Schwann cell-specific ablation of laminin gamma1 causes apoptosis and prevents proliferation

Abstract

To investigate the function of laminin in peripheral nerve development, we specifically disrupted the laminin gamma1 gene in Schwann cells. Disruption of laminin gamma1 gene expression resulted in depletion of all other laminin chains known to be expressed in Schwann cells. Schwann cells lacking laminin do not extend processes required for initiating axonal sorting and mediating axon-Schwann cell interaction. They fail to downregulate Oct-6 and arrest at the premyelinating stage. The impaired axon-Schwann cell interaction prevents phosphorylation of beta-neuregulin-1 receptors and results in decreased cell proliferation. Postnatally, laminin-null Schwann cells exhibit reduced phosphatidylinositol 3 (PI3)-kinase activity and activation of caspase cascades, leading to apoptosis. Injection of a laminin peptide into mutant sciatic nerves partially restores PI3-kinase activity and reduces apoptotic signals. These results demonstrate the following: (1) that laminin initiates axonal sorting and mediates axon-Schwann cell interactions required for Schwann cell proliferation and differentiation, and (2) that laminin provides a PI3-kinase/Akt-mediated Schwann cell survival signal.

Figure 1.

Schwann cells lacking laminin γ1 expression are severe hypomyelination and fail to extend processes to initiate axonal sorting. A, PCR analysis of genomic DNA from various tissues of wild-type, homozygous fLAM γ1 mice (f/f; control) and P₀/Cre:fLAM γ1 mice (f/f, P₀-Cre; mutant). The primers used amplified the wild-type (1.3 kb), unrecombined (3.2 kb), and recombined (2.3 kb) fLAM γ1 alleles. B, Transverse sections of control and mutant sciatic nerves at P0 were double stained for laminin γ1 (red) and MBP (green). In the mutant nerve, laminin γ1 expression was absent in the endoneurium (asterisks) and only remained in the perineurium (arrowheads), and MBP expression was not detected. C, Transverse semithin sections from P28 sciatic nerves show that mutant nerves have large unsorted axonal bundles and few Schwann cells with myelin sheaths near the perineurium (arrows). D, Electron micrographs of P1 sciatic nerves show that mutant Schwann cells (arrows) do not extend cytoplasmic processes and leave axons unsorted. E, Electron micrograph of P28 sciatic nerves show that mutant nerves have large bundles of unsorted axons with some Schwann cells located outside (SC). F, Higher magnification of the boxed region in E shows that the mutant Schwann cell closely associated with unsorted axonal bundles lacks a continuous basal lamina (compare fuzzy materials indicated by arrows and denuded areas indicated by arrowheads) and does not extend processes between axons. Scale bar: B, 76 μm; C, 18.5 μm; D, 5.6 μm; E, 1.7 μm; F, 0.3 μm. wt, Wild type; ct, control; mt, mutant; Lnγ1, laminin γ1.

Figure 2.

Disruption of laminin γ1 gene expression resulted in depletion of all other laminin chains known to be expressed in Schwann cells. Adjacent transverse sections of control and mutant sciatic nerves at P0 were stained for different laminin subunits, including α1, α2, α4, β1, and γ1. In the mutant nerves, disruption of laminin γ1 gene expression resulted in concurrent depletion of other laminin chains. Note that the laminin α1 chain, which is expressed in the perineurium in mature nerves, and the laminin α4 chain, which is expressed at low level in adult nerves, are both nearly undetectable at the P0 stage. Scale bar, 25 μm.

Figure 3.

