Brain-derived neurotrophic factor is an anterograde survival factor in the rat visual system

BACKGROUND: The neurotrophins, which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5 and NT-6, are a family of proteins that play fundamental roles in the differentiation, survival and maintenance of peripheral and central neurons. Much research has focused on the role of neurotrophins as target-derived, retrogradely transported trophic molecules. Although there is recent evidence that BDNF and NT-3 can be transported in an anterograde direction along peripheral and central axons, there is as yet no conclusive evidence that these anterograde factors have direct post-synaptic actions. RESULTS: We report that BDNF travels in an anterograde direction along the optic nerve. The anterogradely transported BDNF had rapid effects on retinal target neurons in the superior colliculus and lateral geniculate nucleus of the brain. When endogenous BDNF within the developing superior colliculus was neutralised, the rate of programmed neuronal death increased. Conversely, provision of an afferent supply of BDNF prevented the degeneration of geniculate neurons after removal of their cortical target. CONCLUSIONS: BDNF released from retinal ganglion cells acts as a survival factor for post-synaptic neurons in retinal target fields.     

Expression of neurotrophins and their receptors in peripheral lung cells of mice

Neurotrophins play an essential role in nerve systems. Recent reports indicated that neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5)] have numerous effects on non-neural cells, especially on immune cells. However, whether lung cells express neurotrophins and/or their receptors (TrkA for NGF, TrkB for BDNF and NT-4/5, and TrkC for NT-3) has never been systematically investigated. We investigated constitutive expression of neurotrophin family and their Trk receptor family in alveolar macrophages and other peripheral lung cells of mice. New findings were: (1) RT-PCR for neurotrophins and their receptors detected NT-3 and NT-4/5 in alveolar macrophages, BDNF, NT-4/5, trkA, the truncated form of trkB, and trkC in lung homogenate, but no trks in alveolar macrophages, (2) immunohistochemistry for neurotrophin receptors detected TrkA in capillary cells, the truncated form of TrkB, and TrkC in interstitial macrophages, (3) immunoelectron microscopy for TrkC revealed expression of TrkC on the surface of interstitial macrophages, and (4) in situ hybridization for neurotrophins detected BDNF in interstitial macrophages and alveolar type I cells, NT-3 in alveolar macrophages, and NT-4/5 in alveolar and interstitial macrophages. These findings indicate that a previously unknown signal trafficking occurs through neurotrophins in peripheral lung.     

Immunohistochemical distribution of NGF, BDNF, NT-3, and NT-4 in adult rhesus monkey brains.

Immunohistochemical distribution and cellular localization of neurotrophins was investigated in adult monkey brains using antisera against nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Western blot analysis showed that each antibody specifically recognized appropriate bands of approximately 14.7 kDa, 14.2 kDa, 13.6 kDa, and 14.5 kDa, for NGF, BDNF, NT-3, and NT-4, respectively. These positions coincided with the molecular masses of the neurotrophins studied. Furthermore, sections exposed to primary antiserum preadsorbed with full-length NGF, BDNF, NT-3, and NT-4 exhibited no detectable immunoreactivity, demonstrating specificities of the antibodies against the tissues prepared from rhesus monkeys. The study provided a systematic report on the distribution of NGF, BDNF, NT-3, and NT-4 in the monkey brain. Varying intensity of immunostaining was observed in the somata and processes of a wide variety of neurons and glial cells in the cerebrum, cerebellum, hippocampus, and other regions of the brain. Neurons in some regions such as the cerebral cortex and the hippocampus, which stained for neurotrophins, also expressed neurotrophic factor mRNA. In some other brain regions, there was discrepancy of protein distribution and mRNA expression reported previously, indicating a retrograde or anterograde action mode of neurotrophins. Results of this study provide a morphological basis for the elucidation of the roles of NGF, BDNF, NT-3, and NT-4 in adult primate brains.     

Changes in neurotrophin responsiveness during the development of cerebellar granule neurons.

