Brain-derived Neurotrophic Factor Prodomain Research Articles

ProBDNF and Brain-Derived Neurotrophic Factor Prodomain Differently Modulate Acetylcholine Release in Regenerating and Mature Mouse Motor Synapses.

The effects of brain-derived neurotrophic factor (BDNF) processing by-products (proBDNF and BDNF prodomain) on the activity of mouse neuromuscular junctions (NMJs) were studied in synapses formed during the reinnervation of extensor digitorum longus muscle (m. EDL) and mature synapses of the diaphragm. The parameters of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were analyzed in presence of each of the BDNF maturation products (both – 1 nM). In newly formed NMJs, proBDNF caused an increase in the resting membrane potential of muscle fibers and a decrease in the frequency of MEPPs, which was prevented by tertiapin-Q, a G-protein-coupled inwardly rectifying potassium channels (GIRK) blocker but not by p75 receptor signaling inhibitor TAT-Pep5. proBDNF had no effect on the parameters of EPPs. BDNF prodomain in newly formed synapses had effects different from those of proBDNF: it increased the amplitude of MEPPs, which was prevented by vesamicol, an inhibitor of vesicular acetylcholine (ACh) transporter; and reduced the quantal content of EPPs. In mature NMJs, proBDNF did not influence MEPPs parameters, but BDNF prodomain suppressed both spontaneous and evoked ACh release: decreased the frequency and amplitude of MEPPs, and the amplitude and quantal content of EPPs. This effect of the BDNF prodomain was prevented by blocking GIRK channels, by TAT-Pep5 or by Rho-associated protein kinase (ROCK) inhibitor Y-27632. At the same time, the BDNF prodomain did not show any inhibitory effects in diaphragm motor synapses of pannexin 1 knockout mice, which have impaired purinergic regulation of neuromuscular transmission. The data obtained suggest that there is a previously unknown mechanism for the acute suppression of spontaneous and evoked ACh release in mature motor synapses, which involves the activation of p75 receptors, ROCK and GIRK channels by BDNF prodomain and requires interaction with metabotropic purinoreceptors. In general, our results show that both the precursor of BDNF and the product of its maturation have predominantly inhibitory effects on spontaneous and evoked ACh release in newly formed or functionally mature neuromuscular junctions, which are mainly opposite to the effects of BDNF. The inhibitory influences of both proteins related to brain neurotrophin are mediated via GIRK channels of mouse NMJs.

Pro-BDNF Knockout Causes Abnormal Motor Behaviours and Early Death in Mice

Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family, best characterized for its survival and differentiative effects in the central nervous system. Pro-BDNF, known as the precursor of BDNF, is believed to have opposite functions to mature BDNF (mBDNF). The opposing effects of Pro-BDNF and mBDNF have led researchers to propose a ‘yin’ (Pro-BDNF) and ‘yang’ (mBDNF) model of which, the specific mechanism of its opposing functions is unclear and requires further investigation. In order to elucidate pro-BDNF’s explicit role, we established a pro-BDNF knockout (KO) mouse model. This BDNF pro-domain KO mouse model showed significant weight loss, impaired righting reflex, abnormal motor behaviours and short lifespan (less than 22 days), mimicking a Huntington’s disease (HD)-like phenotype. ELISA results showed BDNF pro-domain KO not only blocked pro-BDNF, but also significantly affected the level of mBDNF. Abnormal morphologic changes were found in the dentate gyrus (DG) of the hippocampus in pro-BDNF KO mice, and western blot confirmed significant cell apoptosis in pro-BDNF KO mice brains. Furthermore, the expression of glutamic acid decarboxylase 65/67 (GAD65/67) was significantly reduced in pro-BDNF KO mice, indicating impaired inhibitory neurotransmission. Heterozygous (Het) mice showed impaired learning and memory capability and depressive-like behaviours, compared with wild type (WT) mice. Overall, these results support that pro-domain of BDNF is an indispensable part of the BDNF gene; without the proper formation of pro-BDNF, mBDNF cannot be produced successfully and function correctly on its own. Our study also supports the BDNF hypothesis in the pathogenesis of HD.

Sequence specificity despite intrinsic disorder: How a disease-associated Val/Met polymorphism rearranges tertiary interactions in a long disordered protein.