Mutant Schwann cells fail to upregulate Krox-20 and downregulate Oct-6. A, Whole embryo sections at E17.5 and longitudinal sciatic nerve sections at P3 were stained for Oct-6 (green) and laminin γ1 (Lnγ1; red), and images of Oct-6/laminin γ1 at P3 were merged. E17.5 embryo sections were stained for neurofilament (NF; blue) to identify nerves. These images indicate that initiation of Oct-6 does not require laminin γ1. B, Transverse control and mutant sciatic nerve sections at P15 and P28 were stained for Oct-6 (green) and neurofilament (red), and the images from mutant nerves were merged. During postnatal development, Oct-6 fails to be downregulated in mutant Schwann cells. C, Whole embryo sections at E19.5 and transverse sciatic nerve sections at P5 were stained for Krox-20 (green) and laminin γ1 (red), and the images of Krox-20/laminin γ1 were merged. E19.5 embryo sections were stained for neurofilament (blue) to identify nerves. Although initiation of Krox-20 does not require laminin γ1, high level expression of Krox-20 in Schwann cells is impaired during the postnatal development. Scale bar, 50 μm. D, Oct-6 expression in control and mutant sciatic nerves at P3, P15, and P28 (n = 15 per genotype per age) was assessed by Western blots with antibodies against Oct-6. β-Actin was the loading control. Mutant Schwann cells were unable to downregulate Oct-6 and showed aberrant consistent expression of Oct-6. c, Control; m, mutant.

Figure 4.

Impaired interactions of laminin γ1-null Schwann cells and axons result in severe reduction of Schwann cell proliferation. A, Plot of the total cell number in distal transverse sections (mean ± SEM) at various ages. Total cell number in distal transverse sections is significantly reduced in mutant sciatic nerves (open bars) at all ages compared with control nerves (filled bars) (n = 6 per genotype per age; ^**p < 0.001). B, Longitudinal sections of control and mutant sciatic nerves at E19. 5 were triple stained for BrdU (green), neurofilament (red), and DAPI (blue) after a 1 h pulse of BrdU, and the images of BrdU/neurofilament and BrdU/DAPI were merged. Mutant Schwann cells show reduced nuclei BrdU incorporation compared with control Schwann cells. Scale bar, 60 μm. C, Plot of the percentage (mean ± SEM) of BrdU-positive nuclei at various ages. The percentages of BrdU-incorporated nuclei are significantly reduced in mutant nerves (open bars) at E15.5, E17.5, E19.5/P0, and P5 compared with control nerves (filled bars) (n = 6 per genotype per age; ^*p < 0.01; ^**p < 0.001). D, The response of Schwann cells to NRG-1 at P0 and P2 was assessed on immunoblots with antibodies recognizing p-ErbB2 and p-ErbB3. β-Actin is the loading control. Mutant Schwann cells show severe reduction in response to axonal mitogens. c, Control; m, mutant.

Figure 5.

Schwann cell proliferation is not impaired in CaMKII/Cre:fLAMγ1 mutant mice. Longitudinal sections of control (fLAMγ1), CaMKII/Cre:fLAMγ1, and P₀/Cre:fLAMγ1 mutant sciatic nerves at E19.5 were triple stained for BrdU (red), laminin γ1 (green), and DAPI (blue) after a 1 h pulse of BrdU, and the images of BrdU/laminin γ1 and BrdU/DAPI were merged. Laminin γ1 expression was disrupted in some small patches in the sciatic nerves of CaMKII/Cre:fLAMγ1 mutant mice (stars), and Schwann cell proliferation was not impaired [quantitative analysis was shown previously (Chen and Strickland, 2003)]. However, laminin γ1 expression was almost completely disrupted in the Schwann cells in P₀/Cre:fLAMγ1 mutant sciatic nerves (stars), and cell proliferation was dramatically reduced (quantitative analysis shown in Fig. 4). Laminin γ1 expression in the epineurium of P₀/Cre: fLAMγ1 mutant sciatic nerves was normal (arrows).

Figure 6.

Schwann cells that lack laminin γ1 show an increased percentage of cell death. A, Representative transverse sciatic nerve sections of control and mutant mice at P15 were stained with TUNEL (red) and counterstained with DAPI (blue), and the images were merged. Apoptotic cells can be detected in mutant but not control nerves. B, Plot of the percentage of TUNEL-positive nuclei (mean ± SEM) at various ages. The percentages of TUNEL-positive nuclei are higher in mutant (open bars) than in control nerves (filled bars) at P0, P5, P15, and P28 (n = 6 per genotype per age; ^*p < 0.01; ^**p < 0.001). C, P15 mutant sciatic nerve sections were stained with DAPI (blue), S100 (red), and TUNEL (green), and images were merged. The staining indicates that the cell with TUNEL-positive nuclei is a Schwann cell. D, P15 mutant sciatic nerve sections were stained with DAPI (blue), laminin γ1 (Lnγ1; red), and TUNEL (green), and images were merged. The staining shows the TUNEL-positive cell does not express laminin γ1 (arrows), whereas the rest of TUNEL-negative cells still expressed laminin γ1 (arrowheads). Scale bars: A, 20 μm; C, D, 8 μm.