Neurotrophins and their receptors are widespread in the developing and mature CNS. Identifying the differentiation state of neurotrophin-responsive cells provides a basis for understanding the developmental functions of these factors. Studies using dissociated and organotypic cultures of rat cerebellum demonstrated that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) affect developing granule cells at distinct stages in differentiation. While early granule neurons in the external germinal layer responded to BDNF, more mature granule cells responded to NT-3. BDNF, but not NT-3, enhanced survival of granule cells in cultures of embryonic cerebella. Thus, BDNF and NT-3 have distinct sequential functions that are likely to be critical in the development of the cerebellum. BDNF may promote the initial commitment, while NT-3 may direct the subsequent maturation of granule cells.     

Differential Regulation of Hippocampal Neurotrophins During Aging in Rats

Abstract: Neurotrophins are a family of neurotrophic factors with considerable structural homology. We used sensitive and specific two-site enzyme immunoassays to assess age-associated changes in levels of three neurotrophins—nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3)—in the hippocampus of Fischer 344 rats. Expressions of these proteins and their mRNAs were compared in the same animals. More than 200 ng of BDNF per gram of tissue was detected in the hippocampus of 2-month-old rats. This amount was two and 100 times greater than that of NT-3 and NGF, respectively. The levels of BDNF and NT-3 increased further 2–6 months after birth, whereas NGF content declined during this period, and the altered protein levels of all three neurotrophins were maintained 6–18 months postnatally. In contrast to the patterns of protein expression, BDNF mRNA levels increased during both of these periods, and the NT-3 mRNA levels appeared to decline. Changes in the expression of BDNF mRNA and NGF protein were opposite to those reported to occur in Alzheimer's disease. These results suggest that, during normal aging in rats, neurotrophin expression is regulated independently at both the mRNA and posttranslational levels. Any deficiency in their regulation might contribute to neurodegenerative disorders.     

Cutting edge: clonally restricted production of the neurotrophins brain-derived neurotrophic factor and neurotrophin-3 mRNA by human immune cells and Th1/Th2-polarized expression of their receptors.

Neurotrophins, such as neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), are potent regulators of neuronal functions. Here we show that human immune cells also produce NT-3 mRNA, secrete BDNF, and express their specific receptors trkB and trkC. The truncated trkB receptor, usually expressed in sensory neurons of the central nervous system, was also constitutively expressed in unstimulated Th cells. Full-length trkB was detectable in stimulated PBMC, B cell lines, and Th1, but not in Th2 and Th0 cell clones. Clonally restricted expression was also observed for trkC, until now not detected on blood cells. The Th1 cytokine IL-2 stimulated production of trkB mRNA but not of trkC, whereas the Th2 cytokine IL-4 enhanced NT-3 but not BDNF mRNA expression. Microbial Ags, which influence the Th1/Th2 balance, could therefore modulate the neurotrophic system and thereby affect neuronal synaptic activity of the central nervous system.     

Analysis of neurotrophins in human serum by immunoaffinity capillary electrophoresis (ICE) following traumatic head injury

Neurotrophins, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and β-nerve growth factor (β-NGF), play an active role in the development, maintenance and survival of cells of the central nervous system (CNS). Previous research has indicated that a decrease in concentrations of these neurotrophins is often associated with cell death and ultimately patient demise. However, much of the research conducted analyses of samples taken directly from the CNS, i.e., of samples that are not readily available in clinical trauma centers. In an attempt to obtain a method for evaluating neurotrophins in a more readily accessible matrix, i.e., serum, a precise and accurate immunoaffinity capillary electrophoresis (ICE) method was developed and applied to measure neurotrophins in serum from patients with various degrees of head injury. The five neurotrophins of interest were extracted and concentrated by specific immunochemically immobilized antibodies, bound directly to the capillary wall, and eluted and separated in approximately 10 min. NT-3, BDNF, CNTF and β-NGF showed a marked decrease in concentration as the severity of the head injury increased: mild versus severe: 91% decrease for NT-3; 93 % decrease for BDNF; 93 % decrease for CNTF; and a 87 % decrease for β-NGF. This decrease in concentration is consistent with the neuro-protective roles that neurotrophins play in the maintenance and survival of neuronal cells. The results obtained by the ICE method were closely comparable with those generated by a commercially available ELISA method.     

