p><p></p

(A and B) Cotransfection of Krox-20/GFP with Jun(Asp) or with Jun(Ala) inhibits Krox-20–mediated induction of periaxin and P

K20/EV represents cells cotransfected with Krox-20 and control vector. (C–F) P in situ experiment showing that cotransfection of Krox-20/GFP with Jun(Ala) inhibits Krox-20–mediated induction of mRNA. C and D are controls, and the arrows show a cell coexpressing Krox-20 and a control vector where Krox-20 has induced P mRNA. Arrows in E and F show a cell coexpressing Krox-20 and Jun(Ala) where Jun(Ala) has inhibited Krox-induced expression. Bar, 15 μm. (G) Percentages of GFP-positive cells that also express mRNA in cells cotransfected with the constructs indicated. Error bars show one standard deviation of the mean.<p><b>Copyright information:</b></p><p>Taken from "c-Jun is a negative regulator of myelination"</p><p></p><p>The Journal of Cell Biology 2008;181(4):625-637.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386103.</p><p></p

(A) Western blot showing that MKK7, presumably by activating JNK, inhibits Krox-20–induced myelin protein expression

The cells were coinfected with adenoviruses expressing the constructs indicated. (B) Western blot of cells infected with adenoviruses expressing control LacZ or activated MKK7 to activate JNK. Note that periaxin induced by 2 d of exposure to 1 mM of db-cAMP in LacZ control cells is inhibited by MKK7 expression. (C–F) MKK7 activates c-Jun even in the presence of Krox-20. C and E show that Krox-20 coinfected with a control LacZ-expressing adenovirus suppresses c-Jun levels. D and F show that when Krox-20 is coexpressed with MKK7, high c-Jun levels are maintained. (G) MKK7-mediated suppression of myelin gene expression depends on c-Jun. In normal cells ( con), Krox-20–induced periaxin expression (K20/LacZ) is suppressed by MKK7 (K20/MKK7). This suppression does not occur when this experiment is repeated in cells without c-Jun ( null). Error bars show one standard deviation of the mean. (H–K) Reactivation of JNK/c-Jun in Krox-20–expressing cells that already synthesize P abolishes P protein expression. Retrovirally infected cells already expressing Krox-20 and P were infected with either LacZ control (H and I) or MKK7-expressing (J and K) adenoviruses. Cells were labeled with either LacZ and P (H and I) or MKK7 and P (J and K) antibodies. Note down-regulation of P protein in Krox-20–expressing cells infected with MKK7 adenovirus. Bars, 15 μm.<p><b>Copyright information:</b></p><p>Taken from "c-Jun is a negative regulator of myelination"</p><p></p><p>The Journal of Cell Biology 2008;181(4):625-637.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386103.</p><p></p

Laminin 211 inhibits protein kinase A in Schwann cells to modulate neuregulin 1 type III-driven myelination

<div><p>Myelin is required for proper nervous system function. Schwann cells in developing nerves depend on extrinsic signals from the axon and from the extracellular matrix to first sort and ensheathe a single axon and then myelinate it. Neuregulin 1 type III (Nrg1III) and laminin α2β1γ1 (Lm211) are the key axonal and matrix signals, respectively, but how their signaling is integrated and if each molecule controls both axonal sorting and myelination is unclear. Here, we use a series of epistasis experiments to show that Lm211 modulates neuregulin signaling to ensure the correct timing and amount of myelination. Lm211 can inhibit Nrg1III by limiting protein kinase A (PKA) activation, which is required to initiate myelination. We provide evidence that excessive PKA activation amplifies promyelinating signals downstream of neuregulin, including direct activation of the neuregulin receptor ErbB2 and its effector Grb2-Associated Binder-1 (Gab1), thereby elevating the expression of the key transcription factors Oct6 and early growth response protein 2 (Egr2). The inhibitory effect of Lm211 is seen only in fibers of small caliber. These data may explain why hereditary neuropathies associated with decreased laminin function are characterized by focally thick and redundant myelin.</p></div

Model depicting how laminin α2β1γ1 (Lm211) and neuregulin 1 type III (Nrg1III) signaling are integrated during SC development.

<p>In immature SCs, Lm211, via one or more of its basal lamina receptors (Int = integrins, Dystroglycan = Dyst, Gpr126), inhibits protein kinase A (PKA) and prevents Nrg1III from triggering myelination during radial sorting. In promyelinating cells, after radial sorting is finished and the 1:1 relationship with axons larger than 1 μm has been achieved, PKA is activated by Gpr126 independently of Lm211 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001408#pbio.2001408.ref045" target="_blank">45</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001408#pbio.2001408.ref057" target="_blank">57</a>] and contributes to Nrg1III signaling and to the expression of Oct6. In large myelinating fibers (above), Lm211 inhibition is overcome, PKA is fully active, and cooperates with Nrg1III to activate ErbB2, Grb2-Associated Binder-1 (Gab1), and Egr2. In small myelinated fibers (bottom), Lm211 inhibition of PKA persists and prevents excessive Nrg1III-driven myelination.</p

Loss of laminin α2β1γ1 (Lm211) does not affect myelin thickness, but rescues hypomyelination due to neuregulin 1 type III (Nrg1III) haploinsufficiency.

