Characterization of retrograde axonal transport of antibodies in central and peripheral neurons

Retrograde axonal transport of antibodies against synaptic membrane glycoproteins was studied in the hypoglossal nerve and several CNS pathways of the rat. Injection into the tongue of polyclonal antibodies against synaptic membrane glycoproteins produced immunocytochemically labeled cells in the hypoglossal nucleus 4-5 hr later. Immunoreactive staining increased through 48 hr after injection and then declined. Injections of Fab preparations of the antibody gave labeling patterns indistinguishable from those of the whole antibody. The specificity of this method is shown by control studies in which antibodies against antigens that are not known to be present on the surface of presynaptic membranes were injected and gave no retrograde labeling. Retrograde labeling was also demonstrated in CNS pathways. However, labeling was never as intense as that seen in the hypoglossal nucleus, and some CNS pathways failed to show any retrograde labeling. Furthermore, retrograde labeling after control injections could be demonstrated in some cases. To determine if antibodies were also transported anterogradely, injections were made into the vitreous body of the eye, and the superior colliculus was processed for immunocytochemistry. Unlike wheat-germ agglutinin and several other tracers, antibodies were not found to be anterogradely transported in the optic nerve.     

Monocytes of allergics and non-allergics produce, store and release the neurotrophins NGF, BDNF and NT-3

INTRODUCTION: Recent studies have shown that neurotrophins (NTs) are involved in inflammatory processes. Elevated plasma levels of NTs were found allergic diseases with the highest levels in allergic asthma. However, the exact cellular sources involved in the regulation and release of neurotrophins in allergic inflammation are still not well defined. OBJECTIVE: The aim of this study was to assess whether monocytes of allergic and non-allergic subjects produce, store and release the neurotrophins NGF, BDNF and NT-3. METHODS: Monocytes of allergic and non-allergic donors were purified by immunomagnetic selection. APAAP-staining for the presence of NTs and their receptors was performed. RT-PCR and Western blot evaluated the production and storage of NTs. Monocytes were incubated and supernatants were collected for measurement of neurotrophic factors after stimulation with lipopolysaccharide (LPS) as inflammatory stimulus. The neurotrophin content in lysates and cell culture supernatants was determined by ELISA. RESULTS: Human monocytes express the neurotrophins NGF, BDNF and NT-3 but also their specific receptors TrkA, TrkB and TrkC. RT-PCR amplification of isolated mRNA demonstrated expression of the examined neurotrophins. Proteins were detectable by Western blot. NTs were found in the monocyte lysates and supernatants at different levels in allergic and non-allergic donors. Cell stimulation with LPS leads to release of NGF and NT3. CONCLUSIONS: Monocytes, produce, store and release NGF, BDNF and NT-3. They are a possible source of elevated neurotrophin levels found in allergy and asthma.     

Novel roles for neurotrophins are suggested by BDNF and NT-3 mRNA expression in developing neurons.

The results of our in situ hybridization experiments demonstrate that sensory neurons, sympathetic neurons, and motoneurons express brain-derived neurotrophic factor and/or neurotrophin-3 mRNAs during development in mouse. In accordance with previous data, we also find neurotrophins in the targets of sensory neurons (skin) and motoneurons (muscle) and the neurotrophin receptors p75, trkA, and trkB in sensory and sympathetic ganglia. These results suggest that neurotrophins have roles other than being target-derived factors that support neuron survival during developmental cell death (neurotrophic hypothesis), but may be transported in an orthograde fashion in neurons and released from axon terminals. We discuss several novel roles for neurotrophins, including autocrine/paracrine regulation of neuron survival, regulation of Schwann cell activity, and neuron to target signaling.     

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.     

A common mechanism for recombinant human NGF, BDNF, NT-3, and murine NGF slow unfolding

