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BDNF – Second Best?

By Alon Meir, Ph.D.

Brain Derived Neurotrophic Factor (BDNF) is the second best characterized neurotrophin (following NGF) and acts as a key contributor to neuronal development as well as neuronal plasticity. BDNF is a homo-dimer protein which is secreted to affect cells that express the BDNF receptors – TrkB and p75NTR. Each receptor mediates a different signaling cascade resulting in different physiological outputs. The vast research and knowledge concerning BDNF, includes studies exploring neuronal populations and mechanisms responsible for protein synthesis and secretion. In addition, a great effort has been made in characterizing target neuronal populations and the mechanisms resulting from BDNF binding. In this report we describe recent use of Alomone Labs’ Recombinant human BDNF protein, Anti-BDNF Antibody and signaling modulators and their contributions to the growing understanding regarding the role played by BDNF in physiology and disease.

Introduction

The BDNF protein complex is a large intercellular signaling molecule, which is synthesized, and exocytosed from a defined population of neurons in order to affect neighboring and remote cells. The affected cells respond in a variety of signaling cascades at the molecular level. The complexity of the signaling system affected by neurotrophins in general and BDNF in particular, arises also from the notion that the precursor protein (proBDNF) is also a signaling mediator. In addition, the TrkB and p75NTR receptors might be stimulated by more than one neurotrophin. Cellular signaling, following BDNF binding, may lead to many physiological outcomes at the cellular and systemic levels. These include, synapse formation and modulation of synaptic elements during neuronal development, injury, repair or plasticity associated with sleep control, pain perception, learning and memory, as well as with related diseases. BDNF binding may also lead to the inhibition of neuronal apoptosis, which might be involved in the control of neuronal death and injury repair, strongly associated with neuro degenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s diseases (for review see reference 31).

The BDNF effects on cell migration are involved in embryonic development of the nervous system as well as in malignant brain diseases.

Perhaps due to a combination of all or some of the actions mentioned above, BDNF is, for some cellular populations, an agent promoting neuronal differentiation and survival both in vivo and in culture.

BDNF Processing, Secretion and Receptor Binding

BDNF is expressed in neurons and its synthesis and secretion are highly regulated (for review see, reference 15).

In order to study the differential vesicular targeting and time course of synaptic secretion of mammalian BDNF in cultured hippocampal neurons3, Recombinant human BDNF protein (#B-250) was used to compare its activity with that of expressed GFP-BDNF. Heterologously expressed GFP-BDNF behaved similarly to Recombinant human BDNF protein-treated cells.

In a study initiated to explore the involvement of BDNF polymorphism in Huntington’s disease, Anti-BDNF Antibody (#ANT-010) was used to investigate intracellular trafficking and release of mutant BDNF9. In this study it was demonstrated that the gene mutated in Huntington’s disease (htt), differentially affects the trafficking and release of two BDNF polymorphs, and as a consequence, differentially affects cell survival, depending on the BDNF polymorph9 (Figure 1).

As mentioned above, following its release to the extracellular space, BDNF binds to TrkB and to a lesser extent p75NTR on the cell surface membrane6. It was demonstrated, using K252a (#K-150) (a known kinase inhibitor, which also inhibits TrkB), that BDNF exerts its effect by directly activating TrkB17,19,36,2. Accordingly, in a different study in hippocampal neurons, the role of a truncated form of TrkB receptor, TrkB.T1, was studied. It was demonstrated that the truncated receptor is constitutively active and the induction of filopodia, is independent of BDNF binding18.

Figure 1. Mutant Huntington’s (mhtt) Reduces the Number of Val-BDNF but not MetBDNF Vesicles.
A) BDNF-containing vesicles were counted as follows: each stack of images was thresholded, and Anti-BDNF Antibody (#ANT-010) staining was marked in red. B) All of the marked spots comprising 2–10 pixels were counted, showing a reduction in Val-BDNF-containing vesicles in mhtt cells compared with wild type cells. C) Colocalization between Val-BDNF or Met-BDNF and secretogranin II (SecII). D) Val-BDNF in mhtt cells showed a reduced colocalization with secretogranin II compared with wild type cells.
Adapted from reference 9 with permission of The Society for Neuroscience.

