, 1995, Fuhrer et al., 1997 and Moransard et al., 2003). This interaction was abolished or attenuated in muscle-specific, double, and null LRP4 mutant muscles, but not in motoneuron-specific www.selleckchem.com/products/ABT-737.html mutant muscles ( Figures S7A and S7B).
Finally, motoneuron LRP4 may be unable to generate the retrograde signals for presynaptic differentiation. Results of this study highlight distinct functions of motoneuron and muscle LRP4 for NMJ formation and maintenance (Figure 8). In a working model, motoneuron LRP4 contributes to formation of primitive AChR clusters and initial postsynaptic differentiation. This effect is probably mediated by ecto-LRP4, released by extracellular cleavage, which acts as agrin’s receptor in trans to stimulate AChR clustering. Motoneuron LRP4 is also necessary for well-being of motor axons. On the other hand, muscle BAY 73-4506 cost LRP4 plays a dominant role in NMJ formation. It instructs where nerve terminals stop and where muscle
fibers form AChR clusters, is essential for NMJ maturation, and regulates presynaptic differentiation. These observations may have an implication for understanding how LRP4-like “receptors” work in other contexts including CNS synapse formation and plasticity. Chemicals were purchased from Sigma-Aldrich Company unless otherwise indicated. Alexa Fluor 350 phalloidin (A22281; 1:200 for staining) and Alexa Fluor 594 α-bungarotoxin (BTX) (B-13423; 1:3,000 for staining) were purchased from Invitrogen. Matrix metalloproteinase (MMP) inhibitor N-[(2R)-2(hydroxamideocarbonylmethyl)-4-methylpantanoyl]-L-tryptophan Sodium butyrate methylamide (GM6001) (364206) and β-Secretase inhibitor IV (565788) were purchased from Calbiochem (EMD Millipore). Information of antibodies was as follows: neurofilament (Millipore) (AB1991; 1:1,000 for staining); synaptophysin (Dako) (A0010; 1:2,000 for staining); LRP4 (ECD) clone N207/27 (UC Davis/NIH NeuroMab Facility) (75-221; 1:1,000 for western
blotting); Flag (M2) (Sigma-Aldrich) (F3165; 1:2,000 for western blotting); 4G10 (Millipore) (05-1050X; 1:2,000 for western blotting); GFP (Abcam) (ab6556; 1:2,000 for western blotting); β-III-Tubulin (Covance) (MMS-435P; 1:1,000 for immunostaining); HB9 antibodies (C-terminal 307–403, gift from Dr. Samuel Pfaff; 1:4,000 for staining) (Thaler et al., 1999); synapsin (sc-20780; 1:500 for immunostaining); SV2 (Developmental Studies Hybridoma Bank) (1:1,000 for immunostaining); α-Tubulin (sc-23948; 1:3,000 for western blotting); β-actin (Novus) (NB600-501; 1:3,000 for western blotting); and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) (A-11034, 1:1,000 for staining). Polyclonal horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (32260), goat anti-mouse IgG (32230), and goat anti-rat IgG (31470) secondary antibodies were purchased from Pierce (Thermo Scientific) (1:3,000 for western blotting). Anti-MuSK, anti-rapsyn, and anti-AChRα antibodies were described previously (1:1,000 for western blotting) (Luo et al., 2002).