The recombinant human nerve growth factor (hNGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF) dimers all undergo rapid unfolding and dissociation to monomer in GdnHCl. Fluorescence spectroscopy, reversed-phase high-performance liquid chromatography, and size-exclusion chromatography were used to show that this monomer M1 converts slowly to a more fully unfolded monomer, M2, by a first order process with half-lives of 22, 2.5, 1.6, and 0.73 h for hNGF, mNGF, NT-3, and BDNF, respectively, at 25 degrees C. Linear Arrhenius plots for the conversion of M1 to M2 yielded activation energies of 27, 22, 24, and 24 kcal/mol for hNGF, mNGF, NT-3, and BDNF, respectively. The refolding of these neurotrophins from 5 M GdnHCl was also first order with NT-3 the slowest to refold and BDNF the fastest. Threading of the N-terminus out through the cystine-knot loop present in each of these proteins is proposed as the slow step in unfolding. The number of amino acids in the cystine-knot loop (14 for hNGF, mNGF, NT-3, and BDNF; 21 for NT4/5), and the number and position of the proline residues in this loop (2 for hNGF; 1 for mNGF, NT-3, BDNF, and NT4/5) correlate with the relative rates of unfolding. The smaller the loop and the greater the number of prolines, the more hindered and slower the unfolding.     

NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression.

To obtain insight into the site and stage specificity of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) action in vivo, we compared the expression patterns of the genes for these three related neurotrophic factors as well as for the NGF receptor in developing and adult rats. Initial embryonic expression of these related neurotrophic factors approximately coincides with the onset of neurogenesis. However, the levels at which the three factors are expressed at this time and throughout the developing nervous system are dramatically different. NT-3 is by far the most highly expressed in immature regions of the CNS in which proliferation, migration, and differentiation of neuronal precursors is ongoing. NT-3 expression dramatically decreases with maturation of these regions. By contrast, BDNF expression is low in developing regions of the CNS and increases as these regions mature. NGF expression varies during the development of discrete CNS regions, but not in any consistent manner compared with NT-3 and BDNF. Despite the dramatic variations, NT-3, BDNF, and NGF do share one striking similarity--high level expression in the adult hippocampus. Our observations are consistent with the idea that NT-3, BDNF, and NGF have paralleled as well as reciprocal roles in vivo.     

NGF receptor-mediated reduction in axonal NGF uptake and retrograde transport following sciatic nerve injury and during regeneration.

Injury to the rat sciatic nerve leads to the induction of nerve growth factor (NGF) receptors on the denervated Schwann cells and their disappearance on the regenerating axons of the axotomized, normally NGF-sensitive sensory and sympathetic neurons. This disappearance in the axonal expression and retrograde transport of NGF receptors is associated with a similarly dramatic reduction in the axonal uptake and retrograde transport of NGF following axotomy and during regeneration. In view of the massive NGF synthesis occurring in the injured nerve, these results suggest that, while sensory and sympathetic neurons are the primary targets of NGF in the normal peripheral nervous system, the denervated Schwann cells may become its primary target in the aftermath of nerve injury.     

Spatiotemporal Changes of NGF,BDNF and NT-3 in the Developing Spinal Cords of Embryonic Chicken

Spatiotemporal changes of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the spinal cords of chick embryonic stage day 7 (E7) and day 14 (E14) were examined by using immunohistochemistry and Western blot. Intensive NGF immunoreaction (IR) was detected in the white matter of the spinal cords, while BDNF-IR in perikaryon and neurite, and NT-3-IR in the nucleus and cytoplasm were seen in the neurons of the ventral horn in the gray matter. Comparatively, the expressions for three growth factors have expanded largely into the dorsal horn at E14, and the level of proteins for these growth factors increased significantly in the spinal cords from E7 to E14. Morphological observation showed that the lumbar spinal cords of E7 appeared rectangular, whereas it gave a butterfly shape in the gray matter consisting of the typical ventral horn, dorsal horn and intermediate zone at E14. The present findings indicated that the spatiotemporal changes of NGF, BDNF and NT-3 could be associated to the morphological changes of developing spinal cords, suggesting the possible roles of three growth factors in the development of spinal cords.     

Enhanced detection and retrograde axonal transport of PrPc in peripheral nerve

Neuroinvasion of the CNS during orally acquired transmissible spongiform encephalopathies (TSEs) may involve the transport of the infectious agent from the periphery to the CNS via the peripheral nerves. If this occurs within axons, the mechanism of axonal transport may be fundamental to the process. In studies of peripheral nerve we observed that the cellular prion protein (PrPc) is highly resistant to detergent extraction. The implication of this is an underestimation of the abundance of PrPc in peripheral nerve. We have developed nerve extraction conditions that enhance the quantification of the protein in nerve 16-fold. Application of these conditions to evaluate the accumulation of PrPc distal to a cut nerve now reveals that PrPc is retrogradely transported from the axon ending. These results provide a potential cellular mechanism for TSE infectivity to gain entry to the CNS from the periphery.     

Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals. However, the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood. Here, we report that the signals of the purinergic (P)2X(3) receptor, an ATP-gated ion channel, are retrogradely transported in dorsal root ganglion (DRG) neuron axons. We found that Rab5, a small GTPase, controls the early sorting of P2X(3) receptors into endosomes, while Rab7 mediates the fast retrograde transport of P2X(3) receptors. Intraplantar injection and axonal application into the microfluidic chamber of α, β-methylene-ATP (α, β-MeATP), a P2X selective agonist, enhanced the endocytosis and retrograde transport of P2X(3) receptors. The α, β-MeATP-induced Ca(2+) influx activated a pathway comprised of protein kinase C, rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK), which associated with endocytic P2X(3) receptors to form signaling endosomes. Disruption of the lipid rafts abolished the α, β-MeATP-induced ERK phosphorylation, endocytosis and retrograde transport of P2X(3) receptors. Furthermore, treatment of peripheral axons with α, β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability. Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α, β-MeATP-induced retrograde signals. These results indicate that P2X(3) receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels.     

Cytofluorometric quantitation of retrograde axonal transport in single dorsal root ganglia neurons

A technique is described by which neurons from mouse dorsal root ganglia can be dispersed in single-cell suspensions suitable for quantitative cytochemical analyses. The neurons were intact as controlled by trypan blue exclusion test, and the cell size distribution of the dispersed neurons corresponded to that of untreated, intact ganglia. Horseradish peroxidase and Evans blue applied to cut sciatic nerve, were transferred by somatopetal intra-axonal transport and accumulated in corresponding dorsal root ganglia neurons. The tracers were retained during the preparation of cell suspensions. The accumulation of the fluorescent tracer Evans blue was quantitated by cytofluorometric measurements on individual neurons.     

The kinematics of turnaround and retrograde axonal transport

Rapid axonal transport of a pulse of 35S-methionine-labelled material was studied in vitro in the sensory neurons of amphibian sciatic nerve using a position-sensitive detector. For 10 nerves studied at 23.0 +/- 0.2 degrees C it was found that a pulse moved in the anterograde direction characterized by front edge, peak, and trailing edge transport rates of (mm/d) 180.8 +/- 2.2 (+/- SEM), 176.6 +/- 2.3, and 153.7 +/- 3.0, respectively. Following its arrival at a distal ligature, a smaller pulse was observed to move in the retrograde direction characterized by front edge and peak transport rates of 158.0 +/- 7.3 and 110.3 +/- 3.5, respectively, indicating that retrograde transport proceeds at a rate of 0.88 +/- 0.04 that of anterograde. The retrograde pulse was observed to disperse at a rate greater than the anterograde. Reversal of radiolabel at the distal ligature began 1.49 +/- 0.15 h following arrival of the first radiolabel. Considerable variation was seen between preparations in the way radiolabel accumulated in the end (ligature) regions of the nerve. Although a retrograde pulse was seen in all preparations, in 7 of 10 preparations there was no evidence of this pulse accumulating within less than 2-3 mm of a proximal ligature; however, accumulation was observed within less than 5 mm in all preparations.     

Facilitated ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase in peripheral nerve

p-Phenylenediamine/pyrocatechol mixture (PPD-PC) was evaluated as a reagent for the ultracytochemical demonstration of retrograde axonal transport of horseradish peroxidase (HRP). HRP crystals were applied to the proximal stumps of the severed infraorbital nerves in rats. After 48 h the rats were sacrificed by perfusion, and the trigeminal ganglia ipsilateral to the severed nerves were processed for HRP cytochemistry and then prepared for electron microscopy. PPD-PC was rapidly oxidized in HRP-labeled neurons to form a dark brown-black osmiophilic reaction product which was more readily visible than the DAB product in the sections. This facilitated selection by light microscopy of areas in the epoxy wafers for ultrathin sectioning. In thin sections viewed under the electron microscope, the osmicated electron opaque PPD-PC reaction product was present in membrane-bound structures including smooth endoplasmic reticulum and granules of various sizes. The PPD-PC reaction product formed after 10-min incubation appeared to be more electron opaque than the DAB reaction product formed after 20 min. PPD-PC was found to be much less readily oxidized than DAB by endogenous hemoproteins. This methodology facilitated the ultracytochemical localization of HRP in neurons following retrograde axonal transport.     

