PMC

Cleavage of Nrg1 by ADAM10 and ADAM17. Recombinant Nrg1 protein spanning residues 1–246 was incubated with purified ADAM10 (A) or ADAM17 (B) in separate reaction mixtures for 24 h and then separated by SDS-PAGE. Recombinant Nrg1 was cleavable by ADAM10 and produced a band near 30 kDa, whereas ADAM17 cleaved Nrg1 at multiple sites.

Inhibition of ADAM and BACE1 during in vitro myelination. In vitro myelination was established by co-culturing rat Schwann cells and DRG neurons for 36 days. GM6001 or BACE1 inhibitor IV was added to the co-cultures after Schwann cells and DRG neurons were mixed in cultures, and fresh inhibitors were added to the cultures every 48 h. Co-cultures on the coverslips were subjected to confocal staining by standard methods using anti-MBP antibody to mark myelin and anti-Tuj1 antibody to mark axons. Treatment of co-cultures with BACE1 inhibitor IV (but not GM6001) reduced or delayed in vitro myelination. Scale bar = 40 μm.

Mapping the cleavage site of Nrg1 by ADAM10. A, an in vitro reaction mixture containing ADAM10 and Nrg1 peptide spanning amino acids 228–237 was separated by reverse-phase HPLC. Two new peaks were present in the sample containing both ADAM10 and the peptide, indicating cleavage of this peptide by ADAM10. B and C, the reaction mixture was also analyzed by MALDI-TOF mass spectrometry. The molecular weights of peptides in the reaction mixture were determined before (B) and after (C) enzymatic cleavage. The molecular weight of the original peptide is 1472. The molecular weight of the cleaved product is 1006, matching the cleaved product with the corresponding sequences.

Knockdown of ADAM10 expression in an in vitro myelination model. A, co-cultures of Schwann cells and DRG neurons were infected by lentiviral vector-mediated siRNA. Lentivirus infected both dividing and non-dividing cells. Protein lysates were prepared for Western blot analysis to measure the expression of ADAM10 48 h or 5 weeks after transfection. B, after treatment with lentivirus for 7 weeks, the co-cultures on the coverslips were examined by confocal staining with anti-MBP and anti-neurofilament antibodies. Knockdown of ADAM10 protein levels had no obvious effect on myelination. Scale bar = 40 μm.

Knocking down the expression of ADAM10 and ADAM17. A, HEK-293 derivative cells stably expressing type I Nrg1 were treated with the indicated siRNA duplex targeting either ADAM10 (ADAM10-Si) or ADAM17 (ADAM17-Si). Each siRNA duplex has specific sequences, with two targeting ADAM10 and two targeting ADAM17. Forty-eight h after transfection, equal amounts of prepared lysates were examined by Western blot analysis. The effect of lower expression of ADAM10 or ADAM17 on APP processing was detected using APP C terminus-specific antibody (A8717), whereas Nrg1 C terminus-specific antibody was used to assess Nrg1 processing by ADAM proteases. Nonspecific bands are indicated by the arrowhead. B and C, the relative levels of Nrg1-ctfα were calculated from three independent transfection experiments, and the results reflect the average of two treated siRNA oligonucleotides. *, p < 0.05; **, p <0.01 (Student's t test). Nrg1-fl, full-length Nrg1; con-Si, control siRNA.

Cleavage of Nrg1 variants by BACE1 and ADAM10. A, type I Nrg1 and its various mutants were transiently expressed in HM cells stably expressing BACE1 for 48 h, and prepared lysates were resolved by electrophoresis. Under these conditions, Nrg1-ctfs were separated into two bands on Western blots. Mutation of the BACE1 cleavage site in Nrg1, such as deletion of residues ME (Nrg1-βΔME) or replacing F↓M with K↓K (Nrg1-βKK) or A↓A (Nrg1-βAA), reduced the faster migrating bands, whereas the slower migrating band, specified as Nrg1-ctfα, was increased. Nonspecific bands are indicated by the arrowhead. B, in separate transfection experiments, HM cells were treated with BACE1 inhibitor IV (β-i) or GM6001 (α-i) during the transfection. BACE1 inhibition essentially abolished the production of Nrg1-ctfβ in the case of Nrg1-βKK, further confirming that the faster migrating band is due to BACE1 cleavage. BACE1 inhibition also elevated the levels of full-length Nrg1 in cells transfected with both wild-type and mutant Nrg1.

Activation of signaling molecules by the BACE1- or ADAM10-cleaved Nrg1-ntf. A, schematic illustration of the processing of type III β1 Nrg1. The cleavage sites of Nrg1 by ADAM10 and BACE1 are specified by arrows; the corresponding N-terminal fragments (type III Nrg1-ntfα or type III Nrg1-ntfβ) are indicated. The putative cleavage site of Nrg1 by γ-secretase is likely within the lipid bilayer based on its cleavage of APP. CRD, cysteine-rich domain. B, human breast cancer MCF-7 cells were transfected with type I or III Nrg1 expression constructs. After 48 h of transfection, protein lysates were prepared for Western blot analyses with antibodies to total and phosphorylated (p) Akt and ERK. C, human breast cancer MCF-7 cells were transfected with plasmid DNA expressing type III β1 Nrg1-ntfα, Nrg1-ntfβ, or full-length (fl) Nrg1 for 48 h, and cell lysates were prepared for Western blot analyses. Nrg1-ntfα and Nrg1-ntfβ were not easily separable under these electrophoresis conditions, likely due to glycosylation. Like full-length Nrg1, both fragments similarly activated Akt and ERK by increasing their phosphorylation. Anti-Nrg1 antibody also recognizes bands in all cells, and these nonspecifically reacted proteins served as natural loading controls.