Characterization of the Responses of Purkinje Cells to Neurotrophin Treatment

Abstract: The ability of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) to promote neuronal survival and phenotypic differentiation was examined in dissociated cultures from embryonic day 16 rat cerebellum. BDNF treatment increased the survival of neuron-specific enolase-immunopositive cells by 250 and 400% after 8 and 10 days in culture, respectively. A subpopulation of these neurons, the Purkinje cells, identified by calbindin staining, was increased to an equivalent extent, ∼200%, following BDNF, NT-4/5, or NT-3 treatment. The number of GABAergic neurons, identified by GABA immunoreactivity, was greatly increased by treatment with BDNF (470%) and moderately by NT-4/5 (46%), whereas NT-3 was without effect. NGF failed to increase the number of either Purkinje cells or GABAergic neurons. Addition of BDNF within 48 h of cell plating was required to obtain a maximal increase in Purkinje cell number after 8 days. In contrast, the NT-3 responses were nearly equivalent even if treatment was delayed for 96 h after plating. BDNF, NT-4/5, and NT-3, but not NGF, induced the rapid expression of the immediate early gene c- fos . Immunocytochemical double-labeling with antibodies to c-fos and calbindin was used to identify Purkinje cells that responded to neurotrophin treatment by induction of c-fos. After 4 days in vitro, both BDNF and NT-3 induced the formation of c-fos protein in calbindin-immunopositive neurons, whereas NT-4/5 did not. The latter results suggest that although BDNF and NT-4/5 have been shown to act through a common receptor, TrkB, it appears that the effects of BDNF and NT-4/5 are not identical.     

The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons.

The pattern of retrograde axonal transport of the target-derived neurotrophic molecule, nerve growth factor (NGF), correlates with its trophic actions in adult neurons. We have determined that the NGF-related neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are also retrogradely transported by distinct populations of peripheral and central nervous system neurons in the adult. All three 125I-labeled neurotrophins are retrogradely transported to sites previously shown to contain neurotrophin-responsive neurons as assessed in vitro, such as dorsal root ganglion and basal forebrain neurons. The patterns of transport also indicate the existence of neuronal populations that selectively transport NT-3 and/or BDNF, but not NGF, such as spinal cord motor neurons, neurons in the entorhinal cortex, thalamus, and neurons within the hippocampus itself. Our observations suggest that neurotrophins are transported by overlapping as well as distinct populations of neurons when injected into a given target field. Retrograde transport may thus be predictive of neuronal types selectively responsive to either BDNF or NT-3 in the adult, as first demonstrated for NGF.     

TH and NPY in sympathetic neurovascular cultures: role of LIF and NT-3

The sympathetic nervous system is an important determinant of vascular function. The effects of the sympathetic nervous system are mediated via release of neurotransmitters and neuropeptides from postganglionic sympathetic neurons. The present study tests the hypothesis that vascular smooth muscle cells (VSM) maintain adrenergic neurotransmitter/neuropeptide expression in the postganglionic sympathetic neurons that innervate them. The effects of rat aortic and tail artery VSM (AVSM and TAVSM, respectively) on neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were assessed in cultures of dissociated sympathetic neurons. AVSM decreased TH (39 +/- 12% of control) but did not affect NPY. TAVSM decreased TH (76 +/- 10% of control) but increased NPY (153 +/- 20% of control). VSM expressed leukemia inhibitory factor (LIF) and neurotrophin-3 (NT-3), which are known to modulate NPY and TH expression. Sympathetic neurons innervating blood vessels expressed LIF and NT-3 receptors. Inhibition of LIF inhibited the effect of AVSM on TH. Inhibition of neurotrophin-3 (NT-3) decreased TH and NPY in neurons grown in the presence of TAVSM. These data suggest that vascular-derived LIF decreases TH and vascular-derived NT-3 increases or maintains NPY and TH expression in postganglionic sympathetic neurons. NPY and TH in vascular sympathetic nerves are likely to modulate NPY and/or norepinephrine release from these nerves and are thus likely to affect blood flow and blood pressure. The present studies suggest a novel mechanism whereby VSM would modulate sympathetic control of vascular function.     

