Search

K-252 compounds, which share a common polyaromatic aglycon structure, are rather general and potent inhibitors of various protein kinases, including protein kinase C and tyrosine-specific protein kinases, and possibly act by interfering at or near the ATP binding site. However, chemical modifications in their sugar moiety can result in high specificity of the inhibitory action and, furthermore, can induce other stimulatory and inhibitory effects on nerve cells. These compounds are of particular interest because, in intact cells, they inhibit the actions of NGF and other neurotrophins without diminishing comparable actions of other growth factors. This effect seems to reflect a direct inhibitory action on trk neurotrophin receptor proteins. At concentrations lower than those necessary to inhibit neurotrophin actions, K-252a and K-252b have been shown to potentiate the stimulatory effects of NT-3 on different neurons in culture and on PC12 cells. The structural requirements for this effect seem to be different from those for the inhibition of neurotrophin actions. These findings raise the possibility of development of compounds of high selectivity, able to inhibit or potentiate the transduction mechanisms of individual neurotrophins, and identify K-252a and K-252b as lead compounds for the development of such selective molecules. Specific inhibitors and stimulators of neurotrophins would be valuable tools to investigate biological functions of the neurotrophins in vitro and in vivo. Furthermore, it is possible that, in the future, highly selective drugs with agonistic or antagonistic actions on neurotrophin mechanisms could become therapeutically useful in the treatment of neurological disease and injury.

Ca 2+ currents and their modulation by neurotrophin-4/5 were studied in cultured mesencephalic neurons. Tyrosine hydroxylase-positive neurons consistently had larger somas than tyrosine hydroxylase-negative neurons. Neurons with larger somas were therefore targeted for recording. In both control and neurotrophin-4/5-treated cultured neurons, isolation of Ca 2+ currents in cultured mesencephalic neurons revealed prominent low- and high-voltage-activated currents. These currents were separable based upon their voltage dependence of activation, the response to replacement of Ca 2+ with Ba 2+ and the response to Ca 2+ channel blockers. Replacement of Ca 2+ with Ba 2+ resulted in a slight reduction of low-voltage-activated currents and a significant enhancement of high-voltage-activated currents. Cd 2+ blocked a larger fraction of the high-voltage-activated current than Ni 2+ . The synthetic conotoxins SNX-124 and SNX-230 selectively blocked high-voltage-activated currents. Morphological analysis of mesencephalic cultures pretreated with neurotrophin-4/5 revealed an increase in soma size and dendritic length in tyrosine hydroxylase-positive neurons. In agreement with the neurotrophin-4/5 induction of growth, neurotrophin-4/5 also increased cell capacitance in whole-cell recordings. Neurotrophin-4/5 significantly enhanced both low- and high-voltage-activated currents, but normalization for changes in capacitance revealed only a significant increase in high-voltage-activated current density. This study demonstrates the existence of low-voltage-activated and multiple classes of high-voltage-activated calcium currents in cultured mesencephalic neurons. Morphological and physiological data demonstrate that the increases in calcium currents due to neurotrophin-4/5 pretreatment are associated with somatodendritic growth, but an increase in high-voltage-activated Ca 2+ channel expression also occurred.

In the spontaneous ataxic mutant mouse stargazer, there is a selective reduction of brain-derived neurotrophic factor (BDNF) mRNA expression in the cerebellum. BDNF protein levels in the cerebellum are reduced by 70%. Despite normal levels of full-length and truncated TrkB receptor, constitutive and neurotrophin-4/5-induced tyrosine phosphorylation was significantly reduced in several signal transduction molecules, including phospholipase-Cgamma1, erk1, and erk2. Morphological examination revealed an increased number of external granule cells at postnatal day 15 and the presence of abnormal neurons resembling immature granule cells in the adult. These abnormalities are associated with a severe impairment in the acquisition of classical eyeblink conditioning, indicating cerebellar malfunction. Our data suggest that normal BDNF expression and TrkB signal transduction in the cerebellum are necessary for learning and plasticity in this model.

