Interleukin-1ß enhances neuronal vulnerability to proNGF-mediated apoptosis by increasing surface expression of p75(NTR) and sortillin.

Many types of injury such as seizure, ischemia, and oxidative stress cause upregulation of the p75 neurotrophin receptor (p75(NTR)) in brain neurons, where it promotes apoptosis, however the mechanism by which p75(NTR) is regulated under these conditions is not well understood. Proinflammatory cytokines such as interleukin-1ß (IL-1ß) are highly produced under these injury conditions and, in particular, are expressed rapidly in the rat hippocampus after seizure. IL-1ß is known to increase neuronal vulnerability under many conditions, although it does not directly induce neuronal death. Recently, we have shown that these cytokines regulate p75(NTR) induction both in neurons and astrocytes in vitro. Here, we show that IL-1ß infusion into the brain induces p75(NTR) in neurons of the CA1 area of the hippocampus. While IL-1ß induction of p75(NTR) is not sufficient to induce cell death, we demonstrate that IL-1ß primes the neurons by recruiting p75(NTR) and its coreceptor sortilin to the cell surface, making the neurons more vulnerable to subsequent challenge by proNGF. These results suggest a mechanism by which IL-1ß exacerbates neuronal death following injury.

The development of an intuitive understanding of entropy.

Previous studies of the concept of entropy have indicated that before 7 to 9 years of age, children have difficulty understanding the effects of randomizing forces and often predict highly improbable events, such as a return to the original ordered state. In the present research, 306 children between 3 and 11 years of age were asked directly whether undifferentiated forces, such as the wind or objects being thrown into the air, could create order or disorder in a set of objects. The results showed that even 4-year-olds are sensitive to the asymmetrical effects of such events. Older children apply this principle more consistently and are able to use it in explaining their answers.

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.

The development of children's knowledge of the times of future events.

Four studies with 261 children were conducted to describe 4- through 10-year-olds' ability to differentiate the future distances of events. Distances ranged from later the same day through nearly a year in the future. Judgment methods included pointing to parts of a spatial scale representing future distances and answering open-ended questions. Although 4-year-olds failed to differentiate future distances, 5-year-olds were able to distinguish events that would occur in the coming weeks and months from those that would not occur for many months. However, like young children in earlier studies of memory for time, they confused the near future with the recent past. Children 6 through 8 years of age made more differentiated judgments but collapsed the distances of events more than a few months in the future. By 8 to 10 years of age, children accurately judged distances by using mental representations of the times of events in the annual cycle.

Neurotrophins induce death of hippocampal neurons via the p75 receptor.

Nerve growth factor (NGF) and related neurotrophins influence neuronal survival and differentiation via interactions with the trk family of receptors. Recent studies have demonstrated that neurotrophins may also induce cell death via the p75 receptor. The importance and generality of neurotrophin-induced death in the brain have not been defined but may play a critical role during development and in disease-associated neuronal death. Here we demonstrate for the first time that all four members of the neurotrophin family directly elicit the death of hippocampal neurons via the p75 receptor. The hippocampus is a complex structure with many different neuronal subpopulations, and signals that influence neuronal death during development may have a critical impact on the mature function of this structure. In these studies we show that each neurotrophin causes the death of hippocampal neurons expressing p75 but lacking the cognate trk receptor. Neurotrophin-induced neuronal death is mediated by activation of Jun kinase. These studies demonstrate that neurotrophins can regulate death as well as survival of CNS neurons.

Caspase-2 mediates neuronal cell death induced by beta-amyloid.

beta-amyloid (Abeta) has been proposed to play a role in the pathogenesis of Alzheimer's disease (AD). Deposits of insoluble Abeta are found in the brains of patients with AD and are one of the pathological hallmarks of the disease. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. In our current study, treatment with nitric oxide generators protected against Abeta-induced death, whereas inhibition of nitric oxide synthase afforded no protection, suggesting that formation of peroxynitrite is not critical for Abeta-mediated death. Previous studies have shown that aggregated Abeta can induce caspase-dependent apoptosis in cultured neurons. In all of the neuronal populations studied here (hippocampal neurons, sympathetic neurons, and PC12 cells), cell death was blocked by the broad spectrum caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone and more specifically by the downregulation of caspase-2 with antisense oligonucleotides. In contrast, downregulation of caspase-1 or caspase-3 did not block Abeta(1-42)-induced death. Neurons from caspase-2 null mice were totally resistant to Abeta(1-42) toxicity, confirming the importance of this caspase in Abeta-induced death. The results indicate that caspase-2 is necessary for Abeta(1-42)-induced apoptosis in vitro.