Physiological Outcomes of Cellular Activation by BDNF Signaling

The physiological effects of elevated BDNF levels are clearly demonstrated by in vivo injections, followed by behavioral as well as other functional examinations of the injected animals. Such studies, suggest that BDNF plays a role in processes ranging from neuroprotection to memory storage.

It was demonstrated that BDNF has a protective role in induced neonatal excitotoxicity, by inhibiting apoptotic pathways, when injected in vivo. This protective effect was dependent on the activation and type of glutamate receptor, on lesion localization and on the developmental stage21 (Figure 2).

Depression could be associated with reduced expression of BDNF in the hippocampus. In order to validate the reduced expression of BDNF in specific brain regions following shRNA, Anti-BDNF was used in in vivo studies43. It was concluded that knockdown of BDNF in specific brain regions brings on behaviors associated with depression and reduced neurogenesis43 (Figure 3).

In in vivo cortical unilateral microinjections of BDNF, EEG sleep-related parameters in rats are affected, suggesting that BDNF may also have a role in the homeostatic regulation of sleep12. In addition, intra-hippocampal delivery of BDNF reverses the deficit in memory persistence caused by inhibition of hippocampal protein synthesis. Thus, BDNF is necessary and sufficient to induce a late post-acquisition phase in the hippocampus, essential for persistence of long term memory storage1 (Figure 4).

Figure 2. Effects of Intracerebral BDNF on Cortical Gray Matter, DNA Fragmentation and Caspase 3 Activation Induced by Ibotenate Injection on P5.
Analyses were performed on P6. Tunel-stained sections showing DNA fragmentation induced by ibotenate alone (A) or Recombinant human BDNF protein (#B-250) labeled with ibotenate (B). C) Quantitative analysis of Tunel-stained nuclei in the cortical gray matter. Cleaved caspase 3 immunostained sections showing caspase 3 activation induced by ibotenate alone (D) or ibotenate-BDNF (E). F) Quantitative analysis of cleaved caspase 3-immunolabeled cells in the cortical gray matter.
Adapted from reference 21 with permission of Oxford University Press.
Figure 3. Validation of LV-shBDNF Infection and BDNF Knockdown in vitro and in vivo
BDNF protein expression using Anti-BDNF Antibody (#ANT-010) was measured in C6 rat glioma cell line and in vivo in response to infection with lentiviral vector (LV) expressing either green fluorescent protein (GFP) only (LV-GFP; infection control), scrambled shRNA (LV-shSCR; shRNA control) or shRNA complementary to the coding exon of the rat BDNF gene (LV-shBDNF; active sequence). A) BDNF levels measured in a C6 cell medium 48 h after infection. Data are presented as percentages of BDNF secreted from non-infected control cells. B) BDNF levels in the dentate gyrus (DG) measured by immunohistochemistry and epifluorescence microscopy. C) BDNF levels in the dDG measured by ELISA and normalized per total protein. D) Representative micrographs of hippocampal slices including the dDG from rats injected with LV-shSCR (control) or LV-shBDNF (BDNF KD) (upper panels). The micrographs in the left represent the infection spread based on GFP expression (green) within the sections. BDNF protein expression was visualized by immunohistochemical staining (middle panels). Note the significant reduction in BDNF expression induced by the LV-shBDNF microinjection. Cell nuclei were visualized using Hoechst staining (lower panels). The micrographs on the right show a higher magnification of an infected hippocampal region. The upper panels shows GFP expression as an infection marker, whereas the middle panels shows BDNF protein staining. The lower panels shows superimposition of the upper and middle panels to highlight cells expressing both GFP and BDNF. The arrows highlight that while the rat brain infected with LV-shSCR shows BDNF expression within infected (GFP) cells, none of the infected cells in the LV-shBDNF rat brain show BDNF expression.
Adapted from reference 43 with permission of Macmillan Publishers Ltd.
Figure 4. BDNF is Necessary and Sufficient for Memory Persistence.
A) Recombinant human BDNF protein (#B-250) rescues the impairment in memory persistence caused by inhibition of protein synthesis. Anisomycin caused amnesia 7 days after training when infused 12 h after learning (dark gray). This effect was reversed by infusion of Recombinant human BDNF protein (0.25 μg per side) 15 min later (light gray). B) Intrahippocampal infusion of BDNF antisense oligonucleotide (BDNF ASO) late after IA training blocks memory retention at 7, but not at 2 days after training. BDNF ASO, but not BDNF missense oligonucleotide (BDNF MSO) infusion, 10 h after training hinders memory persistence at 7 days but leaves memory intact 2 days after training. The dorsal hippocampus of the animals was infused with BDNF MSO (white bars) or BDNF ASO (gray bars) 10 h after training.
Adapted from reference 1 with permission of The National Academy of Sciences of the USA (Copyright 2008).

