Wilma J. Friedman

NT-3 stimulates sympathetic neuroblast proliferation by promoting precursor survival.

Although proliferation is fundamental to the generation of neuronal populations, little is known about the function of trophic mechanisms during neurogenesis. We now describe a novel role for neurotrophin-3 (NT-3): the neurotrophin stimulates proliferation of sympathetic neuroblasts through trophic mechanisms. NT-3 promotes survival of the dividing precursors, but does not directly stimulate mitosis. NT-3 trophic effects differ markedly from those of the sympathetic mitogen, insulin. Furthermore, whereas NT-3 exhibits trophic activity for dividing neuroblasts, nerve growth factor characteristically promotes survival of postnatal sympathetic neurons. The stage-specific activity of NT-3 and nerve growth factor in culture parallels the sequence of trkC and trkA receptor gene expression detected in vivo. Thus, neurotrophins apparently serve as trophic factors during ontogeny, acting sequentially during establishment of individual populations.

Interaction of Survival and Death Signaling in Basal Forebrain Neurons: Roles of Neurotrophins and Proneurotrophins

Proneurotrophins bind with high affinity to p75 neurotrophin receptor (p75NTR) and lack the capacity to bind Trk receptors, suggesting that proneurotrophins can elicit apoptosis via p75NTR even in cells expressing survival-promoting Trk receptors. In the CNS, basal forebrain (BF) neurons are particularly vulnerable to degeneration in Alzheimer’s disease, and are among the few populations of brain neurons that express p75NTR throughout life. These neurons also express Trk receptors and may be concomitantly exposed to both proneurotrophins and mature neurotrophins during development, disease, or after injury. We investigated the interaction of mature and proneurotrophin signaling in these CNS neurons. Kainic acid-induced seizures elicited production of pro-NGF by BF astrocytes before caspase activation in p75NTR-positive BF neurons, demonstrating local production of proneurotrophins under pathological conditions and suggesting apoptotic signaling in vivo. Mechanisms of proneurotrophin-induced death were analyzed in cultured BF neurons, and required both p75NTR and its coreceptor sortilin. Surprisingly, exposure to both mature neurotrophins and proneurotrophins demonstrated that Trk phosphorylation did not prevent pro-NGF-induced apoptosis via p75NTR. However, activation of PI3K (phosphatidylinositol 3-kinase)/Akt and MEK (mitogen-activated protein kinase kinase)/Erk pathways prevented pro-NGF-induced apoptosis, revealing a novel critical checkpoint in survival versus apoptotic signaling downstream of Trk activation, and suggesting that pro-NGF blocks survival signaling by preventing Akt and Erk activation. This study shows that proneurotrophins are produced in the brain under pathological conditions, and can elicit apoptosis of BF neurons even when Trk receptors are activated.

NGF AND BDNF ARE DIFFERENTIALLY MODULATED BY VISUAL EXPERIENCE IN THE DEVELOPING GENICULOCORTICAL PATHWAY

Neuronal activity and trophic factors have been implicated in shaping the connectivity of functional synaptic circuits. We studied the development and regulation by sensory input of the neurotrophins NGF, BDNF and NT-3 in the developing rat visual system. In the occipital cortex, NT-3 mRNA was transiently expressed in the neonate. In contrast, BDNF and NGF mRNAs increased during postnatal development, and reached mature levels around 3 weeks of age. BDNF mRNA was ten times more abundant than NGF mRNA. In the lateral geniculate nucleus (LGN), NT-3 mRNA was also transiently expressed, whereas NGF and BDNF mRNAs did not vary significantly during development. The high-affinity neurotrophin receptors trkB and trkC were expressed both in the developing LGN and occipital cortex. These receptors for BDNF and NT-3, respectively, were expressed at birth, with little change during development. In contrast, trkA mRNA, which encodes the high-affinity NGF receptor, was undetectable in either region. Visual experience differentially modulated expression of NGF and BDNF mRNAs. NGF mRNA was slightly increased after 3 weeks of light-deprivation. In contrast, BDNF mRNA expression in visual cortex was significantly lower than normal in rats dark-reared from birth. Decreased BDNF expression after sensory deprivation was reversible by exposure to light. Thus, all three neurotrophins were detected in visual cortex and LGN. Differences in abundance developmental profiles, and regulation imply distinct functions for each factor in the visual system.