The role of electrostatic interactions and mutations that change charge states in intrinsically disordered proteins (IDPs) is well-established, but many disease-associated mutations in IDPs are charge-neutral. The Val66Met single nucleotide polymorphism (SNP) in precursor brain-derived neurotrophic factor (BDNF) is one of the earliest SNPs to be associated with neuropsychiatric disorders, and the underlying molecular mechanism is unknown. Here we report on over 250 μs of fully-atomistic, explicit solvent, temperature replica-exchange molecular dynamics (MD) simulations of the 91 residue BDNF prodomain, for both the V66 and M66 sequence. The simulations were able to correctly reproduce the location of both local and non-local secondary structure changes due to the Val66Met mutation, when compared with NMR spectroscopy. We find that the change in local structure is mediated via entropic and sequence specific effects. We developed a hierarchical sequence-based framework for analysis and conceptualization, which first identifies “blobs” of 4-15 residues representing local globular regions or linkers. We use this framework within a novel test for enrichment of higher-order (tertiary) structure in disordered proteins; the size and shape of each blob is extracted from MD simulation of the real protein (RP), and used to parameterize a self-avoiding heterogenous polymer (SAHP). The SAHP version of the BDNF prodomain suggested a protein segmented into three regions, with a central long, highly disordered polyampholyte linker separating two globular regions. This effective segmentation was also observed in full simulations of the RP, but the Val66Met substitution significantly increased interactions across the linker, as well as the number of participating residues. The Val66Met substitution replaces β-bridging between V66 and V94 (on either side of the linker) with specific side-chain interactions between M66 and M95. The protein backbone in the vicinity of M95 is then free to form β-bridges with residues 31-41 near the N-terminus, which condenses the protein. A significant role for Met/Met interactions is consistent with previously-observed non-local effects of the Val66Met SNP, as well as established interactions between the Met66 sequence and a Met-rich receptor that initiates neuronal growth cone retraction.

BDNF pro-peptide: physiological mechanisms and implications for depression.

Most growth factors are synthesized as precursors and biologically active forms are generated by proteolytic cleavage of the pro-domain. However, the biological functions of pro-domains are ill-defined. New roles were recently reported for the pro-domain of brain-derived neurotrophic factor (BDNF), a well-known growth factor in the brain. Interestingly, the pro-domain of BDNF (BDNF pro-peptide) is localized at presynaptic termini, where it facilitates long-term depression (LTD) in hippocampal slices, implicating it as a novel synaptic modulator. BDNF binds its pro-peptide with high affinity in a pH-dependent manner and when bound to BDNF, the BDNF pro-peptide cannot facilitate hippocampal LTD, representing a new mechanism of regulation. The BDNF pro-peptide is present in human cerebrospinal fluid (CSF) and levels were significantly lower in patients with major depressive disorder (MDD) than in controls. Notably, male MDD patients exhibit significantly lower levels of CSF pro-peptide than females. These findings demonstrate that the BDNF pro-peptide is a biologically important synaptic modulator and is associated with MDD, particularly in males.

A Novel Peptide Interfering with proBDNF-Sortilin Interaction Alleviates Chronic Inflammatory Pain.

Rationale: Brain-derived neurotrophic factor (BDNF) is a key mediator in the development of chronic pain. Sortilin is known to interact with proBDNF and regulate its activity-dependent secretion in cortical neurons. In a rat model of inflammatory pain with intraplantar injection of complete Freund's adjuvant (CFA), we examined the functional role of proBDNF-sortilin interaction in dorsal root ganglia (DRG).Methods: Expression and co-localization of BDNF and sortilin were determined by immunofluorescence. ProBDNF-sortilin interaction interface was mapped using co-immunoprecipitation and bimolecular fluorescence complementation assay. The analgesic effect of intrathecal injection of a synthetic peptide interfering with proBDNF-sortilin interaction was measured in the CFA model.Results: BDNF and sortilin were co-localized and their expression was significantly increased in ipsilateral L4/5 DRG upon hind paw CFA injection. In vivo adeno-associated virus-mediated knockdown of sortilin-1 in L5 DRG alleviated pain-like responses. Mapping by serial deletions in the BDNF prodomain indicated that amino acid residues 71-100 supported the proBDNF-sortilin interaction. A synthetic peptide identical to amino acid residues 89-98 of proBDNF, as compared with scrambled peptide, was found to interfere with proBDNF-sortilin interaction, inhibit activity-dependent release of BDNF in vitro and reduce CFA-induced mechanical allodynia and heat hyperalgesia in vivo. The synthetic peptide also interfered with capsaicin-induced phosphorylation of extracellular signal-regulated kinases in ipsilateral spinal cord of CFA-injected rats.Conclusions: Sortilin-mediated secretion of BDNF from DRG neurons contributes to CFA-induced inflammatory pain. Interfering with proBDNF-sortilin interaction reduced activity-dependent release of BDNF and might serve as a therapeutic approach for chronic inflammatory pain.