Neurotrophin signaling via Trks and p75.

This review focuses on recent advances in our understanding of receptor-mediated signaling by the neurotrophins NGF, BDNF, NT3, and NT4/5. Two distinct receptor types have been distinguished, Trks and p75. The Trks are receptor tyrosine kinases that utilize a complex set of substrates and adapter proteins to activate defined secondary signaling cascades required for neurotrophin-promoted neuronal differentiation, plasticity, and survival. A specialized aspect of Trk/neurotrophin action in neurons is the requirement for retrograde signaling from the distal periphery to the cell body. p75 is a universal receptor for neurotrophins that is a member of the TNF receptor/Fas/CD40 superfamily. p75 appears to modify Trk signaling when the two receptor types are coexpressed. When expressed in the absence of Trks, p75 mediates responses to neurotrophins including promotion of apoptotic death. The mechanisms of p75 receptor signaling remain to be fully understood.

Caspase-dependent and -independent death of camptothecin-treated embryonic cortical neurons.

This study investigates the mechanisms underlying death of cultured embryonic cortical neurons exposed to the DNA-damaging agent camptothecin and in particular the interdependence of the roles of cyclin-dependent kinases (Cdks), caspases, and mitochondrial function. Camptothecin evokes rapid neuronal death that exhibits nuclear features of apoptosis. This death is accompanied by loss of cytochrome c and mitochondrial transmembrane potential as well as by induction of caspase-3-like activity and caspase-2 processing. The Cdk inhibitor flavopiridol provides long-term rescue from death and prevents loss of cytochrome c and mitochondrial transmembrane potential as well as caspase activation and processing. General caspase inhibitors rescue neurons from this rapid apoptotic death but do not prevent them from undergoing delayed death in which nuclear features of apoptosis are absent. Moreover, the caspase inhibitors do not affect early cytochrome c release and delay but do not prevent the loss of transmembrane potential. Agents that directly disrupt mitochondrial function without inducing cytochrome c release lead to a caspase-independent death. These observations favor a model in which (1) DNA damage leads to Cdk activation, which lies upstream of release of cytochrome c and caspase activation; (2) cytochrome c release is caspase-independent and may occur upstream of caspase activation; (3) early apoptotic death requires caspases; and (4) delayed nonapoptotic death that occurs in the presence of caspase inhibitors is a consequence of prolonged loss of mitochondrial function. These findings shed light on the mechanisms by which DNA damage kills neurons and raise questions regarding the general utility of caspase inhibitors as neurotherapeutic agents.

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.

Changes in the subjective properties of autobiographical memories with the passage of time.

This study was an investigation of how the subjective properties of autobiographical memories change over time. Over the 25 weeks after Thanksgiving, undergraduates rated the quality of their memories for Thanksgiving dinner on three global scales and six other scales referring to specific kinds of information (conversations, people, food, and clothing). Global ratings declined rapidly in the first 12 weeks but showed little change in the subsequent 12 weeks. The highest ratings and the least decline were found for information that could be reconstructed from general knowledge of Thanksgivings (food and people present). Ratings for non-schematic information showed patterns of decline consistent with previous studies and with humans' ability to discriminate the temporal distances of past events.

Neuroprotective actions of dipyridamole on cultured CNS neurons.

