Functional analysis of BDNF protein using Proteomics techinology

ProBDNF通过激活p75 NTR发挥与成熟BDNF（mBDNF）相反的功能。因此，proBDNF向mBDNF的蛋白水解转化决定了BDNF作用的方向。在这里，我们描述了两个罕见的人类SNP（R125 M和R127 L），取代两个切割位点附近的碱基。SNPs显著抑制proBDNF的内切蛋白水解切割。由此产生的抗切割proBDNF促进小脑颗粒神经元（CGNs）的凋亡和抑制培养的基底前脑胆碱能神经元（BFCNs）的轴突生长。敲入小鼠（BDNF pro/pro）与精氨酸取代表现出较小的小脑，增强CGN凋亡和减少CGN前体细胞增殖。在基底前脑，抑制proBDNF裂解不影响BFCNs的生存，但抑制胆碱能传入海马。这些效应是由proBDNF的增加而不是mBDNF的减少引起的，因为将BDNF pro/pro与p75 NTR突变小鼠杂交挽救了细胞表型。这些结果揭示了proBDNF的体内功能，并强调了proBDNF切割的重要性。

ProBDNF, through activation of p75NTR, exerts functions opposite to those of mature BDNF (mBDNF). Thus, proteolytic conversion of proBDNF to mBDNF determines the direction of BDNF actions. Here we characterize two rare human SNPs (R125M and R127L) that substitute two arginines adjacent to the cleavage site. The SNPs markedly inhibited the endo-proteolytic cleavage of proBDNF. The resulting cleavage-resistant proBDNF facilitated apoptosis of cerebellar granule neurons (CGNs) and suppressed neurite outgrowth of cultured basal forebrain cholinergic neurons (BFCNs). Knock-in mice (BDNF pro/pro) with the arginine substitutions exhibited a smaller cerebellum, enhanced CGN apoptosis and decreased proliferation of CGN precursors. In basal forebrain, inhibition of proBDNF cleavage did not affect BFCNs survival but suppressed cholinergic afferents to the hippocampus. These effects were caused by an increase in proBDNF rather than the decrease in mBDNF, since crossing BDNF pro/pro with p75NTR mutant mice rescued the cellular phenotypes. These results reveal the in vivo function of proBDNF and underscore the importance of proBDNF cleavage.