Design of a Novel Gene Therapy Construct to Achieve Sustained Brain-Derived Neurotrophic Factor Signaling in Neurons.

Brain-derived neurotrophic factor (BDNF) acting through the tropomyosin-related receptor-B (TrkB) is an important signaling system for the maintenance and survival of neurons. Gene therapy using either recombinant adeno-associated virus (AAV) or lentiviral vectors can provide sustained delivery of BDNF to tissues where reduced BDNF signaling is hypothesized to contribute to disease pathophysiology. However, elevation in BDNF at target sites has been shown to lead to a downregulation of TrkB receptors, thereby reducing the effect of chronic BDNF delivery over time. A novel gene sequence has been designed coding both the ligand (BDNF) and the TrkB receptor in a single transgene separated by a short viral-2A sequence. The single transgene is efficiently processed intracellularly in vitro and in vivo to yield the two mature proteins, which are then independently transported to their final cellular locations: TrkB receptors to the cell surface, and BDNF contained within secretory vesicles. To accommodate the coding sequences of both BDNF and TrkB receptors within the narrow confines of the AAV vectors (4.7 kb pairs), the coding region for the pro-domain of BDNF was removed and the signal peptide sequence modified to improve production, intracellular transport, and secretion of mature BDNF (mBDNF). Intracellular processing and efficacy was shown in HEK293 cells and SH-SY5Y neuroblastoma cells using plasmid DNA and after incorporating the TrkB-2A-mBDNF into an AAV2 vector. Increased BDNF/TrkB-mediated intracellular signaling pathways were observed after AAV2 vector transfection while increased TrkB phosphorylation could be detected in combination with neuroprotection from hydrogen peroxide–induced oxidative stress. Correct processing was also shown in vivo in mouse retinal ganglion cells after AAV2 vector administration to the eye. This novel construct is currently being investigated for its efficacy in animal models to determine its potential to progress to human clinical studies in the future.

NMR backbone resonance assignments of the prodomain variants of BDNF in the urea denatured state.

Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family of proteins which plays a central role in neuronal survival, growth, plasticity and memory. A single Val66Met variant has been identified in the prodomain of human BDNF that is associated with anxiety, depression and memory disorders. The structural differences within the full-length prodomain Val66 and Met66 isoforms could shed light on the mechanism of action of the Met66 and its impact on the development of neuropsychiatric-associated disorders. In the present study, we report the backbone 1H, 13C, and 15N NMR assignments of both full-length Val66 and Met66 prodomains in the presence of 2M urea. These conditions were utilized to suppress residual structure and aid subsequent native state structural investigations aimed at mapping and identifying variant-dependent conformational differences under native-state conditions.

BDNF Binds Its Pro-Peptide with High Affinity and the Common Val66Met Polymorphism Attenuates the Interaction.

Most growth factors are initially synthesized as precursors then cleaved into bioactive mature domains and pro-domains, but the biological roles of pro-domains are poorly understood. In the present study, we investigated the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which promotes neuronal survival, differentiation and synaptic plasticity. The BDNF pro-peptide is a post-processing product of the precursor BDNF. Using surface plasmon resonance and biochemical experiments, we first demonstrated that the BDNF pro-peptide binds to mature BDNF with high affinity, but not other neurotrophins. This interaction was more enhanced at acidic pH than at neutral pH, suggesting that the binding is significant in intracellular compartments such as trafficking vesicles rather than the extracellular space. The common Val66Met BDNF polymorphism results in a valine instead of a methionine in the pro-domain, which affects human brain functions and the activity-dependent secretion of BDNF. We investigated the influence of this variation on the interaction between BDNF and the pro-peptide. Interestingly, the Val66Met polymorphism stabilized the heterodimeric complex of BDNF and its pro-peptide. Furthermore, compared with the Val-containing pro-peptide, the complex with the Met-type pro-peptide was more stable at both acidic and neutral pH, suggesting that the Val66Met BDNF polymorphism forms a more stable complex. A computational modeling provided an interpretation to the role of the Val66Met mutation in the interaction of BDNF and its pro-peptide. Lastly, we performed electrophysiological experiments, which indicated that the BDNF pro-peptide, when pre-incubated with BDNF, attenuated the ability of BDNF to inhibit hippocampal long-term depression (LTD), suggesting a possibility that the BDNF pro-peptide may interact directly with BDNF and thereby inhibit its availability. It was previously reported that the BDNF pro-domain exerts a chaperone-like function and assists the folding of the BDNF protein. However, our results suggest a new role for the BDNF pro-domain (or pro-peptide) following proteolytic cleave of precursor BDNF, and provide insight into the Val66Met polymorphism.

