Age-dependent time course of cerebral brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3 in APP23 transgenic mice.

Neurotrophins, including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3), have repeatedly been shown to be involved in the pathophysiology of Alzheimer's disease (AD). Recent studies have claimed that these neurotrophic factors are important tools for therapeutic intervention in neurodegenerative diseases. So far, little is known about the age- and disease-modulated time course of cerebral neurotrophins. Therefore, we have studied protein concentrations of BDNF, NGF, and NT-3 in different brain areas and sciatic nerve, a neurotrophin-transporting peripheral nerve, in a well-characterized AD model of amyloid precursor protein-overexpressing rodents (APP23 mice) at the ages of 5.0, 10.5, and 20.0 months. In APP23 mice, there was a significant increase of BDNF and NGF in the frontal and occipital cortices (for BDNF also in the striatum) of old 20.0-month-old mice (with respect to median values up to 8.2-fold), which was highly correlated with amyloid concentrations of these brain areas. Median values of NGF and NT-3 showed up to a 6.0-fold age-dependent increase in the septum that was not detectable in APP23 mice. Hippocampus, olfactory bulb, and cerebellum (except NT-3) did not show substantial age- or genotype-related regulation of neurotrophins. In the sciatic nerve, BDNF and NGF levels are increased in5-month-old APP23 mice and decrease with age to control levels. In conclusion, APP23 mice show a genotype-dependent increase of cortical BDNF and NGF that is highly correlated with amyloid concentrations and may reflect an amyloid-related glia-derived neurotrophin secretion or an altered axonal transport of these neurotrophic factors.

GABA(B) receptor expression and cellular localization in gerbil hippocampus after transient global ischemia.

Using in situ hybridization, the expression of the GABA receptor subtype B subunit 1 (GABA(B) R1) and subunit 2 (GABA(B) R2) following transient global ischemia in the gerbil hippocampus was investigated. In sham-operated animals, mRNAs of both subunits were mainly detected in hippocampal pyramidal cells and interneurons with lower expression levels of the GABA(B) R2 in the CA1 field. Four days after transient cerebral ischemia, neuronal message decreased in conjunction with neuronal death and both receptor subunits disappeared from the pyramidal cell layer. However, GABA(B) R1 and GABA(B) R2 were still expressed in a few cells. In situ hybridization of the GABA synthesizing enzyme glutamic acid decarboxylase 67 (GAD67) remained unchanged after the ischemic insult. Double-labeling experiments revealed that in the postischemic hippocampus GABA(B) R1 and GABA(B) R2 were not present in GFAP-reactive astrocytes, but that the surviving parvalbumin-containing interneurons possessed GABA(B) R1 and GABA(B) R2 mRNA.

Beta-amyloid modulates tyrosine kinase B receptor expression in SHSY5Y neuroblastoma cells: influence of the antioxidant melatonin.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in humans and is characterized by neuronal loss, neurofibrillary tangles and beta-amyloid deposition. The interaction between neurotrophins and their tyrosine kinase (trk) receptors is important for cellular differentiation and survival. Interestingly, marked reductions in neurotrophins and receptors have been reported in AD. The cause of the decrease in these molecules remains unclear. However, the role of beta-amyloid (A beta) appears central in understanding the mechanisms controlling neurotrophin/trk expression. In this study we exposed SHSY5Y neuroblastoma cells to A beta or hydrogen peroxide and measured the expression of trk B/truncated trk B, and brain-derived neurotrophic factor (BDNF)/NT4 at the protein and molecular level. We show that A beta or hydrogen peroxide (H(2)O(2)) induces oxidative stress and cell cytotoxicity. The exposure of cells to A beta results in an increased trk B expression with a concurrent reduction in truncated trk B levels. H(2)O(2) exposure decreased both trk B and truncated trk B levels at the cell surface. At the molecular level trk B RNA increased in the presence of A beta and was unaffected by H(2)O(2). Similarly, BDNF and NT4 levels increased in the presence of A beta. Pre-treatment of cells with the anti-oxidant melatonin returns trk receptor expression, mRNA and BDNF/NT4 secretion to normal levels. These results are significant as they can help in the planning and implementation of AD treatment strategies involving neurotrophins.