Expression of neurotrophin receptors trkB and trkC and their ligands in rat adrenal gland and the intermediolateral column of the spinal cord

Neurotrophins and their trk receptors constitute major classes of signaling molecules with important actions in the developing and adult nervous system. With regard to the sympathoadrenal cell lineage, which gives rise to sympathetic neurons and chromaffin cells, neurotrophin-3 (NT-3) and nerve growth factor (NGF) are thought to influence developing sympathetic neurons. Neurotrophin requirements of chromaffin cells of the adrenal medulla are less well understood than those for NGF. In order to provide the bases for understanding of putative functions of neurotrophins for the development and maintenance of chromaffin cells and their preganglionic innervation, in situ hybridization has been used to study the expression of brain-derived neurotrophic factor (BDNF) and NT-3, together with their cognate receptors trkB and trkC, in the adrenal gland and in the intermediolateral column (IML) of the spinal cord. BDNF is highly expressed in the embryonic adrenal cortex and later in cells of the cortical reticularis zone. Adrenal medullary chromaffin cells fail to express detectable levels of mRNAs for BDNF, NT-3, and their cognate receptors trkB and trkC. Neurons in the IML express BDNF and trkB, and low levels of NT-3 and trkC. Our data make it unlikely that BDNF and NT-3 serve as retrograde trophic factors for IML neurons but suggest roles of BDNF and NT-3 locally within the spinal cord and possibly for sensory nerves of the adrenal cortex.     

Activity-dependent and hormonal regulation of neurotrophin mRNA levels in brain–implications for neuronal plasticity

The neurotrophins exhibit neurotrophic effects on specific, partially overlapping populations of neurons both in the peripheral and the central nervous system (CNS). In the periphery, they are synthesized by a variety of nonneuronal cells, and their synthesis seems to be independent of the neuronal input. In contrast, in the CNS all neurotrophins are expressed under physiological conditions primarily by neurons. The production of NGF and BDNF is controlled by neuronal activity: up-regulation by glutamate and acetylcholine, down-regulation by gamma-aminobutyric acid. In contrast, NT-3 regulation is independent of neuronal activity, but it is up-regulated by thyroid hormones and BDNF. The latter observation suggests that NT-3 might be controlled indirectly by neuronal activity via BDNF. In peripheral nonneuronal tissues, glucocorticoid hormones down-regulate NGF mRNA levels both in vitro and in vivo. In contrast, in the CNS, neuronal production of NGF is enhanced by glucocorticoids. The rapid regulation of NGF and BDNF by subtle physiological stimuli together with the recent demonstration that the neurotrophin release neurotransmitters such as acetylcholine opens up interesting perspectives for the function of neurotrophins as mediators of neuronal plasticity. 1994 John Wiley & Sons, Inc.     

Dopamine D2 Receptor-Mediated Regulation of Serotonin Extracellular Concentration in the Dorsal Raphe Nucleus of Freely Moving Rats

Abstract: The neuropeptide-inducing activity of neurotrophic factors was tested in cultured cerebral cortical neurons. Brain-derived neurotrophic factor (BDNF) specifically increased contents of the neuropeptides somatostatin (SOM) and neuropeptide Y (NPY), but its effect on contents of cholecystokinin octapeptide and GABA was much less significant. The maximal induction of NPY content (15-fold increase) was achieved by 20 ng/ml of BDNF. These changes were also reproduced at the mRNA level. In contrast, neurotrophin-3 was much less potent at increasing NPY and SOM contents, and nerve growth factor had no effect on them. The expression of mRNA for NPY and SOM was fully dependent on the presence of BDNF in culture but irrelevant to the survival-promoting activity of BDNF, which has been reported previously. Most of the NPY immunoreactivity induced by BDNF was colocalized with glutamate decarboxylase immunoreactivity in cultured cortical neurons. These results suggest that BDNF regulates the peptidergic expression of GABAergic neurons in the cerebral cortex.     

