Expression and cell localization of brain-derived neurotrophic factor and TrkB during zebrafish retinal development.

Brain-derived neurotrophic factor (BDNF) signaling through TrkB regulates different aspects of neuronal development, including survival, axonal and dendritic growth, and synapse formation. Despite recent advances in our understanding of the functional significance of BDNF and TrkB in the retina, the cell types in the retina that express BDNF and TrkB, and the variations in their levels of expression during development, remain poorly defined. The goal of the present study is to determine the age-dependent changes in the levels of expression and localization of BDNF and TrkB in the zebrafish retina. Zebrafish retinas from 10 days post-fertilization (dpf) to 180 dpf were used to perform PCR, Western blot and immunohistochemistry. Both BDNF and TrkB mRNAs, and BDNF and full-length TrkB proteins were detected at all ages sampled. The localization of these proteins in the retina was very similar at all time points studied. BDNF immunoreactivity was found in the outer nuclear layer, the outer plexiform layer and the inner plexiform layer, whereas TrkB immunoreactivity was observed in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer. These results demonstrate that the pattern of expression of BDNF and TrkB in the retina of zebrafish remains unchanged during postembryonic development and adult life. Because TrkB expression in retina did not change with age, cells expressing TrkB may potentially be able to respond during the entire lifespan of zebrafish to BDNF either exogenously administered or endogenously produced, acting through paracrine mechanisms.

Differential localization of Acid-sensing ion channels 1 and 2 in human cutaneus pacinian corpuscles.

Acid-sensing ion channels (ASICs) are the members of the degenerin/epithelial sodium channel (Deg/ENaC) superfamily which mediate different sensory modalities including mechanosensation. ASICs have been detected in mechanosensory neurons as well as in peripheral mechanoreceptors. We now investigated the distribution of ASIC1, ASIC2, and ASIC3 proteins in human cutaneous Pacinian corpuscles using immunohistochemistry and laser confocal-scanner microscopy. We detected different patterns of expression of these proteins within Pacinian corpuscles. ASIC1 was detected in the central axon co-expressed with RT-97 protein, ASIC2 was expressed by the lamellar cells of the inner core co-localized with S100 protein, and ASIC3 was absent. These results demonstrate for the first time the differential distribution of ASIC1 and ASIC2 in human rapidly adapting low-threshold mechanoreceptors, and suggest specific roles of both proteins in mechanotransduction.

The lamellar cells in human Meissner corpuscles express TrkB.

Cutaneous Meissner corpuscles depend for development and survival exclusively on the NT system TrkB/BDNF/NT-4 unlike other types of sensory corpuscles and nerve endings, which have very complex neuronal and growth factor dependence. However, the pattern of expression of TrkB in human Meissner corpuscles is not known. The experiments in these studies were designed to pursue further findings that suggest that BDNF and NT-4 have critical roles in the development and maintenance of Meissner corpuscles by analyzing the pattern of expression of TrkB, their high-affinity receptor, in human glabrous skin. These experiments showed that TrkB is expressed in different patterns by the lamellar cells of Meissner corpuscles and not by the axon. The studies also show that while the percentage of Meissner corpuscles that express TrkB remains constant from birth till 50-year old cases, it decreases approximately 3-fold in subjects older than 50 years. These results are important since the study of Meissner corpuscles from cutaneous biopsies to diagnose some neurological diseases has rapidly become of high interest and therefore the proteins expressed in these corpuscles are potential diagnostic tools.

The expression of ENa(+)C and ASIC2 proteins in Pacinian corpuscles is differently regulated by TrkB and its ligands BDNF and NT-4.

Pacinian corpuscles are innervated by large myelinated Aalpha-beta axons from the large- and intermediate-sized sensory neurons of dorsal root ganglia. These neurons express different members of the degenerin/epithelial Na(+) channel (DEG/ENa(+)C) superfamily of proteins with putative mechanosensory properties, whose expression is regulated by the TrkB-BDNF system. Thus, we hypothesized that BDNF and/or NT-4 signalling through activation of TrkB may regulate the expression of molecules supposed to be necessary for the mechanosensory function of Pacinian corpuscles. To test this hypothesis we analyzed the expression and distribution of ENa(+)C subunits and acid-sensing ion channel 2 (ASIC2) in Pacinian corpuscles from 25 days old mice deficient in TrkB, BDNF and NT-4. Pacinian corpuscles in these animals are normal in number, structure, and expression of several immunohistochemical markers. Using immunohistochemistry we observed that the beta-ENa(+)C and gamma-ENa(+)C subunits, but not the alpha-ENa(+)C subunit, were expressed in wild-type animals, and they were always found in the central axon. ASIC2 immunoreactivity was found in both the central axon and the inner core cells. The absence of TrkB or BDNF abolished expression of beta-ENa(+)C and ASIC2, whereas expression of gamma-ENa(+)C did not change. Expression of beta-ENa(+)C and gamma-ENa(+)C subunits in NT-4 deficient mice was found in the axons but also in the inner core cells whereas levels of expression of ASIC2 were increased in these animals. This study suggests that expression in Pacianian corpuscles of some potential mechanosensory proteins is regulated by BDNF, NT-4 and TrkB.