Oxidative stress modulates tyrosine kinase receptor A and p75 receptor (low-affinity nerve growth factor receptor) expression in SHSY5Y neuroblastoma cells.

The interaction of neurotrophins and their tyrosine kinase receptors (trks) is essential for differentiation and survival of brain cells. In Alzheimer's disease (AD), the number of neurotrophins and receptors is markedly decreased. The cause of this reduction is unclear, but the role of beta-amyloid (Abeta) seems central in understanding the mechanisms controlling neurotrophin and trk expression. In the study reported here, we exposed SHSY5Y neuroblastoma cells to Abeta or hydrogen peroxide (H(2)O(2)) and measured the expression of trk-A and p75 at the protein and molecular levels. Both Abeta and H(2)O(2) induced oxidative stress (measured by a decrease in cellular glutathione), which decreased trk-A levels and increased p75 levels, decreased messenger RNA (mRNA) levels of both receptors, and increased nerve growth factor (NGF) secretion. Pretreatment of cells with the antioxidant melatonin returned levels of protein expression, mRNA, and NGF secretion to normal. These results are significant, as they can help in the planning and implementation of AD treatment strategies involving neurotrophins.

Anti-inflammatory activity of nerve growth factor in experimental autoimmune encephalomyelitis: inhibition of monocyte transendothelial migration.

In order to analyze a putative immunomodulatory effect of NGF in experimental autoimmune encephalomyelitis (EAE) of the Lewis rat, we transduced myelin basic protein (MBP)-specific CD4(+) T cells with a recombinant retrovirus encoding NGF. These T(MBP)NGF cells secreted high levels of NGF, along with an unaltered Th1-like cytokine pattern. Transfer studies showed that T(MBP)NGF cells were unable to mediate clinical EAE, when transferred alone, and, more important, they efficiently suppressed induction of clinical EAE by non-transduced MBP-specific T cells (T(MBP )cells). In contrast, NGF transduced ovalbumin-specific T cells, which secreted high NGF levels, did not affect EAE induction. Suppression of clinical EAE by T(MBP)NGF cells was associated with a general reduction of inflammatory CNS infiltrates, with a most pronounced decrease of the monocyte/macrophage component. Using a culture model of the endothelial blood-brain barrier (BBB), we found that NGF directly acts on blood-derived monocytes via the p75 NGF receptor, thus interfering with monocyte migration through the activated BBB endothelium. Our data establish NGF as an anti-inflammatory mediator interfering with T cell mediated autoimmune disease in the CNS. They further point to monocyte migration through blood vascular endothelium as one possible mechanism of NGF action.

Proteolytic cleavage of inducible nitric oxide synthase (iNOS) by calpain I.

Proteolytic degradation of inducible nitric oxide synthase (iNOS or NOS2; EC 1.14.13.39) is one of the key steps by which the synthetic glucocorticoid dexamethasone controls the amount of iNOS protein and thus the production of nitric oxide (NO) in interferon-gamma-stimulated RAW 264.7 cells. In the present study we examined the role of the calmodulin (CaM)-binding site present within iNOS protein for the proteolytic degradation by the calcium-dependent neutral cysteine protease calpain I (EC 3.4.22.17). Using pulse chase experiments as well as cell-free degradation assays we show that the iNOS monomer is a direct substrate for cleavage by calpain I. Two structural determinants are involved in proteolytic cleavage, the canonical CaM-binding domain present at amino acids 501-532 and a conformational determinant located within iNOS. The access of the CaM-binding region appears to be critical for substrate cleavage as incubation of in vitro synthesized iNOS with purified CaM inhibits iNOS degradation by calpain I. Moreover, cytosolic CaM levels are decreased upon treatment of RAW 264.7 cells with dexamethasone as assessed by immunoprecipitation. The data shown herein provide novel insights into the underlying mechanisms involved in the anti-inflammatory actions of glucocorticoids.