Growth factors and hormones pro-peptides: the unexpected adventures of the BDNF prodomain.

Most growth factors and hormones are synthesized as pre-pro-proteins which are processed to the biologically active mature protein. The pre- and prodomains are cleaved from the precursor protein in the secretory pathway or, in some cases, extracellularly. The canonical functions of these prodomains are to assist in folding and stabilization of the mature domain, to direct intra and extracellular localization, to facilitate storage, and to regulate bioavailability of their mature counterpart. Recently, exciting evidence has revealed that prodomains of certain growth factors, after cleaved from the precursor pro-protein, can act as independent active signaling molecules. In this review, we discuss the various classical functions of prodomains, and the biological consequences of these pro-peptides acting as ligands. We will focus our attention on the brain-derived neurotrophic factor prodomain(pBDNF), which has been recently described as a novel secreted ligand influencing neuronal morphology and physiology.

BDNF Propeptide: A Novel Modulator of Synaptic Plasticity.

One of the most characterized neurotrophic factors is brain-derived neurotrophic factor (BDNF), which regulates neuronal survival and differentiation, and functions in activity-dependent plasticity processes such as long-term potentiation, long-term depression (LTD), and learning memory. Similar to other growth factors, BDNF protein is produced by transcriptional and translational mechanisms. Nevertheless, a posttranslational mechanism, the proteolytic conversion of precursor BDNF into at least two fragments, bioactive BDNF and the prodomain, has not been well elucidated. Recently, we demonstrated that the BDNF prodomain, which is named the BDNF propeptide, was endogenously secreted from neuronal cells and facilitated a cellular mechanism of LTD, suggesting the manner through which this posttranslational mechanism multiplies the biological actions of BDNF. In this chapter, we focus on the BDNF propeptide, especially in synaptic plasticity, and discuss the role of this molecule in the brain. The findings regarding the BDNF propeptide would provide new insights for understanding the mechanisms of action of the propeptides of growth factors as well as the biological roles of neurotrophins.

Definition of a Bidirectional Activity-Dependent Pathway Involving BDNF and Narp.

One of the cardinal features of neural development and adult plasticity is the contribution of activity-dependent signaling pathways. However, the interrelationships between different activity-dependent genes are not well understood. The immediate early gene neuronal-activity-regulated pentraxin (NPTX2 or Narp) encodes a protein that has been associated with excitatory synaptogenesis, AMPA receptor aggregation, and the onset of critical periods. Here, we show that Narp is a direct transcriptional target of brain-derived neurotrophic factor (BDNF), another highly regulated activity-dependent gene involved in synaptic plasticity. Unexpectedly, Narp is bidirectionally regulated by BDNF. Acute BDNF withdrawal results in downregulation of Narp, whereas transcription of Narp is greatly enhanced by BDNF. Furthermore, our results show that BDNF directly regulates Narp to mediate glutamatergic transmission and mossy fiber plasticity. Hence, Narp serves as a significant epistatic target of BDNF to regulate synaptic plasticity during periods of dynamic activity.

The pro-domains of neurotrophins, including BDNF, are linked to Alzheimer's disease through a toxic synergy with Aβ.

