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

Laminin α2β1γ1 (Lm211) leads to myelination only in the presence of neuregulin 1 type III (Nrg1III).

<p>Wild-type (WT) rat Schwann cells (SCs) cocultured with dorsal root ganglia (DRG) neurons from WT (A-F) or <i>Nrg1III</i>^{<i>−/−</i>} (G-L) mouse embryos were maintained in media with or without Lm211 (50 μg/mL), fixed and stained for myelin basic protein (MBP) (green), neurofilament (red), and DAPI (blue). Lm211 induces myelination only when SCs contact <i>Nrg1III</i>^<i>wt</i> neurons. (M) Quantification of the number of SCs per field of view. (N) Number of phosphorylated-histone3 (P-H3) positive nuclei in the cultures. Images are representative of 3 independent experiments. <i>n</i> = 6 coverslips for each treatment. (O-R) Electron micrographs of the cultures show that Lm211 promotes ensheathment of WT axons by SC processes (pseudocolored in pink). <i>Nrg1III</i>^{<i>−/−</i>}axons are not ensheathed, but Lm211 improves the intermingling of SC and their processes (pseudocolored in pink) parallel to axons. Bar = 50 μm in L; 2,5 μm in O. The numerical data used in M–N 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

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

Partial loss of neuregulin 1 type III (Nrg1III) does not impair axonal sorting and does not worsen the axonal sorting defects of laminin α2β1γ1 (Lm211)-deficient mice.

<p>(A) Transverse semithin sections of sciatic nerves from the indicated genotypes at postnatal day 16 (P16). <i>Lama2</i>^{<i>−/−</i>} and <i>Nrg1III</i>^<i>+/−</i><i>//Lama2</i>^{<i>−/−</i>} mice present with bundles of naked axons, which indicate defective radial sorting (arrows). (B) Electron micrograph analysis shows unsorted bundles that contain numerous amyelinated axons with diameter >1 μm (asterisks) in <i>Lama2</i>^{<i>−/−</i>} and <i>Nrg1III</i>^<i>+/−</i><i>//Lama2</i>^{<i>−/−</i>} mice. Nrg1 III^+/− nerves do not contain unsorted axon bundles, but only defective Remak fibers (red asterisk). (C) Quantification of the number of axons contained in each bundle at P16. (D) Quantification of the number of amyelinated axons with diameter >1μm per bundle in sciatic nerves at P16. The number of axons >1 μm is increased in <i>Lama2</i>^{<i>−/−</i>} <i>mice</i> (<i>Lama2</i>^{<i>−/−</i>} 7.91% ± 2.5 versus wild-type (WT) 2.86% ± 1.9); but not in <i>Nrg1III</i>^+/− mice (<i>Nrg1III</i>^+/− 2.26 ± 1.0 versus WT 2.86% ± 1.9); in the <i>Nrg1III</i>^<i>+/−</i><i>//Lama2</i>^{<i>−/−</i>}, the percentage of unsorted axons is comparable to <i>Lama2</i>^{<i>−/−</i>} mice (<i>Lama2</i>^{<i>−/−</i>} 7.91% ± 2.5 versus <i>Nrg1III</i>^+/−//<i>Lama2</i>^{<i>−/−</i>} 7.93 ± 2.63). <i>n</i> = 3 mice per genotype; 1-way ANOVA with Bonferroni posthoc test for individual comparisons. (E) Western blot analysis shows that the levels of the α2 chain of Lm211 are not decreased in sciatic nerves of <i>Nrg1III</i>^<i>+/−</i> mice. Data are represented as mean value ± SD ***<i>p</i> ≤ 0.001, <i>n</i> = 3 mice per genotype; Student <i>t</i> test. Bar = 10μm in A, 2 μm in B. The numerical data used in C, D, and 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