Figure 7.

Laminin γ1-null Schwann cells show impaired PI3-kinase activity and elevated apoptosis/caspase signaling. A, PI3-kinase activity of control and mutant sciatic nerves at P0, P5, P15, P28, and adult (n = 15 per genotype per age) were assessed on immunoblots with antibodies against p-Akt, the downstream effector of PI3-kinase. Mutant sciatic nerves compared with control nerves show reduced PI3-kinase activity at P0, P5, P15, and P28. B, Akt kinase activity of control and mutant sciatic nerves at P5 and P15 were assessed on immunoblots with antibodies recognizing p-GSK-3β. Mutant Schwann cells have reduced Akt kinase activities at P5 and P15 as judged by decreased phosphorylation of GSK-3β. C, Endogenous level of activated (cleaved) caspase-9 in control and mutant sciatic nerves at P5 and P15 was assessed by immunoblots with antibodies recognizing both full length (49kDa) and the large fragment of mouse caspase-9 after cleavage at Asp353 (37 kDa) and Asp368 (39 kDa). The mutant sciatic nerves show activation of caspase-9 at both time points. D, Longitudinal sciatic nerve sections of control and mutant mice at P15 were stained with activated capase-3 and caspase-7 antibodies (red) and counterstained with DAPI (blue), and the images were merged. Mutant sciatic nerves show increased activated caspase-3 and caspase-7. c, Control; m, mutant. Scale bars, 20 μm.

Figure 8.

Restoration of PI3-kinase activity in mutant sciatic nerves suppresses caspase-mediated death signaling. A, The levels of p-Akt in mutant sciatic nerves from Figure 7A were quantified (normalized with total Akt level) and compared with control nerves to obtain the percentage of decreased p-Akt level. The percentages of increased TUNEL-positive nuclei in mutant sciatic nerves were obtained by subtracting the average percentages of TUNEL-positive nuclei in control from mutant nerves in Figure 6 B. A plot of the percentages of increased TUNEL-positive nuclei (black bars) and decreased p-Akt level (black circles) in mutant sciatic nerve against the ages of mice shows that reduced PI3-kinase activities correlated temporally with increased Schwann cell death. B, Control sciatic nerve or laminin peptide (lp)- and control peptide (cp)-injected mutant sciatic nerve extracts were immunoblotted with antibodies recognizing Akt or phosphorylated Akt. Caspase signaling was evaluated on the same blot using antibodies against caspase-9. β-Actin served as a loading control. Compared with control nerves, the PI3-kinase activities were partially restored in mutant sciatic nerves injected with the laminin peptide, resulting in the reduction of activated caspase-9 level. C, Plot of the signal intensity (mean ± SEM) of p-Akt and cleaved caspase-9 from quantitative analysis of Western blots from B. The signal intensity of p-Akt and cleaved caspase-9 in laminin peptide-injected (Ln) mutant sciatic nerves increased 75% and decreased 50%, respectively, compared with contralateral control (con) nerves (n = 20 mice per genotype per lysate, 3 independent experiments; ^*p < 0.05; ^**p < 0.01).

Figure 9.

Proposed mechanism for the function of laminins in PNS development. Laminin deposition guides Schwann cells to extend processes that initiate axonal sorting and mediate axon-Schwann cell interaction. Axon-Schwann cell interaction allows Schwann cells to be exposed to axon-derived signals for proliferation and additional differentiation. β1 integrins expressed on Schwann cells interact with laminins to stabilize the cytoplasmic processes to facilitate completion of the radial sorting of axons. After Schwann cell differentiation, laminins in Schwann cell basal lamina provide PI3-kinase/Akt-mediated survival signal to maintain Schwann cell viability.