<p>(A) Electron micrographs of sciatic nerves from mice of the indicated genotypes at postnatal day 16 (P16). (B) Quantification of g-ratios. At least 150 axons per animal were quantified for 3 animals/genotype, *<i>p</i> < 0.05 by 1-way ANOVA multiple comparison test. (C) Scatter plot displays and distribution (D) of g-ratios of individual fibers as a function of axon diameter shows that in the double mutant, the rescue in myelin thickness mainly occurs on axons with diameters smaller than 2μm. (E) Distribution of diameter of myelinated axons. Bar = 2 μm. The numerical data used in B-E are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001408#pbio.2001408.s001" target="_blank">S1 Data</a>.</p

Increased protein kinase A (PKA), ErbB2, and Grb2-Associated Binder-1 (Gab1) activation in <i>Nrg1III</i>^<i>tg</i><i>//Lama2</i>^{<i>−/−</i>} sciatic nerves.

<p>(A) Schematic representation of the hypothesis that laminin α2β1γ1 (Lm211) inhibits neuregulin 1 type III (Nrg1III) promyelin signaling by negatively regulating PKA. Western blot of PKA phospho-substrates in P16 sciatic nerves. The image is representative of 3 experiments. (B) Measurement of PKA activity in sciatic nerves at P16 from the indicated genotypes. PKA is more active in <i>Lama</i>2^−/− and <i>Nrg1III</i>^<i>tg</i>//<i>Lama</i>2^−/− nerves (<i>n</i> = 3 or more, **p < 0.01, ***p < 0.001 by 1-way ANOVA with Bonferroni multiple comparison test). (C) Treatment with a PKA-selective agonist (6-Bnz-cAMP), but not exchange protein directly activated by cAMP (EPAC) agonist (8-pCPT-2-O-Me-cAMP) for 3 days increases the levels of pErbB2 and pGab1 without Nrg1 treatment in primary SCs. The image is representative of 3 experiments. (D) Representative western blots showing the sensitization of the ErbB2-Gab1 pathway in response to Nrg1 following dbcAMP. Primary Schwann cells (SCs) in the presence (right 7 lanes) or absence (left 7 lanes) of dbcAMP for 3 days were exposed to Nrg1 (50 ng/ml) for the indicated time (h, hour). Where indicated, PKI166 (1 μM) or PP2 (1 μM) was used to pretreat cells before Nrg1 stimulation. Phosphorylation of ErbB2 and Gab1 was significantly enhanced following dbcAMP treatment and suppressed after PKI166 treatment. (E-F) Western blot analysis of ErbB2 (E) or Gab1 (F) phosphorylation in sciatic nerves of the indicated genotypes at P16. ErbB2 and Gab1 phosphorylation are increased only in <i>Nrg1III</i>^<i>tg</i>//<i>Lama</i>2^−/− sciatic nerve. The experiments were repeated at least 3 times on 6 animals per genotype (E) or 3 times on 2 to 6 different animals per genotype (F). *<i>p</i> < 0.05, ***<i>p</i> < 0.001 by 1-way ANOVA with Bonferroni multiple comparison test. The numerical data used in B, E-F are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001408#pbio.2001408.s001" target="_blank">S1 Data</a>.</p

Neuregulin 1 type III (Nrg1III) overexpression worsens the radial sorting defects of Lama2^−/− mice.

<p>(A) Transverse semithin sections of sciatic nerves from mice of the indicated genotypes at postnatal day 16 (P16) are shown. <i>Nrg1III</i>^<i>tg</i><i>//Lama2</i>^{<i>−/−</i>} mice have more bundles of naked axons (unsorted bundles) than <i>Lama2</i>^−/− mice (arrowheads). (B) Electron micrograph analysis shows that in <i>Lama2</i>^{<i>−/−</i>} and <i>Nrg1III</i>^<i>+/−</i><i>//Lama2</i>^{<i>−/−</i>} nerves these unsorted bundles contain amyelinated, naked axons with diameter >1 μm (asterisk), while in wild-type (WT) and <i>Nrg1III</i>^<i>tg</i> nerves, there are only Remak bundles that contain axons ensheathed and smaller than 1 μm (arrow). (C) Number of unsorted bundles per nerve cross semithin section, showing a 3-fold increase in the number of unsorted bundles in <i>Nrg1III</i>^<i>tg</i>//<i>Lama</i>2^−/− (106.33 ± 5.7 versus 35.33 ± 0.5 <i>Lama</i>2^−/− ***<i>p</i> = 0.0005 by Student <i>t</i> test; <i>n</i> = 3). (C’): The number of Remak bundles on ultrathin electron microscopy (EM) sections is decreased in <i>Nrg1III</i>^<i>tg</i>//<i>Lama</i>2^−/− and <i>Lama</i>2^−/− mutants (***<i>p</i> = 0.001 by 1-way ANOVA with Bonferroni multiple comparison test, <i>n</i> = 4). (C”): All mutant nerves have reduced numbers of axons in Remak bundles on ultrathin EM sections (**<i>p</i> = 0.005; ***<i>p</i> = 0.001 by 1-way ANOVA with Bonferroni multiple comparison test, <i>n</i> = 4). (D) Examples of precocious myelination of axons that have not been sorted into a 1:1 relationship (arrows). Bar = 10 μm in A, 2 μm in B and D. The numerical data used in C–C” are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001408#pbio.2001408.s001" target="_blank">S1 Data</a>.</p