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ARTICLE OPEN Extracellular and intracellular cleavages of proBDNF required at two distinct stages of late-phase LTP Petti T Pang1,2 , Guhan Nagappan1,3 , Wei Guo4 and Bai Lu1,4 Although late-phase long-term potentiation (L-LTP) is implicated in long-term memory, its molecular mechanisms are largely unknown.

Here we provide evidence that L-LTP can be divided into two stages: an induction stage (I) and a maintenance stage (II). Both stages require mature brain-derived neurotrophic factor (mBDNF), but involve distinct underlying mechanisms. Stage I requires secretion of existing proBDNF followed by extracellular cleavage by tPA/plasmin. Stage II depends on newly synthesized BDNF. Surprisingly, mBDNF at stage II is derived from intracellular cleavage of proBDNF by furin/PC1. Moreover, stage I involves BDNF-TrkB signaling mainly through MAP kinase, whereas all three signaling pathways (phospholipase C-γ, PI3 kinase, and MAP kinase) are required for the maintenance of L-LTP at stage II. These results reveal the molecular basis for two temporally distinct stages in L-LTP, and provide insights on how BDNF modulates this long-lasting synaptic alternation at two critical time windows. npj Science of Learning (2016) 1, 16003;

doi:10.1038/npjscilearn.2016.3;

published online

11 May

2016 INTRODUCTION Long-term memory is believed to be mediated by long-lasting, protein synthesis-dependent changes in synaptic ef?cacy. The best-studied cellular model is the late-phase long-term potentia- tion (L-LTP) in the hippocampus. L-LTP requires gene transcription and new protein synthesis, and is accompanied by dendritic growth and synaptic remodeling.1 Although tetanic stimulation used to induce L-LTP trigger the expression of many proteins, the speci?c protein synthesis product(s) responsible for the induction and maintenance of L-LTP remain to be established. Although several molecules were reported to be involved in L-LTP, such as dopamine D1/D5 receptors, mGluR and PKA,2C4 the most important candidate is brain-derived neurotrophic factor (BDNF), which is a major secretory neurotrophic factor in the brain.5C7 L-LTP inducing stimuli elicits an increase in hippocampal BDNF mRNA, with a time course well correlated with L-LTP expression and the formation of long-term memory.8C15 Inhibition of BDNF signaling signi?cantly impairs L-LTP as well as long-term memory as assessed by several behavior tests.16C18 In addition, BDNF has been identi?ed as one of the few CREB-dependent proteins critical for the maintenance of L-LTP. In VP16-CREB (constitutively active CREB) over-expressing mice, L-LTP can be induced by weak tetanus and is no longer dependent on protein synthesis.19 Furthermore, when protein synthesis is blocked during the entire course of L-LTP, application of BDNF completely reverses the L-LTP de?cit.5 These results suggest that BDNF is at least one of the key protein synthesis products mediating L-LTP. Similar to other neurotrophins, BDNF is ?rst synthesized as a precursor termed proBDNF, which is then converted to mature BDNF (mBDNF) through the proteolytic removal of the N-terminal fragment by speci?c protease(s).20 Interaction of mature neuro- trophins with Trk receptors leads to cell survival, whereas binding of pro-neurotrophins (proBDNF, proNGF) to p75 NGF receptor (p75NTR ) leads to apoptosis.21,22 Pro-NGFs were initially shown to be processed by intracellular proteases including the serine protease furin (FIN) in the trans-Golgi network and the prohormone convertases (PC1/3) in the secretory granules.23,24 It has also been demonstrated extracellular cleavage of proNGF and proBDNF by matrix metalloproteinases (MMP3 or MMP7) and the serine protease plasmin.21,25 Activation of plasmin, which is initially produced as an inactive zymogen, plasminogen, requires cleavage by tissue plasminogen activator (tPA).26 Given that tPA is a secreted protease selectively involved in L-LTP,27,28 it has been hypothesized that extracellular cleavage of proBDNF by tPA/plasmin may somehow be involved in synaptic modulation by BDNF.20 Indeed, we have shown that tPA, by activating plasminogen, converts proBDNF to mBDNF in the hippocampus, and such conversion is critical for L-LTP expression.5 This work has provided a mechanistic link between tPA and BDNF in L-LTP, and revealed a physiological role of extracellular cleavage of proBDNF. We also have shown that proBDNF, if not processed, selectively enhances NMDA receptor- dependent long-term depression by activating its preferred receptor p75NTR .29 Thus, parallel to the roles of pro/mature NGFs in mediating cell death/survival, proBDNF and mBDNF also elicit opposite effects on long-term synaptic plasticity.25,30,31 Recent work has demonstrated that high-frequency neuronal activity induced the secretion of tPA in extracellular proBDNF to mBDNF conversion.32 Interestingly, this conversion has been shown to mediate activity-dependent synaptic competition during neuromuscular synapse formation.33,34 Cleavage of pro-NGFs by intracellular or extracellular proteases has now emerged as an important mechanism in controlling the direction of NGF regulation. The requirement for proBDNF → mBDNF conversion in L-LTP provides a unique opportunity to investigate the mechanisms underlying L-LTP, which is largely unknown thus far. Is tPA/ plasmin the only enzyme system involved in the cleavage of