Brain-derived neurotrophic factor (BDNF) has a crucial role in learning and memory by promoting neuronal survival and modulating synaptic connectivity. BDNF levels are lower in the brains of individuals with Alzheimer's disease (AD), suggesting a pathogenic involvement. The Drosophila orthologue of BDNF is the highly conserved Neurotrophin 1 (DNT1). BDNF and DNT1 have the same overall protein structure and can be cleaved, resulting in the conversion of a full-length polypeptide into separate pro- and mature-domains. While the BDNF mature-domain is neuroprotective, the role of the pro-domain is less clear. In flies and mammalian cells, we have identified a synergistic toxic interaction between the amyloid-β peptide (Aβ1–42) and the pro-domains of both DNT1 and BDNF. Specifically, we show that DNT1 pro-domain acquires a neurotoxic activity in the presence of Aβ1–42. In contrast, DNT1 mature-domain is protective against Aβ1–42 toxicity. Likewise, in SH-SY5Y cell culture, BDNF pro-domain is toxic only in the presence of Aβ1–42. Western blots indicate that this synergistic interaction likely results from the Aβ1–42-induced upregulation of the BDNF pro-domain receptor p75NTR. The clinical relevance of these findings is underlined by a greater than thirty fold increase in the ratio of BDNF pro- to mature-domains in the brains of individuals with AD. This unbalanced BDNF pro:mature-domain ratio in patients represents a possible biomarker of AD and may offer a target for therapeutic intervention.

Effects of the BDNF Val66Met Polymorphism on Anxiety-Like Behavior Following Nicotine Withdrawal in Mice.

Nicotine withdrawal is characterized by both affective and cognitive symptoms. Identifying genetic polymorphisms that could affect the symptoms associated with nicotine withdrawal are important in predicting withdrawal sensitivity and identifying personalized cessation therapies. In the current study we used a mouse model of a non-synonymous single nucleotide polymorphism in the translated region of the brain-derived neurotrophic factor (BDNF) gene that substitutes a valine (Val) for a methionine (Met) amino acid (Val66Met) to examine the relationship between the Val66Met single nucleotide polymorphism and nicotine dependence. This study measured proBDNF and the BDNF prodomain levels following nicotine and nicotine withdrawal and examined a mouse model of a common polymorphism in this protein (BDNF(Met/Met)) in three behavioral paradigms: novelty-induced hypophagia, marble burying, and the open-field test. Using the BDNF knock-in mouse containing the BDNF Val66Met polymorphism we found: (1) blunted anxiety-like behavior in BDNF(Met/Met) mice following withdrawal in three behavioral paradigms: novelty-induced hypophagia, marble burying, and the open-field test; (2) the anxiolytic effects of chronic nicotine are absent in BDNF(Met/Met) mice; and (3) an increase in BDNF prodomain in BDNF(Met/Met) mice following nicotine withdrawal. Our study is the first to examine the effect of the BDNF Val66Met polymorphism on the affective symptoms of withdrawal from nicotine in mice. In these mice, a single-nucleotide polymorphism in the translated region of the BDNF gene can result in a blunted withdrawal, as measured by decreased anxiety-like behavior. The significant increase in the BDNF prodomain in BDNF(Met/Met) mice following nicotine cessation suggests a possible role of this ligand in the circuitry remodeling after withdrawal.

Val66Met polymorphism of BDNF alters prodomain structure to induce neuronal growth cone retraction

A common single-nucleotide polymorphism in the human brain-derived neurotrophic factor (BDNF) gene results in a Val66Met substitution in the BDNF prodomain region. This single-nucleotide polymorphism is associated with alterations in memory and with enhanced risk to develop depression and anxiety disorders in humans. Here we show that the isolated BDNF prodomain is detected in the hippocampus and that it can be secreted from neurons in an activity-dependent manner. Using nuclear magnetic resonance spectroscopy and circular dichroism we find that the prodomain is intrinsically disordered, and the Val66Met substitution induces structural changes. Surprisingly, application of Met66 (but not Val66) BDNF prodomain induces acute growth cone retraction and a decrease in Rac activity in hippocampal neurons. Expression of p75NTR and differential engagement of the Met66 prodomain to the SorCS2 receptor are required for this effect. These results identify the Met66 prodomain as a new active ligand which modulates neuronal morphology.

Neuronal Brain-derived Neurotrophic Factor Is Synthesized in Excess, with Levels Regulated by Sortilin-mediated Trafficking and Lysosomal Degradation