BDNF and Neuronal Survival

In general, the cell signaling pathways activated by BDNF mostly include that of PI3K-PKB, and in parallel, the activation ERK1/2 MAPK (which could be blocked by K252a)8 leading to cell survival. The activation of the cell survival pathway also leads to the inhibition of the pro-apoptotic kinase, GSK3β.

In a central nervous system trauma model, using adult rat retinal ganglion cells, it was observed that in vivo injection of BDNF enhances neuronal survival by activating the PI3K-PKB pathway, which suppresses apoptosis by inhibiting caspase 328. In cortical neurons, as well as in hippocampal neurons, BDNF was shown to prevent apoptosis and promote survival23 by activating PDK1, ERK1/2 and RSK1/227. In this experiment BDNF was used to stimulate neurons, while several parameters related to the downstream signaling cascades were measured. For instance, the effect and time course of BDNF stimulation on the phosphorylation state of RSK was measured by western blot detection of the phosphorylated versus the non phosphorylated protein. BDNF was found to increase the phosphorylation of RSK27 (Figure 5).

BDNF promotes cell survival in chicken motor neuron culture, via the activation of PKB which is dependent on Ca2+, CaM, membrane depolarization and Calmodulin Kinase IV11,24. In rat and mouse spinal motor neurons, neuronal death elicited in vitro by excitotoxic insult, or the expression of mutant SOD1, or polyQ-expanded androgen receptor was abrogated by the expression of nuclear-targeted FOXO3a transcription factor. The downstream effectors of this pathway are not clear but, BDNF which caused a reduction in FOXO3a phosphorylation did not protect against mutant SOD toxicity38.

Figure 5. PDK1-Mediated Activation of RSK1/2 in BDNF-Stimulated Neurons.
Neurons were stimulated with 10 ng/ml Recombinant human BDNF protein (#B-250) as indicated. A) BDNF increased PDK1- mediated phosphorylation of RSK1/2. B) The BDNF-mediated increase of pSer221/227 required activities of the ERK1/2 pathway and PDK1. Although in neurons treated with either the vehicle (Veh) or the PI3K inhibitor LY294002 (LY), BDNF treatment elevated pSer221/227, the MKK1/2 inhibitor U0126 or the PDK1 pathway inhibitor TPCK blocked that effect.
Adapted from reference 27 with permission of The Society for Neuroscience.

BDNF Effects on Gene Expression

BDNF was shown to affect gene expression either by measuring transcription factor activity or by directly measuring mRNA or protein levels. Such alterations in gene expression may establish the basis of neuronal survival, memory storage and pain perception in response to BDNF. Presented is a list of publications showing usage of Recombinant human BDNF protein and its downstream effects on gene expression.

In cultured neocortical neurons, BDNF-induced CREB phosphorylation/activation which is dependent on MSK1 (Mitogen- and Stress Activated Protein Kinase) and mediated by ERK1/2-activated kinase, was absent in cells deficient for MSK141. In rat cerebral cortical neurons, BDNF (and other neurotrophins) activate PARP-1 to control gene expression45. In developing cortical neurons, BDNF increases glucose utilization and the expression of GLUT34, but does not lead to GLUT8 surface expression in PC12 cells and hippocampal neurons47. In cultured rat hippocampal neurons, BDNF activates NFAT (Nuclear Factor of Activated T-Cells) dependent transcription via TrkB which is inhibited upon K252a treatment and triggers the rapid nuclear translocation of NFATc417. In cultured DRG neurons, axonal levels of the KV3.1a K+ channel mRNA were decreased by NGF and BDNF, while NT-3 increased its expression48. In another experiment in DRG neurons, it was demonstrated that BDNF affected the expression of Damage-Induced Neuronal Endopeptidase (DINE/ECEL) in DRG neurons26.