Expression of interleukin-6 and its receptor in the sciatic nerve and cultured Schwann cells: relation to 18-kD fibroblast growth factor-2.

Expression of interleukin-6 (IL-6) and fibroblast growth factor-2 (FGF-2) in Schwann cells is modulated by external stimuli. To study possible interactions of both factors we have analyzed mutual effects of exogenous IL-6 and FGF-2 on the expression of each other and the corresponding receptor (R) molecules IL-6R and FGFR1 after peripheral nerve lesion in vivo and in vitro using cultured Schwann cells. Using rat Schwann cells we found that IL-6 did not exert any effects on the expression of FGF-2 and FGF receptor type 1 (R1) whereas exogenously applied 18-kD FGF-2 strongly increased the expression of the mRNAs of IL-6 and its receptor. In addition, immortalized Schwann cells over-expressing the 18-kD FGF-2 isoform showed elevated levels of IL-6 and IL-6R whereas immortalized Schwann cells over-expressing the high-molecular-weight isoforms (21 kD and 23 kD) displayed unaltered IL-6 and IL-6R expression levels. According to in situ hybridization studies of intact and crushed sciatic nerves in vivo, Schwann cells seems to be the main source of IL-6 and IL-6R. Following sciatic nerve crush, the FGF-2 and the IL-6 system are upregulated after the first hours. Furthermore, we showed that the early increase of the FGF-2 protein is mainly confined to the 18-kD isoform. These results are consistent with the idea of a functional coupling of FGF-2 and the IL-6 system in the early reaction of Schwann cells to nerve injury.

Increased cerebrospinal fluid levels of neurotrophin 3 (NT-3) in elderly patients with major depression.

Neurotrophin 3 (NT-3) is a member of the neurotrophin gene family which supports the survival of specific neurons. NT-3 was shown to prevent the death of adult central noradrenergic neurons in vivo, a neuronal population which is associated with the pathophysiology of major depression. We quantitated CSF levels of NT-3 in elderly patients with major depression (DE) and compared them to patients with Alzheimer's disease (AD), and mentally healthy control subjects (CTR). CSF levels of NT-3 were markedly and significantly elevated in the DE group, as compared to either the AD or the CTR group (P < 0.01, and P < 0.001, respectively). In terms of diagnostic accuracy, measurement of NT-3 levels in DE resulted in 73.9% sensitivity, and 89.7% specificity. Increased CSF levels of NT-3 may indicate a disturbance of the central noradrenergic system in patients with DE. NT-3 may constitute a biochemical candidate marker for clinical diagnosis and for the evaluation of therapeutic strategies in DE.

Production of neurotrophins by activated T cells: implications for neuroprotective autoimmunity.

Neurotrophins (NTs) promote neuronal survival and maintenance during development and after injury. However, their role in the communication between the nervous system and the immune system is not yet clear. We observed recently that passively transferred activated T cells of various antigen specificities home to the injured central nervous system (CNS), yet only autoimmune T cells specific to a CNS antigen, myelin basic protein (MBP), protect neurons from secondary degeneration after crush injury of the rat optic nerve. Here we examined the involvement of NTs in T-cell-mediated neuroprotection, and the possible significance of the antigen specificity of the T cells in this activity. Analysis of cytokine and NT expression in various rat T cell lines showed that the T cells express mRNA for cytokines of Th1, Th2, and Th3 phenotypes. In addition, the T cells express mRNA and protein specific to nerve growth factor, brain-derived neurotrophic factor, NT-3, and NT-4/5. Antigen activation significantly increased NT secretion. Thus, reactivation of CNS autoimmune T cells by locally presented antigens to which they are specific can lead to enhanced secretion of NTs and possibly also of other factors in injured optic nerves. mRNA for TrkA, TrkB and p75 receptors was expressed in the injured nerve, suggesting that these specific receptors can mediate the effects of the T-cell-derived NTs. The neuroprotective effect of the passively transferred autoimmune anti-MBP T cells in injured optic nerves was significantly decreased after local applicaiton of a tyrosine kinase inhibitor known to be associated with NT-receptor activity. These results suggest that the neuroprotective effect of autoimmune T cells involves the secretion of factors such as NTs by the T cells reactivated by their specific antigen in the injured CNS. T cell intervention in the injured CNS might prove to be a useful means of promoting post-injury CNS maintenance and recovery, possibly via supply of NTs and other factors.