Eya1 acts upstream of Tbx1, Neurogenin 1, NeuroD and the neurotrophins BDNF and NT-3 during inner ear development

Cell fate specification during inner ear development is dependent upon regional gene expression within the otic vesicle. One of the earliest cell fate determination steps in this system is the specification of neural precursors, and regulators of this process include the Atonal-related basic helix-loop-helix genes, Ngn1 and NeuroD and the T-box gene, Tbx1. In this study we demonstrate that Eya1 signaling is critical to the normal expression patterns of Tbx1, Ngn1, and NeuroD in the developing mouse otocyst. We discuss a potential mechanism for the absence of neural precursors in the Eya1-/- inner ears and the primary and secondary mechanisms for the loss of cochleovestibular ganglion cells in the Eya1bor/bor hypomorphic mutant.     

Is the retrograde axonal transport of 3H-5-HT a specific process of serotoninergic neurons?

The specificity of the retrograde axonal transport of 3H-serotonin (3H-5-HT) was radioautographically studied in the afferents to the olfactory bulb (O.B.). Injections of 3H-5-HT of different concentrations (10(-2), 10(-3), 10(-4) and 10(-5) M) were performed into the O.B. of catron pretreated rats. Following injection of 3H-5-HT (10(-2) M), a cytoplasmic perikaryal labeling was observed in the bulk of afferents to the O.B. (aminergic and non-aminergic neurons). When lower concentrations of 3H-5-HT (10(-5) M) were injected into the O.B., the retrograde labeling was only seen in the raphe dorsalis (RD) serotoninergic perikarya. The specificity of the uptake-retrograde transport of 3H-5-HT seems to depend on the selectivity of uptake by nerve terminals.     

Whole-brain patterns of the presynaptic inputs and axonal projections of BDNF neurons in the paraventricular nucleus

Brain-derived neurotrophic factor (BDNF) plays a crucial role in human obesity. Yet, the neural circuitry supporting the BDNF-mediated control of energy homeostasis remains largely undefined. To map key regions that might provide inputs to or receive inputs from the paraventricular nucleus (PVN) BDNF neurons, a key type of cells in regulating feeding and thermogenesis, we used rabies virus-based transsynaptic labeling and adeno-associated virus based anterograde tracing techniques to reveal their whole-brain distributions. We found that dozens of brain regions provide dense inputs to or receive dense inputs from PVN BDNF neurons, including several known weight control regions and several novel regions that might be functionally important for the BDNF-mediated regulation of energy homeostasis.Interestingly, several regions show very dense reciprocal connections with PVN BDNF neurons, including the lateral septum, the preoptic area, the ventromedial hypothalamic nucleus, the paraventricular thalamic nucleus, the zona incerta, the lateral parabrachial nucleus, the subiculum, the raphe magnus nucleus, and the raphe pallidus nucleus. These strong anatomical connections might be indicative of important functional connections. Therefore, we provide an outline of potential neural circuitry mediated by PVN BDNF neurons, which might be helpful to resolve the complex obesity network.     

Costorage of BDNF and neuropeptides within individual dense-core vesicles in central and peripheral neurons

Some central and peripheral neurons synthesize brain-derived neurotrophic factor (BDNF), and, after anterograde transport, release it at synapses. By immunocytochemistry, we examined, in rat and mouse, the subcellular localization of BDNF and BDNF/peptide coexistence, under normal conditions or after intrathecal infusion of nerve growth factor. In dorsal root ganglion neurons and afferent terminals, and in the parabrachial projection to amygdala, we show that BDNF is costored in individual dense-core vesicles (DCVs) with the neuropeptides calcitonin gene-related peptide (CGRP) and substance P. At both locations, nerve endings costoring all three peptides were fairly rare. Remarkably however, costorage occurred in a stoichiometric ratio of 0.7 BDNF:1 CGRP:1 substance P, and DCVs contained 31 (spinal cord) -36 (amygdala) times the amount of BDNF detected in agranular vesicles. This is the first direct demonstration in peripheral and central neurons from two different mammals, that a growth factor is selectively packaged together with neuropeptide transmitters within individual DCVs. It provides structural bases for differential release upon stimulation, and has important implications for understanding BDNF transmitter function.