Ablation of Lrp4 in Schwann Cells Promotes Peripheral Nerve Regeneration in Mice

Abstract

Low-density lipoprotein receptor-related protein 4 (Lrp4) is a critical protein involved in the Agrin-Lrp4-MuSK signaling pathway that drives the clustering of acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). Many studies have shown that Lrp4 also functions in kidney development, bone formation, nervous system development, etc. However, whether Lrp4 participates in nerve regeneration in mammals remains unknown. Herein, we show that Lrp4 is expressed in SCs and that conditional knockout (cKO) of Lrp4 in SCs promotes peripheral nerve regeneration. In Lrp4 cKO mice, the demyelination of SCs was accelerated, and the proliferation of SCs was increased in the injured nerve. Furthermore, we identified that two myelination-related genes, Krox-20 and Mpz, were downregulated more dramatically in the cKO group than in the control group. Our results elucidate a novel role of Lrp4 in peripheral nerve regeneration and thereby provide a potential therapeutic target for peripheral nerve recovery.

Keywords: Lrp4; Schwann cells; nerve injury; proliferation; regeneration.

Figure 1

Lrp4 in SCs was successfully deleted in Lrp4 cKO mice. (A) Diagram of the strategy used to generate the Lrp4 cKO mice. (B) Reduced Lrp4 mRNA levels in cKO mice compared with control mice. **** p < 0.0001, control and cKO, 8 mice/group. (C,D) Reduced Lrp4 protein levels in cKO mice compared with control mice. ** p = 0.0083, 3 mice/group. (E) The images show that Lrp4 (red) is localized in SCs (green). The white arrows indicate the costaining of β-Gal and S-100β. Scale bar = 50 μm.

Figure 2

Ablation of Lrp4 in SCs promotes nerve regeneration. (A) Representative images of the sciatic nerve after crush surgery. (B) Biotin staining of sciatic nerve sections from cKO mice revealed more regenerated axons at 7 dpc. Scale bar = 200 μm. (C) The regenerated axons were longer in the cKO mice than in the control mice. **** p < 0.0001, ** p < 0.01, * p < 0.05, control, n = 6 slices; cKO, n = 5 slices, 3 mice/group. (D) Representative images of NMJ regeneration at 10 and 14 dpc. The arrowheads indicate fully reinnervated NMJs. The arrows indicate denervated NMJs. Scale bar = 50 μm. (E) Quantification of the fully reinnervated endplates at 0, 7, 10, 14, and 21 dpc. Control mice (26.9 ± 3.6% and 71.0 ± 2.4% at 10 and 14 dpc, respectively) display fewer reinnervated NMJs at 10 and 14 dpc than the cKO mice (43.4 ± 5.0% and 89.4 ± 1.5% at 10 and 14 dpc, respectively). *** p < 0.001, **** p < 0.0001, control, n = 33, 26, 23, 50, and 18 slices at 0, 7, 10, 14, and 21 days, respectively. cKO, n = 27, 22, 18, 50, and 16 slices at 0, 7, 10, 14, and 21 days, respectively. Three or four mice per group. (F) The fully denervated NMJs were reduced in cKO mice (26.7 ± 3.5% and 5.0 ± 0.9% at 10 and 14 dpc, respectively) 10 and 14 dpc compared with the control mice (42.6 ± 4.9% and 13.5 ± 1.5% for 10 and 14 dpc, respectively). **** p < 0.0001, *** p < 0.001, 3 or 4 mice per group. All the data are presented as mean ± SEM.

Figure 3

mSCs are involved in accelerating regeneration in Lrp4 cKO mice. (A) Schematic representation of the nerve exchange experiment. (B) The TA muscles in the cKO mice exhibited amyotrophy at 56 days after the transplant surgery. (C) Decreased TA muscle fiber numbers in control mice (1437.0 ± 86.9) compared with cKO mice (916.4 ± 55.9). Control and cKO, n = 8 slices, 3 mice/group, *** p = 0.0002. (D) Representative images of NMJ regeneration at 56 days after nerve transplant. The arrowheads indicate fully reinnervated NMJs. The arrows indicate denervated NMJs. Scale bar = 100 μm. (E) Reduced fully reinnervated and increased denervated endplates in cKO after nerve transplantation. **** p < 0.0001, control, n = 75 slices; cKO, n = 93 slices, 6 mice/group. The fully innervated NMJ percentage was 20.3 ± 2.3% in the control mice, compared with 4.2 ± 0.7% in the cKO mice; the partially innervated NMJ percentage was 28.5 ± 2.1% in the control mice, compared with 13.7 ± 1.2% in the cKO mice; and the denervated NMJ percentage was 51.2 ± 3.6% in the control mice, compared with 82.0 ± 1.6% in the cKO mice. All the data are presented as the mean ± SEM.

Figure 4

tSCs are not involved in accelerating regeneration in Lrp4 cKO mice. (A) Representative images of tSCs in Dhh-tdTomato and Dhh-tdTomato-Lrp4^−/− mice at 7 dpc. Scale bar = 25 μm. (B) The number of tSCs per NMJ in the soleus muscle at 7 dpc. Control and cKO, n = 42 NMJs, 3 mice/group. (C) Distribution of the different tSC numbers in the NMJs of the soleus muscle at 7 dpc. Control and cKO, n = 8 slices. (D) The average area of the tSC in the soleus muscle at 7 dpc. Control and cKO, n = 10 cells. (E) Representative images of tSCs in Dhh-tdTomato and Dhh-tdTomato-Lrp4^−/− mice under normal conditions. Scale bar = 25 μm. (F) The number of tSCs per NMJ in the soleus muscle. Control and cKO, n = 42 NMJs, 3 mice/group. (G) Distribution of the different tSC numbers in the NMJs of the soleus muscle. Control, n = 8 slices; cKO, n = 9 slices. (H) The average area of the tSC per NMJ in the soleus muscle. Control and cKO, n = 10 cells. All the data are presented as the mean ± SEM.