BDNF Effects on Morphological Neuro- and Synapto-Genesis

One of the main activities attributed to BDNF as a neurotrophic factor is neuritis and synapse formation, during embryonic and postnatal development as well as in adult plasticity, such as memory storage. In order to demonstrate its role in synapse formation, Recombinant human BDNF protein was injected in vivo in rat brain to initiate re-innervations in the cerebellum. Following Recombinant human BDNF protein injection, VGLUT2-positive climbing fibers (a synaptic marker) were found to contact somatic thorns during late postnatal re-innervations of Purkinje cells30. In a different study it was demonstrated that BDNF promotes growth of filopodia in dendrites and in parallel also destabilizes the spines, each of these, mediated by a different signaling cascade. These different pathways were assessed by selectively blocking key components of the pathway, after BDNF stimulation and examining the effects using microscopy to visualize the dendrites morphology as well as with western blot to measure phosphorylation of key enzymes29 (Figure 6). In addition, it was demonstrated that, signaling by BDNF regulates axon morphogenesis and branching, through β-catenin phosphorylation7. In nonpyramidal neocortical interneurons, in developing organotypic cultures, BDNF mediates depolarization-induced dendritic growth and branching22. In chick DRG neurons, it was demonstrated that the process of neurite outgrowth is initiated by BDNF and is dependent on signaling cations such as zinc40. As mentioned above, BDNF affects many parameters of neuronal growth, proper function and morphology. However, it has no effect on myelination in mouse brain cultures42.

Figure 6. BDNF Promotes Growth of Filopodia and Destabilizes Dendritic Spines, and Short-Term Inhibition of PI3K-Akt-mTOR and Promotes Spine Development.
The micrographs depict close-up views of 15 days in vitro (DIV) dendritic protrusions from hippocampal neurons transfected with GFP on 6 DIV and treated with Recombinant human BDNF protein (#B-250) alone or with drugs for 24 h starting on 14 DIV. GFP control (A), Recombinant human BDNF protein (20 ng/ml) (B), and coapplication of Recombinant human BDNF protein with K252a (200 nM) (C), LY294002 (D; LY), wortmannin (E; Wort), U0126 (F; U0), LY294002 plus U0126 (G), and rapamycin (H; Rap). I) and J) show mean for spine and filopodia densities, respectively. K), L) Western blot analysis to ascertain the efficacy and specificity of treatment with Recombinant human BDNF protein and pharmacological inhibitors for the above morphological experiments. Sister coverslips were harvested and probed for pAkt, pMAPK, and pS6. A representative experiment is shown (K). L) Quantifications of the western blots from three independent experiments.
Adapted from reference 29 with permission of The Society for Neuroscience.

BDNF Effects on Synaptic Transmission

In addition to the observed morphological changes, BDNF directly affects the activity of certain synapses, leading to changes in brain activity and behavior. In general, BDNF inhibits inhibitory synapses and enhances excitatory synapses, leading to increased overall activity. For example, BDNF increases spontaneous network activity in the superficial layers of the mouse superior colliculus by suppressing GABAergic inhibition by the acute downregulation of GABA (A) Receptors, through PKC activation in the postsynaptic cell via TrkB. These effects, were induced by BDNF in Bdnf-/- mice19. BDNF-induced changes in GABA (A) Receptor phosphorylation may provide a dynamic mechanism for modulating the efficacy of fast synaptic inhibition and thereby, neuronal excitability in the brain. In cultured cortical and hippocampal neurons, the mechanisms underlying the BDNF dependent modulation of GABAergic synaptic transmission was studied in detail. Results show that BDNF modulates both GABAergic synaptic currents and GABA (A) Receptor-β3 subunit phosphorylation by selectively targeting PKC, RACK-1, and protein phosphatase 2A (PP2A) to these receptors25.