Alterations in trk A, trk B and trk C receptor immunoreactivities in parietal cortex and cerebellum in Alzheimer's disease.

The neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 bind to the tyrosine kinase (trk) receptors trk A, trk B and trk C, respectively, with high affinity. We investigated the expression of the trk receptors in the parietal cortex (PC) and cerebellum of patients with Alzheimer's disease (AD) and age-matched controls. Cortical layers II-VI displayed a distinct cellular immunoreactivity for trk A and C with an emphasis in the pyramidal neurons of layers III and V. Trk B immunoreactivity was primarily located in the deeper cortical layers with a predominance in layer V. There was a decrease in trk A and C immunoreactivity in the PC of AD cases, while trk B density appeared to be unchanged. In addition, cerebellar Purkinje cells revealed a distinct immunoreactivity for trk C both in control and AD cases, suggesting trk C may be important in the maintenance of these cells in the aged brain.

Alterations in neurotrophins and neurotrophin receptors in Alzheimer's disease.

We demonstrated that nerve growth factor (NGF) levels were increased in hippocampus and cortical areas, as well as in cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD). Such increases may, at least in part, be due to a decreased expression of the NGF high affinity receptor trkA. Measurement of CSF levels of NGF may add to the repertoire of potential biochemical diagnostic markers in living AD patients.

Region-specific neurotrophin imbalances in Alzheimer disease: decreased levels of brain-derived neurotrophic factor and increased levels of nerve growth factor in hippocampus and cortical areas.

BACKGROUND: Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5 (NT-4/5) are members of the neurotrophin gene family that support the survival of specific neuronal populations, including those that are affected by neurodegeneration in Alzheimer disease (AD). OBJECTIVE: To determine whether neurotrophin protein levels are altered in the AD-affected brain compared with control brains. METHODS: We quantitated protein levels of NGF, BDNF, NT-3, and NT-4/5, and calculated neurotrophin/NT-3 ratios in AD-affected postmortem hippocampus, frontal and parietal cortex, and cerebellum, and compared them with age-matched control tissue (patients with AD/controls: hippocampus, 9/9 cases; frontal cortex, 19/9; parietal cortex, 8/5; and cerebellum, 5/7, respectively). We applied highly sensitive and specific enzyme-linked immunosorbent assays in rapid-autopsy-derived brain tissue (mean+/-SD postmortem interval, 2. 57+/-1.75 h, n=71) to minimize postmortem proteolytic activity. RESULTS: Levels of BDNF were significantly reduced in hippocampus and parietal cortex (P<.001, and P<.01) as well as BDNF/NT-3 ratios in frontal and parietal cortices (P<.05, and P<.01) in the group with AD compared with the control group. Levels of NGF and NGF/NT-3 ratio were significantly elevated in the group with AD compared with the control group in the hippocampus and frontal cortex (P<.001). Levels of NT-4/5 and the NT-4/NT-3 ratio were slightly reduced in hippocampus and cerebellum in the group with AD compared with the control group (P<.05). In contrast, the levels of NT-3 were unchanged in all brain regions investigated. CONCLUSION: Decreased levels of BDNF may constitute a lack of trophic support and, thus, may contribute to the degeneration of specific neuronal populations in the AD-affected brain, including the basal forebrain cholinergic system. Arch Neurol. 2000.

Increased CSF levels of nerve growth factor in patients with Alzheimer's disease.

The authors quantitated CSF levels of nerve growth factor (NGF) in patients with AD, nondemented control subjects (CTR), and age-matched patients with major depression (DE). CSF levels of NGF were markedly higher in the AD group than in both the CTR and DE groups (p < 0.01 and p < 0.001). Increased CSF levels of NGF in AD patients may reflect reported accumulation of NGF in the AD brain and may constitute a candidate marker for clinical diagnosis and therapeutic monitoring.