Figure 5

The proliferation of SCs in Lrp4 cKO mice was enhanced after nerve damage. (A) Immunostaining of the sciatic nerve of newly formed SCs at 5 dpc. Scale bar = 200 μm. (B) Representative images of the sham operation control and proximal and distal crushed nerves. The white arrows indicate the costaining of Dhh-tdTomato and BrdU. Scale bar = 50 μm. (C) Enhanced proliferation of SCs in Dhh-tdTomato-Lrp4^−/− mice (1229.0 ± 75.2) sciatic nerves compared with Dhh-tdTomato mice (806.6 ± 27.6) after nerve crush. **** p < 0.0001, control and cKO, n = 10 slices, 3 mice/group. (D) Representative images of mSCs under normal conditions. Scale bar = 50 μm. (E) The mSC numbers under normal conditions did not differ between Dhh-tdTomato and Dhh-tdTomato-Lrp4^−/− mice. Control, n = 20 slices; cKO, n = 21 slices, 3 mice/group. All the data are presented as the mean ± SEM.

Figure 6

Lrp4 ablation in SCs does not influence SC survival or macrophage recruitment after injury. (A) The numbers of TUNEL-positive cells in the sham control and proximal and distal ends of crushed nerve segments did not differ between the control and cKO mice. The white arrows indicate the apoptotic cells. Scale bar = 25 μm (B) The proportion of TUNEL-positive cells was not significantly different between the control and cKO mice. Control and cKO, n = 10 slices, 3 mice/group. (C) Representative pictures of macrophages from control and cKO mice. The macrophage numbers in the sham control and proximal and distal ends of the crushed nerve segments did not differ between the control and cKO mice. The white arrows indicate the macrophages. Scale bar = 25 μm (D) Quantification of the macrophage numbers revealed no significant difference between the control and cKO mice. Control and cKO, n = 10 slices, 3 mice/group.

Figure 7

Lrp4 deficiency promotes demyelination and downregulates Krox-20 after nerve injury. (A) The β-catenin and MAPK pathway-related mRNA levels did not differ between the control and cKO mice (3 mice/group). (B) The mRNA levels of c-Jun, Notch, and Id2 were comparable between the control and cKO mice before injury and at 7 dpc. **** p < 0.0001 (3 mice/group). (C) The mRNA levels of Mpz and Krox-20 were decreased in the cKO mouse sciatic nerves compared with the control mice at 7 dpc. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (3 mice/group). (D) Representative electron micrograph of control and cKO sciatic nerves. The cKO mice showed lower amounts of myelin debris and more regenerated axons. BB indicates the Büngner band, Sc indicates Schwann cells. Scale bar = 5 μm. (E) The size of the myelin debris area was decreased in the cKO mice (13.4 ± 1.6) compared with the control mice (25.5 ± 1.1). **** p < 0.0001, control, n = 10 slices; cKO, n = 9 slices (3 mice/group). (F) The number of newly formed axons was increased in cKO mice (61.5 ± 7.3) compared with control mice. (30.4 ± 3.9). ** p = 0.0021, control, n = 15 slices; cKO, n = 22 slices (3 mice/group). All the data are presented as the mean ± SEM.

Figure 8

Lrp4 deficiency in SCs does not influence NMJ formation or transmission. (A) Representative images of the diaphragms of control and cKO mice at E14.5 and P0. The left ventral regions of the diaphragms of control and mutant mice at P0 and E14.5 were stained in whole-mount NMJ preparations. The arrow indicates the primary branch. The arrowhead indicates the secondary branch. Scale bar = 50 μm. (B) Comparable endplate bandwidths between the control and cKO mice. Control, n = 29 or 31 regions; cKO, n = 9 or 16 regions, 3 to 5 mice/group. (C) Comparable AChR cluster areas between the control and cKO mice. Control, n = 26 or 39 regions; cKO, n = 19 regions, 3 to 5 mice/group. (D) Comparable AChR cluster numbers between the control and cKO mice. Control, n = 12 or 15 regions; cKO, n = 11 regions, 3 to 5 mice/group. (E) Comparable secondary branch lengths between the control and cKO mice. Control, n = 29 or 22 branches; cKO, n = 21 branches, 3 to 5 mice/group. (F) Comparable secondary branch numbers between the control and cKO mice. 3 or 4 mice/group. (G) Comparable AChR cluster length distribution between the control and cKO mice of the same age. Control, n = 5 slices; cKO, n = 4 slices, 3 to 5 mice/group. (H) Enlarged images of a single NMJ from the limb muscle. Scale bar = 10 μm. (I) Comparable nerve coverage ratios between the control and cKO mice. Control and cKO, n = 10 NMJs, 3 or 4 mice/group. (J) Representative mEPP traces of adult control and cKO mice. (K,L) The mEPP frequencies and amplitudes were similar between the control and cKO mice. Control, n = 36 regions of different NMJs; cKOs, n = 30 regions of different NMJs, 3 or 4 mice/group. (M) Representative EPP traces in control and cKO mice. (N) The EPP amplitudes were similar in control and cKO mice. Control, n = 16 regions of different NMJs; cKOs, n = 25 regions of different NMJs, 3 or 4 mice/group. All the data are presented as the mean ± SEM.