TrkB is necessary for the normal development of the lung.

Normal development of the lung requires coordinated activation of cascades of signaling pathways initiated by growth factors signaling through their receptors. TrkB and its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4, belong to the neurotrophin family of growth factors, which are expressed in a large variety of non-neuronal tissues including the lung. Aberrant neurotrophin signaling underlies the pathogenesis of several lung-related pathologies, including asthma and lung cancer, however, little is known about the role of neurotrophins in the embryonic development of the lung. To fill this gap in knowledge, we analyzed the pattern of TrkB expression in the murine lung and we observed that TrkB is expressed in alveolar macrophages, type II pneumocytes, neuroepithelial bodies and nerves. Analysis of the structure of lung from mice deficient in TrkB revealed that absence of TrkB signaling results in thinner bronchial epithelium and apparent larger air space, and, more importantly, lack of neuroepithelial bodies, an important reduction in the density of nerve fibres in the bronchial smooth muscle, submucous plexus in bronchioles, and pulmonary artery walls. These findings suggest TrkB is essential for the normal development of the lung and the nervous system in the lung.

Enteric glial cells express full-length TrkB and depend on TrkB expression for normal development.

The embryonic development of the enteric nervous system (ENS) from neural crest precursor cells requires neurotrophic signaling. Neurotrophins (NTs) are a family of growth factors that bind Trk receptors to signal diverse functions, including development and maintenance of different cell populations in the peripheral nervous system. In this study we investigated the expression and cell localization of TrkB, the high affinity receptor for brain-derived neurotrophic factor and NT-4, in the murine ENS using Western blot and immunohistochemistry. The results demonstrate that enteric glial cells within the ENS express full-length TrkB at all stages tested. The ENS of TrkB deficient mice have reduced expression of glial cell markers, and a disarrangement of glial cells and the plexular neuropil. These results strongly suggest TrkB has essential roles in the normal development and maintenance of glial cells in the ENS.

Characterization of sensory deficits in TrkB knockout mice.

The sensory deficit in TrkB deficient mice was evaluated by counting the neuronal loss in lumbar dorsal root ganglia (DRG), the absence of sensory receptors (cutaneous--associated to the hairy and glabrous skin - muscular and articular), and the percentage and size of the neurocalcin-positive DRG neurons (a calcium-binding protein which labels proprioceptive and mechanoceptive neurons). Mice lacking TrkB lost 32% of neurons, corresponding to the intermediate-sized and neurocalcin-positive ones. This neuronal lost was accomplished by the absence of Meissner corpuscles, and reduction of hair follicle-associated sensory nerve endings and Merkel cells. The mutation was without effect on Pacinian corpuscles, Golgi's organs and muscle spindles. Present results further characterize the sensory deficit of the TrkB-/- mice demonstrating that the intermediate-sized neurons in lumbar DRG, as well as the cutaneous rapidly and slowly adapting sensory receptors connected to them, are under the control of TrkB for survival and differentiation. This study might serve as a baseline for future studies in experimentally induced neuropathies affecting TrkB positive DRG neurons and their peripheral targets, and to use TrkB ligands in the treatment of neuropathies in which cutaneous mechanoreceptors are primarily involved.

The Trk tyrosine kinase inhibitor K252a regulates growth of lung adenocarcinomas.

The neurotrophin family of growth factors and their receptors support the survival of several neuronal and non-neuronal cell populations during embryonic development and adult life. Neurotrophins are also involved in malignant transformation. To seek the role of neurotrophin signaling in human lung cancer we studied the expression of neurotrophin receptors in human lung adenocarcinomas and investigated the effect of the neurotrophin receptor inhibitor K252a in A549 cell survival and colony formation ability in soft agar. We showed that human lung adenocarcinomas express TrkA and TrkB, but not TrkC; A549 cells, derived from a human lung adenocarcinoma, express mRNA transcripts encoding nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), TrkA, TrkB, and p75, and high protein levels of TrkA and TrkB. Stimulation of cells using NGF or BDNF activates the anti-apoptotic protein Akt. Interestingly, inhibition of neurotrophin receptor signaling using K252a prevents Akt activation in response to NGF or BDNF, induces apoptotic cell death, and diminishes the ability of A549 cells to growth in soft agar. The data suggest that neurotrophin signaling inhibition using k252a may be a valid therapy to treat patients with lung adenocarcinomas.

