Biosynthesis and Post-translational Processing of the Precursor to Brain-derived Neurotrophic Factor

Abstract

We examined the biosynthesis and post-translational processing of the brain-derived neurotrophic factor precursor (pro-BDNF) in cells infected with a pro-BDNFencoding vaccinia virus. Metabolic labeling, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis reveal that pro-BDNF is generated as a 32-kDa precursor that is N-glycosylated and glycosulfated on a site, within the pro-domain. Some pro-BDNF is released extracellularly and is biologically active as demonstrated by its ability to mediate TrkB phosphorylation. The precursor undergoes N-terminal cleavage within the trans-Golgi network and/or immature secretory vesicles to generate mature BDNF (14 kDa). Small amounts of a 28-kDa protein that is immunoprecipitated with BDNF antibodies is also evident. This protein is generated in the endoplasmic reticulum through N-terminal cleavage of pro-BDNF at the Arg-Gly-Leu-Thr 57 -2-SerLeu site. Cleavage is abolished when Arg 54 is changed to Ala (R54A) by in vitro mutagenesis. Blocking generation of 28-kDa BDNF has no effect on the level of mature BDNF and blocking generation of mature BDNF with ␣ 1 -PDX, an inhibitor of furin-like enzymes, does not lead to accumulation of the 28-kDa form. These data suggest that 28-kDa pro-BDNF is not an obligatory intermediate in the formation of the 14-kDa form in the constitutive secretory pathway. Brain-derived neurotrophic factor (BDNF)1 along with nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) are members of the neurotrophin family of trophic factors (1). The neurotrophins play essential roles in the development, survival, and function of a wide range of neurons in both the peripheral and central nervous systems.The neurotrophins have a number of shared characteristics, including similar molecular weights (13.2-15.9 kDa), isoelectric points (in the range of 9 -10), and ϳ50% identity in primary structure. They exist in solution as noncovalently bound dimers. Six cysteine residues conserved in the same relative positions give rise to three intra-chain disulfide bonds (2, 3). The neurotrophins interact with two cell surface receptors, the low affinity P75 receptor (4) and the Trk family of high affinity tyrosine kinase receptors (5). NGF preferentially binds TrkA, BDNF and NT4/5 bind TrkB, and NT-3 binds TrkC (and TrkA to a lesser extent).Sequence data predict that mature neurotrophins are generated through the proteolytic processing of higher molecular weight precursors (31-35 kDa), a process that has been extensively studied with respect to the production of NGF (6, 7). Almost nothing is known, however, about the biosynthesis and post-translational processing of the other members of the neurotrophin family. Recent data from our laboratory show that cells with a regulated secretory pathway, including central nervous system neurons, release mature (i.e. fully processed) NGF (8) and NT-3 (9) via the constitutive secretory pathway, whereas mature BDNF is packaged in vesicles and released through the regulated pathway (8). Furthermore, BDNF i...

Chronic Duloxetine Treatment Induces Specific Changes in the Expression of BDNF Transcripts and in the Subcellular Localization of the Neurotrophin Protein

“…With the exception of ECT, most antidepressant drugs have limited or no effect on BDNF protein levels (Altar et al, 2003;Jacobsen and Mork, 2004;Tardito et al, 2006). BDNF is present within the cell in two forms (Mowla et al, 2001)Fthe precursor and the matureFand can also be localized in different subcellular compartments, where its distribution may be influenced by genetic variants (Egan et al, 2003;Lessmann et al, 2003;Chen et al, 2004;Bramham and Messaoudi, 2005). We found that rats chronically treated with duloxetine have significantly higher levels of mBDNF in the crude synaptosomal fraction of the frontal cortex, with a concomitant reduction in the cytosolic fraction.…”

Prenatal opiate exposure impairs radial arm maze performance and reduces levels of BDNF precursor following training

“…There has been growing interest in the distinct role that the BDNF precursor plays as compared to the better-studied secreted form of BDNF. BDNF precursor can be localized to synaptic regions where it may serve to stabilize synapses or it can be secreted (Agassandian, 2006;Mowla et al, 2001;Zhou et al, 2004). BDNF precursor can be transported in the axons and has been found to be both an anterograde and retrograde signaling molecule (Wang et al, 2006).…”

“…BDNF mRNA is translated into a pre-proprotein that is packaged in secretory vesicles before cleavage to the mature, secreted protein (Mowla et al, 2001). The precursor form of BDNF may actually play a different role in the brain than the mature form (Teng et al, 2005), including affecting learning and memory and drug abuse (Cheng et al, 2005;Hariri et al, 2003;Silhol et al, 2007).…”

A BDNF Coding Variant is Associated with the NEO Personality Inventory Domain Neuroticism, a Risk Factor for Depression

“…Our results, together with the neurotrophic hypothesis of depression (Duman et al, 1997), predict that the Met allele of BDNF produces higher activity or more efficient processing of BDNF. Given the proximity of this variant to a BDNF cleavage site, it is possible that this variant affects the efficiency of cleavage at this site (Mowla et al, 2001). The variant may also be involved in determining the efficiency of mature BDNF folding.…”