Control of extracellular cleavage of ProBDNF by high frequency neuronal activity

Abstract

Pro-and mature neurotrophins often elicit opposing biological effects. For example, mature brain-derived neurotrophic factor (mBDNF) is critical for long-term potentiation induced by highfrequency stimulation, whereas proBDNF facilitate long-term depression induced by low-frequency stimulation. Because mBDNF is derived from proBDNF by endoproteolytic cleavage, mechanisms regulating the cleavage of proBDNF may control the direction of BDNF regulation. Using methods that selectively detect proBDNF or mBDNF, we show that low-frequency stimulation induced predominant proBDNF secretion in cultured hippocampal neurons. In contrast, high-frequency stimulation preferentially increased extracellular mBDNF. Inhibition of extracellular, but not intracellular cleavage of proBDNF greatly reduced high-frequency stimulationinduced extracellular mBDNF. Moreover, high-frequency, but not low-frequency stimulation selectively induced the secretion of tissue plasminogen activator, a key protease involved in extracellular proBDNF to mBDNF conversion. Thus, high-frequency neuronal activity controls the ratio of extracellular proBDNF/mBDNF by regulating the secretion of extracellular proteases. Our study demonstrates activity-dependent control of extracellular proteolytic cleavage of a secretory protein, and reveals an important mechanism that controls diametrically opposed functions of BDNF isoforms.BDNF ͉ hippocampus ͉ LTP ͉ proteases ͉ tPA B rain-derived neurotrophic factor (BDNF) plays a critical role in activity-dependent processes, such as synapse development and plasticity (1-3). Synthesized as a precursor, proBDNF is then sorted into one of the two secretory pathways: constitutive (passive) or regulated (induced or active) (4-6). Until recently, only mature BDNF (mBDNF) was considered biologically active. This view was challenged by findings that exogenous proBDNF induces apoptosis in peripheral neurons (7) and facilitates long-term depression (LTD) in the hippocampus (8). ProBDNF, is believed to be cleaved intracellularly by furin in trans-Golgi or by pro-protein convertase 1/3 (PC1/3) in secretory granules (9, 10) to generate mBDNF. Recently, Lee et al. demonstrated that proBDNF could be converted to mBDNF by extracellular proteases, such as plasmin and matrixmetalloprotease-7 (11). The physiological significance of extracellular proBDNF cleavage became evident when proBDNF 3 mBDNF conversion through the tissue plasminogen activator (tPA)/plasminogen cascade was shown to be essential for late phase long-term potentiation (L-LTP) in the hippocampus (12). Given the opposing biological effects of proBDNF and mBDNF, mechanisms controlling the cleavage of proBDNF have emerged as important mechanisms in controlling the direction of BDNF regulation (13).Neuronal release of endogenous proBDNF remains controversial. While studies using neuronal cultures in absence of glial cells and in the presence of cell impermeant ␣2 antiplasmin inhibitors successfully detected proBDNF in the extracellular culture medium (7), a recent study by Mat...

Role of pro-brain-derived neurotrophic factor (proBDNF) to mature BDNF conversion in activity-dependent competition at developing neuromuscular synapses

“…We applied glutamate, instead of high K + , to selectively depolarize neurons, because muscle cells do not express glutamate receptors (34). After neuronal depolarization, cultures were subsequently fixed and processed for surface immunostaining using an mBDNF-specific antibody (27). In control conditions, there was no mBDNF staining (Fig.…”

“…The muscle cell cultures were depolarized by high-K + treatment (50 mM) for 5 min, fixed, and processed for cell surface immunofluorescence staining under membrane impermeable conditions. A proBDNF-specific monoclonal antibody was used for cellsurface staining of secreted proBDNF (27). Although proBDNF was barely detectable on the muscle cell surface at rest (control), the proBDNF immunoreactivity increased dramatically (88%) upon depolarization (Fig.…”

“…Because of unavailability of a proBDNF-specific ELISA and limited number of muscle cells in the Xenopus cocultures (<1,000 myocytes), it was not feasible to measure proBDNF secretion from muscle cells using existing biochemical techniques. Thus, we used cell surface immunostaining to measure proBDNF secretion, given that proBDNF is positively charged at physiological pH and could be associated with a negatively charged cell membrane upon secretion (27,33). The muscle cell cultures were depolarized by high-K + treatment (50 mM) for 5 min, fixed, and processed for cell surface immunofluorescence staining under membrane impermeable conditions.…”

“…In contrast, mBDNF-TrkB signaling is essential for the early phase of long-term potentiation (E-LTP) (23)(24)(25). Moreover, recent studies indicate that a significant proportion of BDNF in the brain is secreted in the proform (26)(27)(28), and extracellular conversion of proBDNF to mBDNF by the tissue plasminogen activator (tPA)/plasmin protease system is critical for late-phase LTP (29). The expression of proBDNF and p75 NTR in rodents is developmentally regulated, with the highest levels in the first and second postnatal week, correlating well with the timings of synapse formation (28).…”

State of Art of Serum Brain-Derived Neurotrophic Factor in Schizophrenia

“…Mature BDNF (mBDNF) is generated either intracellularly in the trans-Golgi by furin (Mowla et al, 2001, Matsumoto et al, 2008, or extracellularly by plasmin or matrixmetalloprotease-7 (Lee et al, 2001Nagappan et al, 2009). Truncated-BDNF is cleaved by the Membrane-Bound Transcription Factor Site-1 protease (MBTFS-1), also identified as Subtilisin/kexin-isozyme 1 (Seidah et al, 1999).…”