Cleavage of proBDNF to BDNF by a tolloid-like metalloproteinase is required for acquisition of in vitro eyeblink classical conditioning

Abstract

The tolloid/bone morphogenetic protein-1 family of metalloproteinases have an important role in the regulation of embryonic pattern formation and tissue morphogenesis. Studies suggest that they participate in mechanisms of synaptic plasticity in adults, but very little is known about their function. Recently, we isolated a reptilian ortholog of the tolloid gene family designated turtle tolloid-like gene (tTll). Here, we examined the role of tTLL in an in vitro model of eyeblink classical conditioning using an isolated brainstem preparation to assess its role in synaptic plasticity during conditioning. Analysis by real-time reverse transcription-PCR shows that an extracellularly secreted form of tTLL, tTLLs, is transiently expressed in the early stages of conditioning during conditioned response acquisition, whereas a cytosolic form, tTLLc, is not. Short interfering RNA (siRNA)-directed gene knockdown and rescue of tTLL expression demonstrate that it is required for conditioning. Significantly, we show that tTLLs cleaves the precursor proBDNF into mature BDNF in cleavage assay studies, and application of recombinant tTLLs protein alone to preparations results in induction of mature BDNF expression. The mature form of BDNF is minimally expressed in preparations treated with anti-tTLL siRNA, and the synaptic incorporation of both GluR1- and GluR4-containing AMPA receptors is significantly reduced, resulting in suppression of conditioning. This is the first study to demonstrate that expression of an extracellularly secreted tolloid-like metalloproteinase is regulated in the early stages of classical conditioning and functions in the conversion of proBDNF to mature BDNF. The mature form of BDNF is required for synaptic delivery of AMPA receptors and acquisition of conditioned responses.

Figure 1.

Expression of tTLLs mRNA is upregulated in the early stages of conditioning. A, Acquisition curves of the mean percentage of CRs recorded from the same preparations analyzed by RT-PCR that were conditioned (Cond) or pseudoconditioned (Ps) for one, two, or five pairing sessions. Examples of physiological records from the abducens nerve show an unconditioned response alone (top record) and a CR (arrow), followed by the unconditioned response after acquisition (lower record). The CS and US are illustrated at the bottom. B, Real-time RT-PCR analysis of the total level of tTLL mRNA and of the cytosolic (tTLLc) and secreted (tTLLs) splice variants expressed as the mean fold difference compared with pseudoconditioned levels. Data are shown from preparations that were conditioned or pseudoconditioned for one pairing session (Ps1, C1; or a total of 25 min), two pairing sessions (Ps2, C2; or a total of 80 min), or five sessions (Ps5, C5; or ∼4 h). Data are plotted as means ± SEM, except for conditioning data that are expressed as means ± SD. Asterisks indicate significant differences from Ps for all figures.

Figure 2.

Treatment with anti-tTLL siRNA suppresses conditioning and is reversed by application of tTLLs rescue plasmids. A, The battery of siRNAs used here, Negative Control #6 (Nc #6), Negative Control #7 (Nc #7), and the anti-tTLL siRNA (tTLL siRNA), was tested for their effectiveness in knocking down target genes using cell cultures. COS-1 cells were transfected with tTLLs, tTLLc, or tGAPDH expression plasmids and later transfected with one of the siRNAs and examined by Western blot. The Nc #6 and Nc #7 siRNAs had no effect on expression of either splice variant of tTLL or tGAPDH. The anti-tTLL siRNA effectively blocked expression of both tTLLs and tTLLc but had no effect on tGAPDH. B, COS-1 cells were transfected with a tTLLs rescue plasmid that was rendered resistant to the anti-tTLL siRNA by a mutation in the binding region of the siRNA. Cells were transfected with rescue tTLLs, or unmutated tTLLs or tTLLc expression plasmids and then transfected with anti-tTLL siRNA. Western blots showed that tTLLs was expressed only by cells that were transfected by the rescue plasmid demonstrating resistance to the anti-tTLL siRNA. C, Mean percentage of CR acquisition and fold difference in levels of tTLLs or tTLLc mRNA expression for all treatment groups: pseudoconditioned for two pairing sessions (Ps2), conditioned for two sessions (C2), treatment with anti-tTLL siRNA for two sessions (tTLL siRNA), negative control siRNA #6 (Nc #6), negative control #7 (Nc #7), or tTLLs rescue plasmids (tTLLs Resc). Preparations treated with anti-tTLL siRNA showed no CRs and significantly reduced levels of tTLLs mRNA compared with conditioning. In contrast, preparations treated with control siRNAs showed levels of conditioning and tTLLs mRNA expression similar to untreated preparations. Treatment with the rescue plasmids also resulted in expression of CRs and tTLLs mRNA at values similar to untreated preparations. There were no differences in tTLLc among the groups.