Metabolic compromise with systemic 3-nitropropionic acid (3-NP) results in the degeneration of striatal cells, mimicking the pathology of Huntington's disease (HD). Here we show that 10-week- and 8-month-old BALB/c ByJ mice show an unexpected striatal resilience to single and multiple systemic injections of 3-NP, while Sprague–Dawley rats are vulnerable, albeit in a variable manner. Identification of lesions was made by staining of DNA fragmentation with terminal deoxytransferase-mediated dUTP–biotin nick-end labeling (TUNEL) and hematoxylin/eosin, 1–10 days after injection. Quantitative imaging of histochemistry for succinate dehydrogenase (SDH) activity, the target of 3-NP inhibition, revealed that vulnerable rats reached maximal inhibition in brain at 1 day after 3-NP, whereas mice and resilient rats took 7 days to reach maximal inhibition. All groups of animals reached similar maximal decreases in SDH activity in striatum and cortex. Remarkably, only the fast decline in SDH activity seen in vulnerable rats was associated with TUNEL labeling. In addition, vulnerable rats developed a region within striatum where SDH activity was fully depleted and a similarly depleted region in CA1 hippocampus. While mice did not develop this region in striatum, some developed one in CA1. These regions of SDH depletion in both structures were associated with widespread TUNEL staining, with maximal labeling at 3 days after 3-NP. The existence of an animal strain resilient to 3-NP suggests that there are mediating factors involved in the preferential vulnerability of striatum to metabolic lesioning. The identification of these factors could provide strategies for therapeutic intervention in HD.

The laminin-alpha2 chain is a component of brain capillary basement membranes and appears also to be present in neurons of rat, rabbit, pig and non-human primate brain as evidenced by immunohistochemistry. In the present study, we have further characterized this very distinct neuronal laminin-alpha2 chain-like immunoreactivity in the hippocampus of various species. Immunoelectron microscopy with poly- and monoclonal antibodies to the laminin-alpha2 chain G-domain localized laminin-alpha2 chain immunoreactivity in adult rat and rabbit hippocampus to dendritic processes, primarily to dendritic spines. In the developing rat hippocampus, spine-associated laminin-alpha2 chain-like immunoreactivity first appeared at a time corresponding to that of active synaptogenesis. After an entorhinal cortex lesion in adult rats, the time course of denervation-induced loss and reactive reappearance of spines in the molecular layer of the dentate gyrus was correlated closely to the loss and reappearance of laminin-alpha2 chain immunoreactivity. Immunoblot analysis of normal adult rat, rabbit and pig brain revealed a protein similar in size to the reported 80-kDa laminin-alpha2 chain fragment of human placenta as well as 140/160-kDa proteins. These results suggest the presence of proteins with antigenic homology to the laminin-alpha2 chain and/or laminin-alpha2 isoforms in dendrites and dendritic spines in selected areas of the brain, predominately in the hippocampus and other limbic structures. Given the adhesion and neurite promoting functions of laminins, it is possible that neuronal laminin-alpha2 chain-like proteins play a role in synaptic function and plasticity in the CNS.

The inducible transcription factor c-Jun increases in neurons in response to axotomy by unknown mechanisms, and it has been postulated that c-Jun may regulate genes involved in promoting either degeneration or regeneration of axotomized neurons. In this report, we investigated the effect of daily or twice daily intraventricular administration of the neurotrophins nerve growth factor or neurotrophin-4/5 on the decrease in choline acetyltransferase expression and the increase in c-Jun expression in rat medial septum/diagonal band neurons three, seven and 14 days following unilateral, complete, fornix fimbria lesion. We also examined whether medial septum/diagonal band neurons might die by apoptosis within two weeks of fornix fimbria lesion using terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labelling. Our results show that both nerve growth factor and neurotrophin-4/5 maintain the phenotype of basal forebrain cholinergic neurons following axotomy. Furthermore, using double-labelling immunofluorescence, we found that while c-Jun was expressed in cholinergic neurons in control-treated rats seven days following fornix fimbria lesion, cholinergic neurons rescued by either nerve growth factor or neurotrophin-4/5 in neurotrophin-treated rats failed to express c-Jun. At no time-point (three, seven or 14 days post-axotomy) did any neurons in the medial septum/diagonal band stain positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling, suggesting that medial septum/diagonal band neurons do not undergo apoptosis within the first two weeks following axotomy at the time-points observed by us. Therefore, these results show that both nerve growth factor and neurotrophin-4/5 rescue the phenotype of axotomized cholinergic neurons and that these rescued neurons fail to express c-Jun in response to axotomy. In addition, since neither nerve growth factor nor neurotrophin-4/5 induced c-Jun in medial septum/diagonal band cholinergic neurons, it seems unlikely that the neurotrophic effects of nerve growth factor and neurotrophin-4/5 on cholinergic neurons are mediated via c-Jun expression. Furthermore, since axotomy failed to increase terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labelling in septal neurons, it appears unlikely that c-Jun expression in these axotomized neurons is related to neuronal degeneration via apoptosis.