Brain-derived neurotrophic factor (BDNF) regulates neuronal differentiation, synaptic plasticity, and morphology, and modest changes in BDNF levels results in complex behavioral phenotypes. BDNF levels and intracellular localization in neurons are regulated by multiple mechanisms, including use of distinct promoters, mRNA and protein transport, and regulated cleavage of proBDNF to mature BDNF. Sortilin is an intracellular chaperone that binds to the prodomain of BDNF to traffic it to the regulated secretory pathway. However, sortilin binds to numerous ligands and plays a major role in mannose 6-phosphate receptor-independent transport of lysosomal hydrolases utilizing motifs in the intracellular domain that mediate trafficking from the Golgi and late endosomes. Sortilin is modified by ectodomain shedding, although the biological implications of this are not known. Here we demonstrate that ADAM10 is the preferred protease to cleave sortilin in the extracellular stalk region, to release the ligand binding sortilin ectodomain from the transmembrane and cytoplasmic domains. We identify sortilin shedding at the cell surface and in an intracellular compartment. Both sortilin and BDNF are trafficked to and degraded by the lysosome in neurons, and this is dependent upon the sortilin cytoplasmic tail. Indeed, expression of the sortilin ectodomain, which corresponds to the domain released after shedding, impairs lysosomal targeting and degradation of BDNF. These findings characterize the regulation of sortilin shedding and identify a novel mechanism by which sortilin ectodomain shedding acts as a regulatory switch for delivery of BDNF to the secretory pathway or to the lysosome, thus modulating the bioavailability of endogenous BDNF.

Huntingtin-associated Protein-1 Interacts with Pro-brain-derived Neurotrophic Factor and Mediates Its Transport and Release

Brain-derived neurotrophic factor (BDNF) plays a pivotal role in brain development and synaptic plasticity. It is synthesized as a precursor (pro-BDNF), sorted into the secretory pathway, transported along dendrites and axons, and released in an activity-dependent manner. Mutant Huntingtin with expanded polyglutamine (polyQ) and the V66M polymorphism of BDNF reduce the dendritic distribution and axonal transport of BDNF. However, the mechanism underlying this defective transport remains unclear. Here, we report that Huntingtin-associated protein-1 (HAP1) interacts with the prodomain of BDNF and that the interaction was reduced in the presence of polyQ-expanded Huntingtin and BDNF V66M. Consistently, there was reduced coimmunoprecipitation of pro-BDNF with HAP1 in the brain homogenate of Huntington disease. Pro-BDNF distribution in the neuronal processes and its accumulation in the proximal and distal segments of crushed sciatic nerve and the activity-dependent release of pro-BDNF were abolished in HAP1(-/-) mice. These results suggest that HAP1 may participate in axonal transport and activity-dependent release of pro-BDNF by interacting with the BDNF prodomain. Accordingly, the decreased interaction between HAP1 and pro-BDNF in Huntington disease may reduce the release and transport of BDNF.

Differential effects of pro‐BDNF on sensory neurons after sciatic nerve transection in neonatal rats

Brain-derived neurotrophic factor (BDNF) plays a critical role in the development of the central and peripheral nervous systems, and also in neuronal survival after injury. The actions of BDNF are mediated by its high-affinity receptors TrkB and p75NTR. Recent studies have shown that proneurotrophins bind p75NTR and sortilin with high affinity, and trigger apoptosis of neurons in vitro. As proneurotrophins are a dominant form of gene products in developing and adult animals, it is imperative to understand their physiological functions in animals. Here, we showed differential roles of proBDNF in injured and uninjured sensory neurons. proBDNF, p75NTR and sortilin are highly expressed in dorsal root ganglia (DRG) neurons. Recombinant proBDNF induced a dose-dependent death of PC12 cells and the death activity was completely abolished in the presence of antibodies against the prodomain of BDNF. The exogenous proBDNF enhanced the death of axotomized sensory neurons and the neutralizing antibodies to the prodomain or exogenous sortilin-extracellular domain-Fc fusion molecule reduced the death of axotomized sensory neurons. Interestingly, the treatment of neutralizing antibody in vivo increased the number of sensory neurons in the contralateral DRG. We conclude that proBDNF may induce the death of axotomized sensory neurons and suppress neuronal addition in the intact DRG in neonatal rats, and the suppression of endogenous proBDNF may protect neurons after neurotrauma.