Pleiotrophin disrupts calcium-dependent homophilic cell-cell adhesion and initiates an epithelial-mesenchymal transition.

Regulation of the levels of tyrosine phosphorylation is essential to maintain the functions of proteins in different signaling pathways and other cellular systems, but how the steady-state levels of tyrosine phosphorylation are coordinated in different cellular systems to initiate complex cellular functions remains a formidable challenge. The receptor protein tyrosine phosphatase (RPTP)beta/zeta is a transmembrane tyrosine phosphatase whose substrates include proteins important in intracellular and transmembrane protein-signaling pathways, cytoskeletal structure, cell-cell adhesion, endocytosis, and chromatin remodeling. Pleiotrophin (PTN the protein and Ptn the gene) is a ligand for RPTPbeta/zeta; PTN inactivates RPTPbeta/zeta, leaving unchecked the continued endogenous activity of tyrosine kinases that increase phosphorylation of the substrates of RPTPbeta/zeta at sites dephosphorylated by RPTPbeta/zeta in cells not stimulated by PTN. Thus, through the regulation of the tyrosine phosphatase activity of RPTPbeta/zeta, the PTN/RPTPbeta/zeta signaling pathway coordinately regulates the levels of tyrosine phosphorylation of proteins in many cellular systems. We now demonstrate that PTN disrupts cytoskeletal protein complexes, ablates calcium-dependent homophilic cell-cell adhesion, stimulates ubiquitination and degradation of N-cadherin, reorganizes the actin cytoskeleton, and induces a morphological epithelial-mesenchymal transition (EMT) in PTN-stimulated U373 cells. The data suggest that increased tyrosine phosphorylation of the different substrates of RPTPbeta/zeta in PTN-stimulated cells alone is sufficient to coordinately stimulate the different functions needed for an EMT; it is possible that PTN initiates an EMT in cells at sites where PTN is expressed in development and in malignant cells that inappropriately express Ptn.

Thymocyte depletion affects neurotrophin receptor expression in thymic stromal cells.

Thymocytes and thymic stromal cells cross-talk in a bidirectional manner within the thymus, thus contributing to the generation of mature T-cells. The thymic stromal cells in the rat express the high- (TrkA, TrkB) and low-affinity (p75NTR) receptors for neurotrophins. In this study we analysed the regulation of TrkA, TrkB and p75NTR expression in the rat thymus by thymocytes. We induced thymocyte apoptosis by administration of corticoids in rats, and then analysed the expression and distribution of these receptors 1, 4 and 10 days later. Thymocyte death was assessed by the activation of caspase-3 in cells undergoing apoptosis. We observed massive thymocyte apoptosis 1 day after injection and, to a lesser extent, after 4 days, which was parallel with a reduction in the density of thymic epithelial cells normally expressing TrkA and p75NTR. Furthermore, TrkA expression was found in cortical thymic epithelial cells, which normally lack this receptor. The expression of TrkB was restricted to a subset of macrophage-dendritic cells, and remained unchanged with treatment. The normal pattern of neurotrophin receptor expression was almost completely restored by day 10. The results demonstrate that the expression of neurotrophin receptors by thymic epithelial cells, but not by macrophage-dendritic cells, is regulated by thymocytes.

APP processing and the APP-KPI domain involvement in the amyloid cascade.

Alternative APP mRNA splicing can generate isoforms of APP containing a Kunitz protease inhibitor (KPI) domain. KPI is one of the main serine protease inhibitors. Protein and mRNA KPI(+)APP levels are elevated in Alzheimer's disease (AD) brain and are associated with increased amyloid beta deposition. In the last years increasing evidence on multiple points in the amyloid cascade where KPI(+)APP is involved has been accumulated, admitting an outstanding position in the pathogenesis of AD to the KPI domain. This review focuses on the APP processing, the molecular activity of KPI and its physiological and pathological roles and the KPI involvement in the amyloid cascade through the nerve growth factor, the lipoprotein receptor-related protein, the tumor necrosis factor-alpha converting enzyme and the Notch1 protein.

S-100 proteins in the human peripheral nervous system.

This article reviews the distribution of S100 proteins in the human peripheral nervous system. The expression of S100 by peripheral glial cells seems to be a distinctive fact of these cells, independently of their localization and their ability to myelinate or not. S100 proteins expressing cells include satellite cells of sensory, sympathetic and enteric ganglia, supporting cells of the adrenal medulla, myelinating and non-myelinating Schwann cells in the nerve trunks, and the Schwann-related cells of sensory corpuscles. In addition, S100 proteins are expressed in peripheral neurons. Most of them express S100alpha protein, and a subpopulation of sensory neurons in dorsal root ganglia contains S100beta protein or S100alpha plus S100beta proteins.