Improvement of the cholinergic deficit in Alzheimer's disease (AD) by intracerebral application of nerve growth factor (NGF) serves as a paradigmatic example for a novel approach to the treatment of neurodegeneration. The first part of this paper presents and discusses experiments which were performed in our laboratory to study the NGF receptor response after intracerebral NGF treatment in vivo. We found that intraparenchymal NGF elicits prolonged tyrosine phosphorylation of Trk type NGF receptors. Our results indicate that intraparenchymal injections are preferable to intraventricular application for targeting specific neuronal populations with minimal side effects. Besides the cholinergic deficit, severely disturbed brain energy metabolism, particularly in cortical association areas, is another consistent feature of AD. Metabolic hypofunction is observed early in the disease progression and correlates with the cognitive impairment. Cell culture findings are presented which indicate that brain-derived neurotrophic factor (BDNF), and other neurotrophins with activity on the TrkB tyrosine kinase receptor, increase mRNA levels and biochemical activity of enzymes of the glycolytic pathway in brain cells. Treatment with these factors was also found to stimulate glucose utilization in rat embryonic cortex cells in primary cultures. Our observations suggest that selected neurotrophins should become useful not only for the treatment of the cholinergic deficit in AD, but also of the cortical metabolic hypofunction associated with this disease.

Neurotrophins are a family of highly conserved proteins that affect the development and maintenance of distinct neuronal populations. Neurotrophins exist in vivo as homodimers, but we show that neurotrophins can exist as heterodimers in vitro and are pluripotent, being able to bind and to activate different Trk tyrosine kinase receptors as well as promote neuronal differentiation in PC12 cells as effectively as wild type homodimers. These asymmetric neurotrophin dimers allow unique characterization of neurotrophin structure-function relationships with Trk receptors. The chimeric Trk activities of these heterodimers suggest an alternative model of neurotrophin-Trk receptor activation in which the critical Trk-interacting elements may be attributed to a single protomer.

t-Butylhydroperoxide can be used as a model oxidative stress-inducing agent in the brain following intracerebroventricular administration. Mice were treated with saline, t-butanol, or t-butylhydroperoxide. t-Butanol is the major metabolite of t-butylhydroperoxide. t-Butylhydroperoxide had a number of effects, including that it damages dopaminergic, cholinergic, and GABAergic neurons as demonstrated immunohistochemically. Electron microscopic examination demonstrated that astrocytes, oligodendrocytes, endothelial cells, pericytes, and neurons are damaged by t-butylhydroperoxide. Dopamine and its metabolites were affected in a number of brain regions, as were serotonin and its metabolite. Choline acetyl transferase activity was decreased in the striatum. Edema was apparent as assessed by tissue protein levels. There was evidence of lipid peroxidation produced by t-butylhydroperoxide in the midbrain. t-Butylhydroperoxide is a neurotoxin that may be useful in understanding the unexpected ways the brain responds to oxidative stress.