Cell Type-Specific Interleukin-1β Signaling in the CNS

Interleukin-1β (IL-1β) is a potent and pleiotropic inflammatory cytokine that is highly produced in the CNS under conditions of damage, disease, or stress. This cytokine acts on CNS glia to effect inflammatory responses, mediated in part via activation of the nuclear factor-κB (NF-κB) transcription factor, and consequent induction of numerous cytokines. Neurons as well as astrocytes in the hippocampus also express the type 1 IL-1 receptor, indicating that this cytokine can influence neuronal function directly, yet IL-1β does not induce production of cytokines in neurons as it does in glia. In contrast, IL-1β regulates synaptic function of hippocampal neurons. Here we demonstrate that different signaling pathways mediate IL-1β actions in neurons as compared with astrocytes. IL-1β activates the p38 mitogen-activated protein kinase (MAPK) signaling pathway and induces the activation of CREB in hippocampal neurons, in contrast to the activation of NF-κB in hippocampal astrocytes, demonstrating cell type-specific signaling responses to IL-1 in the brain and yielding distinct functional responses.

Distribution of the neurotrophins brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 in the postnatal rat brain: an immunocytochemical study

The neurotrophin family of trophic factors influences survival and function of neurons in both the peripheral and central nervous systems. Critical information regarding physiological function of these factors may be gained by examining their localization in the brain. Here we report the immunocytochemical characterization of antisera directed against brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin 4/5. These antisera provide important tools to localize the bioactive neurotrophin proteins. Correspondence between protein distribution and previously determined messenger RNA expression was observed in some brain regions, such as hippocampus and cortex. However, neurotrophin proteins were also detected in neurons which have no apparent corresponding messenger RNA, indicating that the proteins may be transported from the sites of synthesis in certain populations. Immunocytochemical double-labelling analysis also indicated that a sub-population of neurotrophin-positive cells were labelled with an astrocyte marker (glial fibrillary acidic protein) as well, demonstrating that trophic molecules are localized to glial cells as well as neurons in vivo. Thus, the use of antisera specific for individual neurotrophic factors has indicated potential cellular sites of action.

Cytokines regulate expression of the type 1 interleukin-1 receptor in rat hippocampal neurons and glia.

Interleukin-1 beta is a key mediator of inflammation and stress in the central nervous system (CNS). This cytokine induces CNS glial cells to produce numerous additional cytokines and growth factors under inflammatory conditions. We have investigated regulation of the signal transducing type 1 interleukin-1 receptor (IL-1R1) in the CNS. In vivo, IL-1R1 was not detected in glial cells under basal conditions but was strongly induced after a stab lesion. Cultured astrocytes were used to identify specific signals that regulate expression of the receptor. IL-1R1 mRNA and protein were induced by inflammatory stimuli including tumor necrosis factor (TNF alpha) and IL-1 beta itself. Although expression of the receptor was not detected in glia under basal conditions in vivo, pyramidal neurons in the hippocampus expressed the IL-1 receptor in the normal, unlesioned brain. Cultured embryonic hippocampal neurons were used to investigate specific stimuli that regulate IL-1R1 in neurons. As in astrocytes, IL-1 and TNF alpha induced expression of IL-1R1. The expression of IL-1R1 in hippocampal neurons suggests a possible role for IL-1 in regulating neuronal function, and indicates that these neurons may be directly influenced by cytokines.

Regulation of Nerve Growth Factor mRNA by Interleukin-1 in Rat Hippocampal Astrocytes Is Mediated by NFκB

Cytokines such as interleukin-1β (Il-1) are produced in the brain during development and during inflammatory processes that result from lesions or disease. One function of Il-1 in the brain appears to be the stimulation of astrocytes to proliferate and produce a variety of cytokines and trophic factors, including nerve growth factor. The mechanisms by which Il-1 exerts its actions on astrocytes remain poorly defined. We present evidence that this cytokine elicits activation of the NFκB transcription factor and that this transcription factor mediates effects of Il-1 on nerve growth factor mRNA expression. Elucidation of the processes by which cytokines activate astrocytes and influence trophic factor expression may provide insight into mechanisms governing inflammatory processes within the central nervous system.