We report that dipyridamole is neuroprotective for a variety of rat embryonic CNS neurons cultured in serum-free basal medium lacking trophic factors or other additives. We also describe the mechanism underlying this action. Neurons died rapidly in basal medium but were rescued in large measure by 10 microM dipyridamole. The protective action of dipyridamole seems to be attributable to its antioxidant property. Vitamin E and N-acetylcysteine provided comparable neuroprotection in basal medium, whereas an array of compounds that mimic other actions of dipyridamole (inhibition of phosphodiesterases, blockade of nucleoside and chloride transport, interference with the multidrug resistance protein, and enhancement of prostacyclin synthesis) failed to promote survival. Thus, a major cause of neuronal death in this system seems to be oxidative stress that is relieved by dipyridamole. Iron plays a significant role in generation of such stress, as indicated by the observations that addition of apotransferrin or iron chelators to basal medium or use of iron-free medium also afforded protection. Although oxidative stress was a major determinant of neuronal death, it was not the only factor. Dipyridamole or other antioxidant measures did not provide sustained neuroprotection. However, provision of insulin, which was not protective alone in basal medium, along with dipyridamole significantly enhanced long-term neuronal survival. Hence, optimal protection requires both trophic support and relief from oxidative stress. These findings lend credence to the potential use of dipyridamole or its derivatives in prevention and/or treatment of CNS injuries and degenerative disorders in which oxidative stress is a significant component.

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.

Regulation of nerve growth factor mRNA by interleukin-1 in rat hippocampal astrocytes is mediated by NFkappaB.

Cytokines such as interleukin-1beta (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 NFkappaB 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.

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 mRNA's 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 mRNA's 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 mRNA's. 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.

Synergistic trophic actions on rat basal forebrain neurons revealed by a synthetic NGF/BDNF chimaeric molecule.

Basal forebrain cholinergic neurons, which degenerate in Alzheimer's disease, respond to multiple trophic factors, including the neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). This dual responsiveness prompted us to investigate the effects of a synthetic chimaeric molecule, containing the active domains of both NGF and BDNF. The NGF/BDNF chimaeric factor exhibited synergistic actions, and was 100-fold more potent than wild-type BDNF in enhancing survival of cultured dissociated basal forebrain cholinergic neurons. This effect was apparently due to true BDNF/NGF synergy, since addition of the two wild-type trophins simultaneously reproduced the effect of the chimaera. Synergy was selective for neurons which respond to both factors; substantia nigra dopaminergic neurons, which respond to BDNF but not NGF, exhibited no potentiation. The chimaeric factor thus revealed a synergy that may normally occur in the brain, and constitutes a potentially novel therapeutic agent with greater potency than naturally occurring individual trophins.

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.

Differential actions of neurotrophins in the locus coeruleus and basal forebrain.

The neurotrophin gene family, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4/NT-5, supports the survival of distinct peripheral neurons, however, actions upon central neurons are relatively undefined. In this study we have compared different neurotrophins in the regulation of neuronal survival and function using dissociated embryonic cell cultures from two brain regions, the basal forebrain (BF) and locus coeruleus (LC). In the BF, NGF increased choline acetyl transferase (ChAT) activity, but did not influence cholinergic cell survival. In contrast to NGF, BDNF, NT-3, and the novel neurotrophin, NT-4, all increased ChAT activity and cholinergic cell survival. We also examined embryonic LC neurons in culture. LC neurons are unresponsive to NGF. In contrast, NT-3 and NT-4 elicited significant increases in survival of noradrenergic LC neurons, the first demonstration of trophic effects in this critical brain region. Identification of factors supporting coeruleal and basal forebrain neuronal survival may provide insight into mechanisms mediating degeneration of these disparate structures in clinical disorders.

Mechanisms of nerve growth factor mRNA regulation by interleukin-1 beta in hippocampal cultures: role of second messengers.