BDNF modulates both postsynaptic and presynaptic NMDA Receptors to enhance transmission in brain slices. Such effects are different depending on the brain regions35. In distal basal dendritic regions of neocortical pyramidal neurons, pairing of NMDA spikes and BDNF is necessary for LTP induction14.

In the rat superficial spinal dorsal horn organotypic culture, BDNF differentially affects inhibitory and excitatory synapses, contributing to the overall excitability and central sensitization related to pain sensation33 (Figure 7). Like in the chronic constriction injury (CCI) model of chronic pain, BDNF mimics the effect of activated microglia in reducing inhibitory drive and enhancing excitatory drive34.

Figure 7. Enhanced K+ -Induced Ca2+ Rise in BDNF-Treated Organotypic Culture Slices.
A), B) Sample fluorescent Ca2+ intensity traces during a 90 sec, 20 mM K+ challenge for a cell in control (A), and a Recombinant human BDNF protein (#B-250)-treated organotypic culture slice (B). C), D) Sample fluorescence images of the dorsal region of a control slice (C) and Recombinant human BDNF protein-treated organotypic culture slice (D), loaded with Fluo-4 AM, during a 20 mM K+ solution challenge. E) Comparison of Ca2+ fluorescence signal amplitude over a range of high K+ solutions tested. F) Comparison of area under the Ca2+ fluorescence signal traces over a range of high K+ solutions in control and Recombinant human BDNF protein-treated organotypic culture slices. At all concentrations tested, both area and amplitude of the Ca2+ signal were significantly larger in Recombinant human BDNF protein-treated cells. G), H) Sample baseline recordings from four cells in the same control organotypic culture slice (G) and a Recombinant human BDNF protein-treated organotypic culture slice (H). Dots with dashed lines indicate synchronous oscillations in Ca2+.
Adapted from reference 33 with permission of Blackwell Publishing Ltd.

BDNF Effects on Differentiation and Proliferation

Probably due to a combination of many of the factors mentioned above, BDNF is also considered as a general agent that promotes, in certain cellular populations, neuro-differentiation and proliferation. Below, we list several recent papers in which BDNF was used to maintain neuronal cultures.

Recombinant human BDNF protein is used to differentiate SH-SY5Y cells into neuron like cells10 and does so by ultimately upregulating Bcl-2 which leads to the inhibition of apoptosis46 (Figure 8). Hypoxia-inducible Factor-1 (HIF-1) is a transcriptional activator activated under hypoxic conditions and involved in TRKB gene transcription. Enhanced expression of TrkB could represent a critical switch for the dedifferentiation of neuroblastoma cells under hypoxic conditions. Cell migration under hypoxia and normal O2 levels was reduced significantly in the absence of BDNF36.

In hippocampal granule neurons, BDNF regulation of proliferation and differentiation is mediated by the SMAD pathway32. In cultured septal cholinergic neurons, noradrenaline (NA) rescues cells from degeneration caused by low-level oxidative stress. Neither NGF nor BDNF alone, was able to mimic the protective action of NA, although when combined, both neurotrophins seemed dependent on the presence of the neurotransmitter to reinforce the expression of the cholinergic phenotype44.

BDNF supports survival of cortical projection neurons5, GABAergic cerebellar interneurons13 and motor neuron cultures37, as well as postmortem brain tissue maintained in culture medium39. Recombinant human BDNF protein could also be used to maintain sensory neurons and promote explant geniculation16.

Figure 8. The Dedifferentiated SH-SY5Y-N Cells are Resistant to CamptothecinInduced Cell Death.
A) SH-SY5Y cells were differentiated by sequential treatment with retinoic acid (RA) and Recombinant human BDNF protein (#B-250). The undifferentiated SH-SY5Y (filled circle) and the differentiated SH-SY5Y-N cells (empty circle) were treated for 16 h with various doses of camptothecin (B), etoposide (D), and cisplatin (E) or 30 ng/ ml camptothecin over a time course (C). Cell viability was analyzed by the trypan blue exclusion assay.
Adapted from reference 46 with permission of Macmillan Publishers Ltd.

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