Figure 1
BDNF polymorphic substitutions inhibit the conversion of proBDNF to matBDNF and lead to predominant secretion of proBDNF in CNS neurons. (A) Schematic of human BDNF protein. Amino acid substitutions near the cleavage site in rare SNP variants are represented by single-letter symbols. The amino acid substitutions caused by the SNPs are depicted by white symbols. (B) Bioinformatic prediction of changes in the secondary structure of proBDNF SNPs variants using PONDR. M, L and ML depict the amino acid substitution of R125M, R127L and R125M/R127L, respectively. The disordered regions (PONDR score > 0.5) of wild-type BDNF (aa 25–103 and 120–140) is highlighted by bold lines. (C) Inhibition of the intracellular cleavage of SNP variants in cultured hippocampal neurons. The cultures were infected with Sindbis viruses expressing wild-type (RR), R125M (M), R127L (L), and R125M/R127L (ML) constructs for 12 h and 3 days later processed for Western blot analysis using anti-BDNF (IB: BDNF) and anti-Myc antibodies (IB: Myc). Doublet bands of 29- and 32-kDa were detected with the monoclonal anti-proBDNF antibody (IB: proBDNF), whereas an additional band of 14-kDa (matBDNF) was detected using an antibody against the mature domain (IB: BDNF). The levels of bicistronically expressed GFP, which correlated with the viral infection levels, were similar in all cultures. Note that proBDNF bands were predominant in the lysates of cells expressing the M, L, and ML constructs, while matBDNF expression was residual. In C and F, 1 ng recombinant matBDNF and proBDNF (R125M/R127L-BDNF) were used as positive controls. (D) The ratio of proBDNF to matBDNF was quantified by densitometric analysis on BDNF, and proBDNF bands. **P < 0.01 (Student's t-test) when compared with matBDNF (matBDNFRR + proBDNFRR = 100% as control). n = 3 independent experiments. (E) The entire amount of BDNF was quantified by densitometric analysis on matBDNF, proBDNF, and GFP bands. *P < 0.05 (t-test) when compared with RR (100% as control). n = 3 independent experiments. (F) Secretion of poorly cleaved proBDNF. Cultured cerebral cortical neurons were incubated with Sindbis viruses for 12 h and maintained in serum-containing medium for 3 days. Supernatants were collected and immunoprecipitated using anti-Myc antibody-conjugated agarose beads, followed by Western blot analysis using rabbit antibody against the mature domain (IB: BDNF) and mouse antibody recognizing the prodomain (IB: proBDNF). Cell lysates were blotted with anti-GFP antibody to normalize viral infection (IB: GFP).