Neurotrophin-4/5, brain-derived neurotrophic factor,and neurotrophin-3 promote survival of cultured vestibular ganglion neurons and protect them against neurotoxicity of ototoxins

The ability of neurotrophin-4/5 (NT-4/5), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF) to promote survival of postnatal rat vestibular ganglion neurons (VGNs) was examined in dissociated cell cultures. Of the four neurotrophins, NT-4/5 and BDNF were equally effective but more potent than NT-3 in promoting the survival of VGNs. In contrast, NGF showed no detectable effects. As expected, TrkB-IgG (a fusion protein of extracellular domain of TrkB and Fc domain of human immunoglobulin G) specifically inhibited the survival-promoting effects by NT-4/5 or BDNF and TrkC-IgG fusion protein completely blocked that of NT-3. Immunohistochemistry with TrkB, TrkA, and p75 antisera revealed that VGNs made TrkB and p75 proteins, but not TrkA protein. Ototoxic therapeutic drugs such as cisplatin and gentamicin often induce degeneration of hair cells and ganglion neurons in both auditory and vestibular systems that leads to impairment of hearing and balance. When cisplatin and gentamicin were added to the dissociated VGN culture in which the hair cells were absent, additional cell death of VGNs was induced, suggesting that the two ototoxins may have a direct neurotoxic effect on ganglion neurons in addition to their known toxicity on hair cells. However, if the cultures were co-treated with neurotrophins, NT-4/5, BDNF, and NT-3, but not NGF, prevented or reduced the neurotoxicity of the two ototoxins. Thus, the three neurotrophins are survival factors for VGNs and are implicated in the therapeutic prevention of VGN loss caused by injury and ototoxins. © 1995 John Wiley & Sons, Inc.     

The interaction of neurotrophins with the p75NTR common neurotrophin receptor: a comprehensive molecular modeling study

Neurotrophins are a family of proteins with pleiotropic effects mediated by two distinct receptor types, namely the Trk family, and the common neurotrophin receptor p75NTR. Binding of four mammalian neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), to p75NTR is studied by molecular modeling based on X-ray structures of the neurotrophins and the extracellular domain of p55TNFR, a homologue of p75NTR. The model of neurotrophin/receptor interactions suggests that the receptor binding domains of neurotrophins (loops I and IV) are geometrically and electrostatically complementary to a putative binding site of p75NTR, formed by the second and part of the third cysteine-rich domains. Geometric match of neurotrophin/receptor binding domains in the complexes, as characterized by shape complementarity statistic Sc, is comparable to known protein/protein complexes. All charged residues within the loops I and IV of the neurotrophins, previously determined as being critical for p75NTR binding, directly participate in receptor binding in the framework of the model. Principal residues of the binding site of p75NTR include Asp47, Lys56, Asp75, Asp76, Asp88, and Glu89. The additional involvement of Arg80 and Glu53 is specific for NGF and BDNF, respectively, and Glu73 participates in binding with NT-3 and NT-4/5. Neurotrophins are likely to induce similar, but not identical, conformational changes within the p75NTR binding site.     