Variant Brain-Derived Neurotrophic Factor (Val66Met) Alters Adult Olfactory Bulb Neurogenesis and Spontaneous Olfactory Discrimination

Neurogenesis, the division, migration, and differentiation of new neurons, occurs throughout life. Brain derived neurotrophic factor (BDNF) has been identified as a potential signaling molecule regulating neurogenesis in the subventricular zone (SVZ), but its functional consequences in vivo have not been well defined. We report marked and unexpected deficits in survival but not proliferation of newly born cells of adult knock-in mice containing a variant form of BDNF [a valine (Val) to methionine (Met) substitution at position 66 in the prodomain of BDNF (Val66Met)], a genetic mutation shown to lead to a selective impairment in activity-dependent BDNF secretion. Utilizing knock-out mouse lines, we identified BDNF and tyrosine receptor kinase B (TrkB) as the critical molecules for the observed impairments in neurogenesis, with p75 knock-out mice showing no effect on cell proliferation or survival. We then localized the activated form of TrkB to a discrete population of cells, type A migrating neuroblasts, and demonstrate a decrease in TrkB phosphorylation in the SVZ of Val66Met mutant mice. With these findings, we identify TrkB signaling, potentially through activity dependent release of BDNF, as a critical step in the survival of migrating neuroblasts. Utilizing a behavioral task shown to be sensitive to disruptions in olfactory bulb neurogenesis, we identified specific impairments in spontaneous olfactory discrimination, but not general olfactory sensitivity or habituation to olfactory stimuli in BDNF mutant mice. Through these observations, we have identified novel links between genetic variant BDNF and adult neurogenesis in vivo, which may contribute to significant impairments in olfactory function.

Aminoglycoside-Induced Degeneration of Adult Spiral Ganglion Neurons Involves Differential Modulation of Tyrosine Kinase B and p75 Neurotrophin Receptor Signaling

Aminoglycoside antibiotics induce sensorineural hearing loss by destroying hair cells of the organ of Corti, causing progressive secondary degeneration of primary auditory or spiral ganglion neurons (SGNs). Recent studies show that the p75 neurotrophin receptor (NTR) is aberrantly up-regulated under pathological conditions when the neurotrophin receptor tyrosine kinases (Trks) are presumptively down-regulated. We provide in vivo evidence demonstrating that degenerating SGNs induced an augmented p75NTR expression and a coincident reduction of TrkB expression in their peripheral processes. Nuclear transcription factors c-Jun and cyclic AMP response element-binding protein phosphorylated by p75NTR- and TrkB-activated signal pathways, respectively, also showed a corresponding differential modulation, suggesting an activation of apoptotic pathways, coupled to a loss of pro-survival neurotrophic support. Our findings identified brain-derived neurotrophic factor (BDNF) expression in hair and supporting cells of the adult cochlea, and its loss, specifically the mature form, would impair TrkB-induced signaling. The precursor of BDNF (pro-BDNF) is differentially cleaved in aminoglycoside-deafened cochleae, resulting in a predominant up-regulation of a truncated form of pro-BDNF, which colocalized with p75NTR-expressing SGN fibers. Together, these data suggest that an antagonistic interplay of p75NTR and TrkB receptor signaling, possibly modulated by selective BDNF processing, mediates SGN death in vivo.

Variant brain-derived neurotrophic factor (BDNF) (Met66) alters the intracellular trafficking and activity-dependent secretion of wild-type BDNF in neurosecretory cells and cortical neurons.

Brain-derived neurotrophic factor (BDNF) plays a critical role in nervous system and cardiovascular development and function. Recently, a common single nucleotide polymorphism in the bdnf gene, resulting in a valine to methionine substitution in the prodomain (BDNF(Met)), has been shown to lead to memory impairment and susceptibility to neuropsychiatric disorders in humans heterozygous for the variant BDNF. When expressed by itself in hippocampal neurons, less BDNF(Met) is secreted in an activity-dependent manner. The nature of the cellular defect when both BDNF(Met) and wild-type BDNF (BDNF(Val)) are present in the same cell is not known. Given that this is the predominant expression profile in humans, we examined the effect of coexpressed BDNF(Met) on BDNF(Val) intracellular trafficking and processing. Our data indicate that abnormal trafficking of BDNF(Met) occurred only in neuronal and neurosecretory cells and that BDNF(Met) could alter the intracellular distribution and activity-dependent secretion of BDNF(Val). We determined that, when coexpressed in the same cell, approximately 70% of the variant BDNF forms BDNF(Val).BDNF(Met) heterodimers, which are inefficiently sorted into secretory granules resulting in a quantitative decreased secretion. Finally, we determined the form of BDNF secreted in an activity-dependent manner and observed no differences in the forms of BDNF(Met) or the BDNF(Val).BDNF(Met) heterodimer compared with BDNF(Val). Together, these findings indicate that components of the regulated secretory machinery interacts specifically with a signal in the BDNF prodomain and that perturbations in BDNF trafficking may lead to selective impairment in CNS function.