The expression of the specific trk receptors for nerve growth factor and brain-derived neurotrophic factor ( trk A and trk B) has been assayed by messenger RNA in situ hybridization in adult rats with partial fimbrial transections along with intraventricular treatment of nerve growth factor or brain-derived neurotrophic factor. In the forebrain, specific hybridization labeling for trk A messenger RNA showed an identical pattern to that of choline acetyltransferase messenger RNA, supporting the view that trk A expression is confined to the cholinergic population in the basal forebrain and the cholinergic interneurons in the striatum. After partial unilateral transections of the fimbria there was a progressive loss of choline acetyltransferase and trk A messenger RNA expression in the septal region ipsilateral to the lesion. Daily intraventricular administration of brain-derived neurotrophic factor or nerve growth factor partially prevented the lesion-induced decrease in the levels of both messengers, the latter being more effective than the former. Grain count analysis of individual cells was used to test whether the two factors upregulated choline acetyltransferase or trk A expression in individual cells surviving the lesion. Brain-derived neurotrophic factor treatment failed to induce any change in the levels of both messengers per neuron in the septal area. In contrast, daily intraventricular administration of nerve growth factor upregulated both choline acetyltransferase and trk A messenger RNA expression in individual neurons. This upregulation was evident on ipsilateral and contralateral sides, suggesting that nerve growth factor is able to upregulate these markers in intact and injured cholinergic cells in the basal forebrain. Similar to the situation in the septum, brain-derived neurotrophic factor did not upregulate choline acetyltransferase or trk A expression in the striatum. However, nerve growth factor administration strongly upregulated choline acetyltransferase messenger RNA expression by individual cholinergic neurons of the striatum. A medial to lateral gradient decrease in this upregulation was detected in the striatum ipsilateral to the side of administration, suggesting a limited diffusion of the nerve growth factor protein from the ventricle into brain parenchyma. In contrast to the strong effect on choline acetyltransferase expression, nerve growth factor treatment was ineffective in altering trk A messenger RNA in the striatum. The contrasting findings between septum and striatum suggest different regulatory mechanisms for trk A messenger RNA expression in the two cholinergic populations. Since nerve growth factor was found to upregulate the expression of its trk A receptor, we tested whether brain-derived neurotrophic factor administration had similar effects on the regulation of its trk B receptor. Neither brain-derived neurotrophic factor administration nor fimbrial transection induced any change in the expression of full-length or truncated trk B messenger RNAs. However, in situ hybridization for brain-derived neurotrophic factor messenger RNA detected lower levels in CA2-CA3 following the fimbrial transections in the ipsilateral side, which confirmed that septal cholinergic afferents are necessary for maintaining normal levels of brain-derived neurotrophic factor messenger RNA expression in the hippocampus.

The response of brain tissue to neurotrophins during rat development was examined using a novel in vitro assay for Trk/neurotrophin receptor activity. In this assay, brain tissues were exposed to neutrophins and ligand-induced Trk tyrosine phosphorylation was measured. During the perinatal period, Trk tyrosine phsphorylation in all brain area was induced very similarly by the TrkB and TrkC ligands brain-derived neurotrophic factor (BNDF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT-4/5). In the adult brain, minimal signals were observed after treatment with these three factors, despite the continued presence of full length and truncated TrikB protein. In contrast, responsiveness to the TrkA ligand NGF was absent in the ebmryo and increased during the first 2 weeks after birth in various brain areas, particularly in striatum, basal forebrain, and hippocampus. Our results, showing maximal responsiveness of brain tissue to BDNF, NT-3, and NT-4/5 during early neuronal differentiation and migration, suggest involvement of TrkB in these events. The lack of a significant response to these neurotrophins in the adult brain indicates effective posttranslational mechanisms that control the response of Trk family receptors. Our findings further demonstrate that neurons of the striatum and basal forebrain remain NGF responsive in the adult, confirming at the molecular level results obtained earlier at the cellular level for the basal forebrain cholinergic neurons.

Phospholipase C gamma 1 (PLC-gamma 1) is involved at an early step in signal transduction of many hormones and growth factors and catalyzes the hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate to diacylglycerol and inositol trisphosphate, two potent intracellular second messenger molecules. The transformation of PC12 cells into neuron-like cells induced by nerve growth factor is preceded by a rapid stimulation of PLC-gamma 1 phosphorylation and PI hydrolysis. The present study analyzed the effects of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on phosphorylation of PLC-gamma 1 in primary cultures of embryonic rat brain cells. BDNF and NT-3 stimulated the phosphorylation of PLC-gamma 1, followed by hydrolysis of PI. The stimulation of PLC-gamma 1 phosphorylation occurred within 20 s after addition of BDNF or NT-3 and lasted up to 30 min, with a peak after 4 min. ED50 values were similar for BDNF and NT-3, with approximately 25 ng/ml. Phosphorylation of PLC-gamma 1 by BDNF and NT-3 was found in cultures from all major brain areas. K-252b, a compound known to inhibit selectively neutrophin actions by interfering with the phosphorylation of trk-type neutrophin receptors, prevented the BDNF- and NT-3-stimulated phosphorylation of PLC-gamma 1. Receptors of the trk type were coprecipitated with anti-PLC-gamma 1 antibodies. The presence of trkB mRNA in the cultures was substantiated by northern blot analysis. The action of BDNF and NT-3 seems to be neuron specific because no phosphorylation of PLC-gamma 1 was observed in cultures of nonneuronal brain cells. The results provide evidence that developing neurons of the cerebral cortex and other brain areas are responsive to BDNF and NT-3, and they indicate that the transduction mechanism of BDNF and NT-3 in the brain involves rapid phosphorylation of PLC-gamma 1 followed by PI hydrolysis.

