BDNF Is Expressed in Skeletal Muscle Satellite Cells and Inhibits Myogenic Differentiation

BDNF is low or absent in mature myofibers and not restricted to NMJs. ISH of BDNF probes on transverse sections of adult skeletal muscles. A, C, E, Representative micrographs showing hybridization of BDNF antisense probe on diaphragm, soleus, and EDL, respectively. B, D, F, BDNF sense control on diaphragm, soleus, and EDL, respectively. Arrows point to NMJs (brown staining). G, Representative micrograph showing hybridization of AChE antisense probe on adult diaphragm muscle sections. Arrows point to silver grain densities overlaying mature NMJs. H, Representative micrograph showing hybridization of AChE sense probe. Scale bar, 50 μm. I, A micrograph of coronal sections of neonatal (P10) brain showing hybridization of BDNF antisense probe. Scale bar, 500 μm. Arrows point to CA1–3 hippocampal regions.

BDNF protein is found in junctional and extrajunctional compartments of adult diaphragm skeletal muscle but not in mature myofibers or within the subsynaptic regions of NMJs. A, Levels of BDNF protein, per milligram of wet muscle mass, in junctional (Junc) and extrajunctional (EJunc) regions of diaphragm (n = 4). B, D, Immunofluorescence of BDNF on transverse sections of the diaphragm. Arrows point to subsynaptic areas of NMJs. Insets are 4′,6-diamidino-2-phenylindole staining of myonuclei. C, Same section as in B showing the localization of postsynaptic acetylcholine receptors stained with α-bungratoxin depicting the presence of NMJs. E, Same section as in D but stained for MyHC slow or β/I. Scale bar, 50 μm. Error bars represent SE.

Concentrations of BDNF transcript(s) do not colocalize with NMJs in neonatal diaphragm skeletal muscle. A, Micrograph of a transverse section of a neonatal diaphragm stained for NMJs. This micrograph is devoid of any NMJ. Arrows point to the same regions as in B. B, Serial section to A showing hybridization of BDNF antisense probe. Arrows point to concentrations of silver grains representing BDNF transcripts. C, Serial section to A processed for BDNF sense control. Scale bar, 25 μm. D, A micrograph showing NMJs in the developing diaphragm. E, Serial section to D showing hybridization of BDNF antisense probe. Arrowheads point toward NMJs. F, Serial section to D processed for BDNF sense probe. Scale bar, 25 μm.

Levels of BDNF are highly correlated to those of a muscle progenitor marker, Pax3. A, Arbitrary levels of BDNF mRNA (normalized to α-actin levels) in adult diaphragm (DIA), soleus (SOL), and extensor digitorum longus (EDL) skeletal muscles (n = 3). B, Arbitrary levels of Pax3 (relative to α-actin) in DIA, SOL, and EDL (*p < 0.05 vs DIA; ^#p < 0.05 vs soleus). Error bars represent SE.

BDNF is localized to skeletal muscle satellite cells. A–C, BDNF is colocalized to Pax7+ cells. A, BDNF IF in adult diaphragm skeletal muscle. B, Same section stained for Pax7. C, Overlay of A, B, and 4′,6-diamidino-2-phenylindole (DAPI) staining of nuclei (blue), showing BDNF in cytoplasmic regions of Pax7+ cells (arrows). D, E, Colocalization of BDNF with p75^NTR in adult diaphragm skeletal muscle. D, BDNF IF. E, p75^NTR IF. F, Overlay of D, E, and DAPI (blue), showing the colocalization of BDNF with p75^NTR (arrows). Scale bar, 12.5 μm. G–I, Isolated diaphragm skeletal muscle satellite cells express BDNF in culture. G, BDNF IF. H, Same cell as in G stained for the myogenic marker desmin. I, Overlay of G, H, and DAPI (blue). Scale bar, 12.5 μm.

Repression of BDNF and p75^NTR expression during myogenic differentiation. A, Representative ethidium bromide gels of BDNF, p75^NTR, TrkB, Pax3, and α-actin RT-PCR products from L6 myoblasts (MB) and myotubes (MT). No RNA (CL) was used as negative control. TrkB was not detected in MB or MT samples. B, There was an approximate fivefold reduction in the levels of BDNF mRNA, as normalized to α-actin after myogenic differentiation (*p < 0.05). C, The expression of p75^NTR followed the same trend as BDNF during differentiation (*p < 0.05). D, Pax3 levels are reduced by ∼50% after myogenic differentiation (n = 6; *p < 0.05). E–J, BDNF protein is detected in myoblasts and not in myotubes. E, H, BDNF IF of myoblasts and myotubes, respectively. Scale bar, 50 μm. Insets are staining for all isoforms of MyHC (red). F, I, Same as in E and H, except BDNF antibody was blocked with BDNF before incubation (see Materials and Methods). G, J, No primary control. K, BDNF protein levels per microgram of total protein in MB and MT (n = 3; *p < 0.05). Error bars represent SE.

siRNA design for BDNF. A, Representative map of BDNF NM_012513 sequence. BDNF599 and BDNF948 siRNA fragments are shown to be targeted to the coding regions (exon 5) of BDNF. B, Amplification of siRNA fragments from stably transfected siRNA cell lines. Using psiRNA-hH1neo specific primers (arrows), siRNA fragments (red) were amplified using DNA preparations from stable cell lines. A representative ethidium bromide gel showing amplified PCR products of psiRNAhH1neo in transfected cells. PCR product length is ∼290 bp. siRNA constructs are present in stably transfected clonal cells. L6 extracts and no DNA (BL) were used controls. C, Reduction of BDNF, per microgram of total protein, in BDNF599 (n = 9) and BDNF948 (n = 6) clonal cells compared with LacZ (n = 9) control cells (*p < 0.05 vs 599; ⁁p < 0.05 vs 948). Error bars represent SE.

Suppressing BDNF synthesis enhances myogenic differentiation. A, A Western blot showing expression of MyHC in BDNF599, LacZ, and parental (MB) after 6 d of incubation in growth media. Four day differentiated myotubes (MT) were used as MyHC+ control. Tubulin is shown as a loading control. Right markers show the position of embryonic (EMB) and neonatal (NEO) MyHC bands on the blot. B–D, Representative micrographs of BDNF599, LacZ, and parental cultures, respectively, stained for MyHC (red) after 6 d of incubation in growth media. Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). Scale bar, 100 μm. E, Number of nuclei in BDNF599, LacZ, and parental cultures after 6 d of incubation in growth media (n = 5; *p < 0.05). F, BDNF599 clonal cells exhibit a higher rate of fusion compared with LacZ and parental controls (n = 5; *p < 0.05). Error bars represent SE.

Recombinant BDNF rescues the accelerated myogenic differentiation of BDNF599 siRNA clonal cells. A, A representative micrograph of vehicle-treated BDNF599 cells stained for MyHC (red) after 6 d of incubation in growth media. Nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). B, A micrograph of parallel BDNF599 cultures, which were treated with daily dose of BDNF, stained for MyHC (red). Scale bar, 100 μm.