Figure 3.

Conversion of proBDNF to mature BDNF occurs after the first pairing session of conditioning as shown by Western blot analysis. A, Significant expression of mature BDNF occurs after one complete pairing session of conditioning (C1, or after 25 min) compared with conditioning for 15 min (C₁₅) or pseudoconditioning for the same time periods (Ps₁₅, Ps1). Expression of BDNF declines to control values after five pairing sessions (C5, or after ∼4 h). B, Western blots of the 30, 24, and 18 kDa forms of proBDNF and mature BDNF after early conditioning show relatively stable amounts of proBDNF protein and appearance of the mature form at C1. Quantitative data of the ratio of proBDNF to BDNF for the 30 and 24 kDa proBDNF proteins show that significant conversion to the mature form occurs at C1. Data from the 18 kDa protein are similar to the 30 and 24 kDa proteins shown.

Figure 4.

Synaptic incorporation of GluR1- and GluR4-containing AMPARs on abducens motor neurons observed after conditioning is inhibited by incubation of preparations in antibodies to BDNF (BDNF Ab) or anti-tTLL siRNA (tTLL siRNA). A, Confocal images of punctate staining for GluR1 or GluR4 AMPAR subunits (red) and the presynaptic marker synaptophysin (green) for the different treatment groups. Colocalization of AMPAR subunits with synaptophysin is indicated (arrowheads). B, Quantitative analysis of punctate staining using stereological procedures shows that the conditioning-related increase in colocalization of both GluR1 and GluR4 AMPAR subunits with synaptophysin is significantly inhibited by treatment with BDNF antibodies and tTLL siRNA. Scale bar, 2 μm.

Figure 5.

ProBDNF is cleaved by tTLLs, but not tTLLc, in in vitro cleavage assays using purified recombinant proteins from turtle. A, Recombinant protein was generated to turtle proBDNF and incubated with recombinant proteins to tTLLs or tTLLc, or mutated recombinants to tTLLs or tTLLc that were rendered inactive (mut), or no proteinase. Western blot analysis shows that only samples treated by normal tTLLs resulted in cleavage of turtle proBDNF into mature BDNF. B, To verify this finding, point mutations were made into the cleavage site of proBDNF to render it resistant to cleavage. Incubation procedures like those above show that this manipulation blocked the ability of tTLLs to cleave proBDNF.

Figure 6.

Brainstem preparations incubated in recombinant proteins to tTLLs generate mature BDNF and expression of BDNF during conditioning is inhibited by anti-tTLL siRNA. A, Western blot analysis was performed for BDNF expression in untreated (naive) and conditioned (C2) preparations and those incubated in recombinant proteins to tTLLs or tTLLc alone (1 μg/ml) for the same time period (the equivalent of two sessions) without stimulation. The results show that incubation in tTLLs recombinant protein generates significant levels of mature BDNF, whereas tTLLc does not. B, Analysis of BDNF expression in preparations that were incubated in anti-tTLL siRNA and underwent conditioning for two pairing sessions show significantly reduced levels of mature BDNF compared with conditioning.