To elucidate the role of TGF alpha as a growth factor in the developing brain and to obtain information on signal transduction mechanisms mediating these effects, we measured the hydrolysis of phosphatidylinositol (PI) in cultures from fetal rat brain cells. Stimulation of PI breakdown induced by TGF alpha was observed in cultures of mesencephalic cells containing both neuronal and non-neuronal cells, whereas TGF alpha was ineffective in both pure neuronal and pure glial cultures. These findings are compatible with the view that TGF alpha plays a role during brain development and that its actions on PI hydrolysis, requires the presence of neuronal and glial cells.

Cell culture studies with dissociated primary cultures from embryonic rat brain revealed that brain-derived neurotrophic factor (BDNF) promotes the developmental differentiation of both basal forebrain cholinergic and mesencephalic dopaminergic neurons. These studies suggested that, in the adult brain, BDNF may be able to protect cholinergic and dopaminergic neurons from degenerative changes induced by axotomy, similar to the known protective action of NGF in cholinergic neurons. Testing this hypothesis, we found that intraventricular administration of recombinant human BDNF (rhBDNF) to adult rats with transections of the fimbria significantly reduces axotomy-induced degenerative changes of the cholinergic cells in the basal forebrain. No such effect was seen on the dopaminergic neurons of the ventral mesencephalon after transection of their axons ascending in the medial forebrain bundle. Injected in equal amounts, rhBDNF and recombinant human NGF had quantitatively different effects on the cholinergic neurons. BDNF sustained only part of the population of cholinergic neurons affected by the lesion, whereas the entire population was protected by NGF treatment.

K-252b, a protein kinase inhibitor, has been shown earlier to inhibit nerve growth factor actions on cholinergic neurons of the basal forebrain. In the present study, K-252b was found to prevent trophic actions of two other neurotrophins, brain-derived neurotrophic factor, and neurotrophin-3, on central cholinergic and dopaminergic neurons, peripheral sensory neurons, and PC12 pheochromocytoma cells, when used at greater than 2 microM concentration. Comparable actions of nonneurotrophin growth factors were not affected. Surprisingly, at 0.1-100 nM, K-252b selectively enhanced the trophic action of neurotrophin-3 on central cholinergic neurons, peripheral sensory neurons, and PC12 cells. In PC12 cells, K-252b potentiated the neurotrophin-3-induced tyrosine phosphorylation of trk, a protein kinase responsible for transmitting neurotrophin signals. Of the three structurally related nerve growth factor inhibitors, K-252a, K-252b, and staurosporine, only the first two also mediated neurotrophin-3 potentiation. These findings indicate that K-252b generally and selectively potentiates the neurotrophic action of neurotrophin-3 and suggest that this action involves trk-type neurotrophin receptors.

To enable us to study expression of tyrosine hydroxylase [TH; tyrosine 3-monooxygenase; L-tyrosine tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] as a measure of dopaminergic neuron function in future experiments, methods were developed to quantify TH mRNA levels in cultures of dopaminergic mesencephalic cells. The model of selective dopaminergic toxicity of 1-methyl-4-phenylpyridinium (MPP+) was used to verify the specificity of our methods. Fetal (embryonic day 15) rat ventral mesencephalic cell cultures were treated with 15 microM MPP+ for 48 h, conditions previously shown to reduce the number of TH-immunoreactive neurons, TH activity, and dopamine uptake to 5-10% of control values. This treatment decreased the number of neurons labeled by TH in situ hybridization to 9% of untreated controls and caused a strong reduction of the abundance of TH mRNA in Northern blots. Our findings establish TH mRNA expression as a parameter for future studies of toxic and trophic effects on cultured dopaminergic neurons, and they support the view that MPP+ destroys dopaminergic neurons.