Effect of Electroacupuncture on Neurotrophin Expression in Cat Spinal Cord after Partial Dorsal Rhizotomy

Neuroplasticity of the spinal cord following electroacupuncture (EA) has been demonstrated although little is known about the possible underlying mechanism. This study evaluated the effect of EA on expression of neurotrophins in the lamina II of the spinal cord, in cats subjected to dorsal rhizotomy. Cats received bilateral removal of L1–L5 and L7–S2 dorsal root ganglia (DRG, L6 DRG spared) and unilateral EA. They were sacrificed 7 days after surgery, and the L6 spinal segment removed and processed by immunohistochemistry and in situ hybridization histochemistry, to demonstrate the expression of neurotrophins. Significantly greater numbers of nerve growth factor (NGF) and neurotrophin-3 (NT-3) positive neurons, brain-derived neurotrophic factor (BDNF) immunoreactive varicosities and NT-3 positive neurons and glial cells were observed in lamina II on the acupunctured (left) side, compared to the non-acupunctured, contralateral side. Greater number of neurons expressing NGF mRNA was also observed on the acupunctured side. No signal for mRNA to BDNF and NT-3 was detected. The above findings demonstrate that EA can increase the expression of endogenous NGF at both the mRNA and protein level, and BDNF and NT-3 at the protein level. It is postulated that EA may promote the plasticity of the spinal cord by inducing increased expression of neurotrophins.     

Constitutive phosphorylation of TrkC receptors in cultured cerebellar granule neurons might be responsible for the inability of NT-3 to increase neuronal survival and to activate p21 ras

The neurotrophins brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are both expressed in developing cerebellum in addition to their tyrosine kinase receptors, TrkB and TrkC. In contrast to BDNF, NT-3 has only a negligible or a transient survival activity on cultured cerebellar granule neurons. The granule neurons however, express both TrkC and Trk B receptors which suggests a basic difference in signaling between BDNF and NT-3 in these neurons. Here we have studied whether this difference can be attributed to the presence of alternative TrkC receptor variants on the granule neurons and which signaling pathway is specifically activated by BDNF but not by NT-3 in these neurons. Using RT-PCR it was shown that the cerebellar granule neurons express the full length TrkC receptor, in addition to variant receptors containing small inserts in the receptor tyrosine kinase domain. There was no dramatic change in the relative amounts of different TrkC receptors during development. However, we found the TrkC receptor constitutively phosphorylated even in the absence of added ligand suggesting an interaction of TrkC with endogenously produced NT-3. In addition, NT-3 was able to phosphorylate the BDNF receptor, TrkB but only at higher concentration (50 ng/ml). There were also distinct differences in the activation of intracellular molecules by BDNF and NT-3. Thus, p21 Ras and PLCγ were activated by BDNF but not by NT-3 whereas both BDNF and NT-3 increased calcium and c-fos mRNA in the granule neurons. These results show that differential activation of specific intracellular pathways such as that of p21 Ras determines the specific effects of BDNF and NT-3 on granule neuron survival. In addition, since calcium is increased by NT-3 in the cerebellar granule neurons, this neurotrophin might have some unknown important effects on these neurons. Special issue dedicated to Dr. Hans Thoenen.     

Microarray analysis of cultured rat hippocampal neurons treated with brain derived neurotrophic factor

Brain derived neurotrophic factor (BDNF) has been shown to exert multiple actions on neurons. It plays a role in neuronal growth and maintenance and use-dependent plasticity, such as long-term potentiation and learning. This neurotrophin is believed to regulate neuronal plasticity by modifying neuronal excitability and morphology. There is experimental evidence for both an acute and a long-term effect of BDNF on synaptic transmission and structure but the molecular mechanisms underlying these events have not been completely clarified. In order to study the BDNF-induced molecular changes, the set of genes modulated in cultured hippocampal neurons by BDNF treatment was investigated after subchronic treatment with the neurotrophin. Microarray analysis performed with these cells, revealed increased expression of mRNA encoding the neuropeptides neuropeptide Y and somatostatin, and of the secreted peptide VGF (non acronymic), all of which participate in neurotransmission. In addition, the expression of genes apolipoprotein E (ApoE), delta-6 fatty acid desaturase (Fads2) and matrix metalloproteinase 14 (Mmp14), which play a role in neuronal remodelling, was also enhanced. More studies are needed to investigate and confirm the role of these genes in synaptic plasticity, but the results reported in this paper show that microarray analysis of hippocampal cultures can be used to expand our current knowledge of the molecular events triggered by BDNF in the hippocampus.