GABA(B) receptor antagonists elevate both mRNA and protein levels of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) but not neurotrophin-3 (NT-3) in brain and spinal cord of rats.

In this study we show that single, physiologically-active and non-convulsive doses of the three GABA(B) receptor antagonists CGP 36742, CGP 56433A and CGP 56999A increase NGF and BDNF mRNA levels by 200-400% and protein levels by 200-250% in rat neocortex, hippocampus as well as spinal cord. In all areas examined the increase in NGF protein preceded that of BDNF. Peak levels of both neurotrophins are transient and occur between 24 and 72 h, depending on the region. In contrast, NT-3 protein concentrations in the neocortex and hippocampus were decreased significantly to 50% of control values within 48-96 h. The decrease in the spinal cord was less than 30% and did not reach significant levels. These data clearly demonstrate that GABA(B) receptor antagonists induce a specific neurotrophin expression in the central nervous system at physiologically relevant doses, as opposed to the extreme conditions of seizure paradigms. The results are in line with the concept that neuronal neurotrophin synthesis and release in brain are controlled by afferent nerve activity. GABA(B) receptor antagonists could therefore be a valuable new approach to selectively increase endogenous neurotrophin levels in the central nervous system.

Cytokines and neurotrophins interact in normal and diseased states.

Neurotrophins (NTs) such as nerve growth factor (NGF) as well as cytokines, for example, interleukin-6 (IL-6), are communicators between the nervous and immune systems. There is evidence for mutual interactions between NTs and cytokines. Strategies are being developed to elucidate the molecular mechanism/s of interactions and to understand how cytokines are involved in health and disease. Analysis of underlying signaling pathways in glial cells indicates that different transcription factors, such as NF-kappa B, cAMP-responsive-element binding protein (CREB), and activator protein 1 (AP-1), are involved in NT induction. IL-6 and NTs of the NGF family are coexpressed at sites of nerve injury. Interactions of these factors could modulate both neuronal de- and regeneration: IL-6 in conjunction with its soluble IL-6 receptor induces a specific pattern of NTs in astrocytes in defined brain regions. This indicates that the IL-6 system mediates a local supply of NTs that participate in diverse CNS functions, such as protection of neurons from insults, neuronal survival, and neuroimmune responses.

Neural activities of IL-6-type cytokines often depend on soluble cytokine receptors.

Cytokines of the interleukin-6 (IL-6) family participate in regulatory and inflammatory processes within the nervous system. IL-6, ciliary neurotrophic factor (CNTF) and IL-11 act via specific membrane receptors which, together with their ligands, associate with signal-transducing receptor subunits thereby initiating cytoplasmic signalling. Cells which only express signal-transducing receptor subunits but no ligand binding subunits for IL-6, CNTF and IL-11 are refractory to these cytokines. An unusual feature of the IL-6 cytokine family is that the soluble forms of the ligand binding receptor subunits generated by one cell type in complex with their ligands can directly stimulate the signal-transducing receptor subunits on different cell types which lack ligand binding receptor subunits. This process has been named transsignalling. This article focuses on the importance of transsignalling events in neuronal differentiation and survival responses.

Modulation of mRNA expression of the neurotrophins of the nerve-growth-factor family and their receptors in the septum and hippocampus of rats after transient postnatal thyroxine treatment. II. Effects on p75 and trk receptor expression.