Activation of the p75 Neurotrophin Receptor through Conformational Rearrangement of Disulphide-Linked Receptor Dimers

Ligand-mediated dimerization has emerged as a universal mechanism of growth factor receptor activation. Neurotrophins interact with dimers of the p75 neurotrophin receptor (p75(NTR)), but the mechanism of receptor activation has remained elusive. Here, we show that p75(NTR) forms disulphide-linked dimers independently of neurotrophin binding through the highly conserved Cys(257) in its transmembrane domain. Mutation of Cys(257) abolished neurotrophin-dependent receptor activity but did not affect downstream signaling by the p75(NTR)/NgR/Lingo-1 complex in response to MAG, indicating the existence of distinct, ligand-specific activation mechanisms for p75(NTR). FRET experiments revealed a close association of p75(NTR) intracellular domains that was transiently disrupted by conformational changes induced upon NGF binding. Although mutation of Cys(257) did not alter the oligomeric state of p75(NTR), the mutant receptor was no longer able to propagate conformational changes to the cytoplasmic domain upon ligand binding. We propose that neurotrophins activate p75(NTR) by a mechanism involving rearrangement of disulphide-linked receptor subunits.

Induction of proneurotrophins and activation of p75NTR-mediated apoptosis via neurotrophin receptor-interacting factor in hippocampal neurons after seizures.

Seizure-induced damage elicits a loss of hippocampal neurons mediated to a great extent by the p75 neurotrophin receptor (NTR). Proneurotrophins, which are potent apoptosis-inducing ligands for p75NTR, were increased in the hippocampus, particularly in astrocytes, by pilocarpine-induced seizures; and infusion of anti-pro-NGF dramatically attenuated neuronal loss after seizures. The p75NTR is expressed in many different cell types in the nervous system, and can mediate a variety of different cellular functions by recruiting specific intracellular binding proteins to activate distinct signaling pathways. In this study, we demonstrate that neurotrophin receptor-interacting factor (NRIF) mediates apoptotic signaling via p75NTR in hippocampal neurons in vitro and in vivo. After seizure-induced injury, NRIF−/− mice showed an increase in p75NTR expression in the hippocampus; however, these neurons failed to undergo apoptosis in contrast to wild-type mice. Treatment of cultured hippocampal neurons with proneurotrophins induced association of NRIF with p75NTR and subsequent translocation of NRIF to the nucleus, which was dependent on cleavage of the receptor. Neurons lacking NRIF were resistant to p75NTR-mediated apoptosis in vitro and in vivo. In addition, we demonstrate some mechanistic differences in p75NTR signaling in hippocampal neurons compared with other cell types. Overall, these studies demonstrate the requirement for NRIF to signal p75NTR-mediated apoptosis of hippocampal neurons and that blocking pro-NGF can inhibit neuronal loss after seizures.

Expression of neurotrophins in the adult spinal cord in vivo

Potential roles of trophins in the normal and injured spinal cord are largely undefined. However, a number of recent studies suggest that adult spinal cord expresses neurotrophin receptors and responds to the neurotrophins, brain‐derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3), particularly after injury. The data indicate that trophins may enhance regrowth after damage and may represent a new therapeutic approach to injury. Neurotrophins are reportedly present in the spinal cord, but the cellular localization is unknown. This information is critical to begin delineating mechanisms of actions. To approach this problem, we examined whether spinal cord glia express BDNF and NT3 in vivo and have begun to define cellular distribution. Specific antibodies directed against the neurotrophins were utilized to visualize neurotrophin protein. Initial studies indicated that small cells in the white matter of adult rat spinal cord express BDNF and NT3. Large neurotrophin‐positive neurons were also identified in the ventral cord. To identify the neurotrophin‐positive cells, co‐localization studies were performed utilizing neurotrophin polyclonal antisera together with monoclonal antibodies directed against the astrocyte marker, glial fibrillary acidic protein (GFAP). In the white matter of adult spinal cord, GFAP‐positive and GFAP‐negative cells expressed BDNF and NT3. Our study suggests that astrocyte and non‐astrocyte cells provide trophic support to the adult spinal cord. J. Neurosci. Res. 56:1–7, 1999.