Cytokines such as interleukin-1, which are found in the brain after trauma, regulate expression of nerve growth factor (NGF) mRNA and protein in hippocampal cultures. We have investigated possible mechanisms by which Il-1 beta regulates NGF in hippocampal cells. The induction of NGF mRNA by Il-1 beta was blocked by a receptor antagonist indicating that this effect is receptor mediated. Il-1 beta elicited a dramatic induction of c-fos mRNA and a slight elevation of c-jun mRNA in a time dependent manner which may allow for a role in the induction of NGF mRNA expression. We examined whether specific second messenger pathways were involved in mediating the action of Il-1 beta in the hippocampus. Activation of cAMP with forskolin or treatment with 8-Br-cAMP had no effect on NGF mRNA levels. Moreover, exposure of hippocampal cultures to Il-1 beta evoked no change in cAMP levels, indicating that this second messenger system played little or no role in the regulation of NGF expression by Il-1 beta in these cells. Further, interleukin-1 elicited no change in membrane inositol phosphate turnover, nor did it affect intracellular calcium levels. Treatment of cell cultures with the phorbol ester PMA elicited an increase in NGF mRNA, suggesting that activation of protein kinase C (PKC) may mediate NGF mRNA expression. However, prolonged treatment of cultures with PMA to desensitize PKC did not eliminate the Il-1 beta induction of NGF mRNA. Il-1 beta, therefore, did not appear to activate NGF expression via cAMP, Ca2+, or a PKC isoform that is downregulated by prolonged PMA treatment. However, a phosphorylation event may be involved in the signal transduction mechanism, as treatment with okadaic acid to inhibit protein phosphatase 2a potentiated the induction of NGF mRNA by Il-1 beta. The results presented indicate that Il-1 beta acts via its receptor to induce a rise in NGF expression. Identification of the specific second messenger pathway has remained elusive; however, a phosphorylation event appears to be intermediary. Moreover, the induction of c-fos and c-jun may represent a final common path in activation of NGF gene expression by different signals such as Il-1 beta and PMA.

Temporal and spatial expression of NGF receptor mRNA during postnatal rat brain development analyzed by in situ hybridization.

The expression of nerve growth factor (NGF) receptor mRNA was examined in the rat brain during postnatal development using in situ hybridization. Cells expressing NGF receptor mRNA were detected in the basal forebrain at all ages examined, with a peak in expression at 2 weeks of age. NGF receptor mRNA was further demonstrated to be expressed transiently in several brainstem nuclei. Expression of NGF receptor mRNA was high at postnatal day (P) 1 and 1 week of age in the facial and abducens nuclei, but was undetectable in the facial nucleus by 2 weeks of age. In the abducens nucleus, a few labeled cells were still present at 2 weeks of age, but absent by 3 weeks. In the cerebellum, a strong signal was present at P1 and 1 week of age which clearly diminished by 2 weeks and disappeared by 3 weeks of age. The labeled cells in the cerebellum had the size and morphology of developing Purkinje cells. These data suggest that the population of NGF-responsive cells in the brain is more widespread during development than in the adult, and that the trophic requirements of specific brain regions are altered with maturity.

Transient and persistent expression of NT-3/HDNF mRNA in the rat brain during postnatal development.

Neurotrophin-3 (NT-3) is closely related to two known neurotrophic agents, NGF and brain-derived neurotrophic factor (BDNF), and acts upon overlapping, yet distinct, populations of peripheral ganglia. NT-3 mRNA expression in the adult rat brain is largely confined to the hippocampus. In this study, we have used in situ hybridization to examine expression of this novel neurotrophic factor during postnatal development. The striking observation was made that NT-3 mRNA was transiently expressed at high levels in the cingulate cortex during the first 2 weeks of age. In the hippocampus, the adult pattern of expression, in the CA2, medial CA1, and granule layer of the dentate gyrus, was detected at all ages examined. However, there were two major differences in NT-3 mRNA expression in the developing hippocampus: Labeled cells were detected in the hilar region of the dentate gyrus at postnatal day 1 (P1) and 1 week that were absent by 2 weeks of age. Further, the caudal hippocampus, which has a lower intensity of labeling than the rostral region in the adult, was devoid of NT-3-expressing cells in the P1 and 1-week-old rat brain. These data indicate a substantial plasticity in NT-3 mRNA expression and suggest that the spectrum of neurons supported by NT-3 during development is partially different from that in the mature rat brain.