Early postnatal application of thyroid hormones to rats results in morphological changes of the septo-hippocampal cholinergic and the hippocampal mossy fiber systems. Modulation in the expression of either neurotrophins and/or their receptors is postulated to be involved in these effects. In a recent study, we showed that, after thyroxine application, the mRNA expression of neurotrophins of the nerve-growth-factor (NGF) family is significantly upregulated both in septum and hippocampus. To test whether the neurotrophin receptors (the low-affinity neurotrophin receptor p75 and the specific high-affinity receptors trkA, trkB, and trkC) were also affected by hormone administration, newborn rats were treated daily with subcutaneous injections of thyroxine until postnatal day 12 (P12) at latest. Control animals received corresponding injections of saline. The pups were sacrificed at defined intervals from P9 to P14. The septal areas and the hippocampi were analyzed using the reverse-transcription polymerase chain reaction (RT-PCR) method for quantification of p75, trkA, trkB, and trkC mRNA levels. Analysis of variance over the total investigation period revealed no significant general increases of the gene expressions of either neurotrophin receptor, neither in the septum nor in the hippocampus, although previous results have shown marked changes in neurotrophin levels. On particular postnatal days, significant upregulation could be observed in hippocampus for trkB and trkC. From these and recent data, we conclude that modulation of neurotrophin expression rather than neurotrophin-receptor expression contributes to the morphological modifications within the hippocampal mossy fiber system and the septo-hippocampal cholinergic system.

Role of interleukin-6 and soluble IL-6 receptor in region-specific induction of astrocytic differentiation and neurotrophin expression.

Increasing evidence supports an essential role for interleukin-6 (IL-6) in the development, differentiation, as well as de- and re-generation of neurons in the central nervous system (CNS). Both IL-6 and its specific receptor (IL-6R) are expressed on neurons and glial cells including astrocytes. In this study, we have analyzed the responses of primary rat astrocytes of various brain regions to IL-6 with respect to morphological changes and neurotrophin expression. Since IL-6 alone failed to initiate effects on astrocytes, we have examined whether the soluble IL-6R (sIL-6R) can modulate the responsiveness of to IL-6 in these cells. For this purpose, we used a highly active fusion protein of IL-6 and sIL-6R, which is designated Hyper-IL-6 (H-IL-6). We show that treatment of cultured astrocytes with Hyper-IL-6 promotes region-specific morphological changes of GFAP-positive astrocytes from typical stellate- to fibrous-like cells. In addition, we find that Hyper-IL-6 induces expression of neurotrophins (NTs) of the nerve growth factor (NGF)-family in a dose-dependent manner. Interestingly, astrocytes of various brain regions show differing patterns of cytokine-induced NT expression: NGF is maximally induced in cortex and hippocampus, NT-3 in hippocampus, and NT-4/5 in cortex and cerebellum. In summary, our results indicate that IL-6 in conjunction with sIL-6R regulates specific neurotrophin expression in astrocytes in a brain region dependent manner. Thus, the IL-6 system provides a local supply of neurotrophins that participate in diverse CNS functions such as protection of neurons from insults, neuronal survival, and neuro-immune responses.

Interleukin-6 (IL-6) and its soluble receptor support survival of sensory neurons.

The cytokine interleukin-6 (IL-6) has multiple functions in the immune and hematopoietic systems. IL-6 is related to ciliary neurotrophic factor (CNTF), a trophic factor for motoneurons, sensory dorsal root ganglion (DRG) neurons, and other neuronal subpopulations. Both act via related receptor complexes, consisting of one ligand-specific alpha-receptor subunit (IL-6R and CNTFR, respectively) and two signal-transducing receptor components. Even though IL-6 is expressed by neurons and glia, the functions of IL-6 in the nervous system are poorly understood. Here, we report that exogenous human IL-6 promotes the survival of dissociated newborn rat DRG neurons in vitro if supplemented with soluble human IL-6-alpha-receptor. The dosages of human IL-6 and soluble human IL-6R necessary to achieve neurotrophic effects could be reduced markedly by linking ligand and alpha-receptor component in a designer cytokine. Furthermore, we show that newborn rat DRG neurons express and secrete bioactive IL-6. Endogenously secreted IL-6 does not enhance survival of these neurons in vitro, suggesting that DRG neurons do not sufficiently express cell surface IL-6R. Exogenously added soluble rat IL-6R rendered DRG neurons responsive to secreted IL-6. Our results indicate an autocrine function of IL-6 in DRG neuron survival which depends on membrane-bound or soluble IL-6R as a neurotrophic cofactor.