Cholinergic neurons in the basal forebrain magnocellular complex (BFMC) respond to nerve growth factor (NGF) during development and in adult life, and it has been suggested that the administration of NGF might ameliorate some of the abnormalities that occur in neurological disorders associated with degeneration of this population of neurons. A prerequisite for the introduction of NGF in clinical trials is the availability of active recombinant human NGF (rhNGF). The present investigation was designed to test, in vivo, the efficacy of a preparation of rhNGF. Axons of cholinergic neurons of the BFMC in the rat were transected in the fimbria-fornix; this manipulation alters the phenotype and, eventually, causes retrograde degeneration of these neurons. Our investigation utilized two lesion paradigms (resection and partial transection of fibers in the fimbria-fornix), two different strains of rats, and two delivery systems. Following lesions, animals were allowed to survive for 2 weeks, during which time one group received intraventricular mouse NGF (mNGF), a second group received rhNGF, and a third group received vehicle alone. In animals receiving vehicle, there was a significant reduction in the number (resection: 70%; transection: 50%) and some reduction in size of choline acetyltransferase- or NGF receptor-immunoreactive cell bodies within the medial septal nucleus ipsilateral to the lesion. Treatment with either mNGF or rhNGF completely prevented these alterations in the number and size of cholinergic neurons. The rhNGF was shown to be equivalent in efficacy with mNGF. Thus, rhNGF is effective in preventing axotomy-induced degenerative changes in cholinergic neurons of the BFMC. Our results, taken together with the in vitro effects of rhNGF (42), indicate that an active rhNGF is now available for further in vivo studies in rodents and primates with experimentally induced or age-associated lesions of basal forebrain cholinergic neurons. These investigations provide essential information for the consideration of future utilization of rhNGF for treatment of human neurological disorders, including Alzheimer's disease.

Recombinant human brain-derived neurotrophic factor (rhBDNF) and neurotrophin 3 (rhNT-3), two recently cloned molecules closely related to nerve growth factor (NGF), were produced from human cDNA expressed in human embryonic kidney cells. The recombinant proteins were tested in cultures of dissociated fetal rat brain cells containing basal forebrain cholinergic neurons. rhBDNF stimulated the differentiation of the cholinergic neurons, similar to NGF, which is well established as a neurotrophic factor for these cells. However, rhBDNF was particularly effective during the first few days in vitro, whereas the stimulatory action of rhNGF was more pronounced later in the development of the cultures. This finding indicates the existence of different time periods of responsiveness of the cholinergic neurons to BDNF and NGF. To assess the selectivity of the effect of rhBDNF on cholinergic neurons, its actions were tested in cultures of ventral mesencephalon containing dopaminergic cells. In contrast to NGF, which does not affect central dopaminergic neurons, rhBDNF increased dopamine uptake activity. The findings suggest that BDNF stimulates survival or differentiation of other cells besides the cholinergic neurons.

NGF is a neurotrophic factor for basal forebrain cholinergic neurons and may serve to counteract the cholinergic deficits that are observed in Alzheimer's disease. Prior to the introduction of clinical trials, it is essential that recombinant human NGF (rhNGF) be produced and that its actions on target cells in the CNS be demonstrated. We prepared rhNGF and examined its actions on fetal rat brain neurons in culture including, in particular, the cholinergic neurons of the basal forebrain. rhNGF was more potent in increasing choline acetyltransferase (ChAT) activity in septal cultures than NGF purified from mouse salivary glands (mNGF). ED50s of the beta-NGF dimers were 4.9 pM for rhNGF and 12.4 pM for mNGF. The maximal ChAT activity response was achieved at approximately 35 pM with both NGFs and their efficacies were not significantly different. The two NGFs were not additive in effect. Identical to the results with mNGF, rhNGF strongly enhanced the intensity of ChAT immunostaining in septal cultures. Neither rhNGF nor mNGF affected the appearance of the cultures under phase-contrast illumination. Survival of cells at very low plating density on polyornithine/laminin-coated culture dishes was not affected by rhNGF or mNGF. Protein content and the uptake of GABA were also unaffected. At concentrations of up to 10 micrograms/ml, rhNGF did not significantly increase uptake of dopamine into cultures of ventral mesencephalon. We conclude that rhNGF produces potent and selective actions on cholinergic neurons of the basal forebrain as previously shown for mNGF.

Excess neuronal activity upregulates the expression of two neurotrophins, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in adult hippocampus. Nerve growth factor has been shown to contribute the induction of aberrant hippocampal mossy fiber sprouting in the inner molecular layer of the dentate gyrus, however the role of prolonged brain-derived neurotrophic factor exposure is uncertain. We examined the distribution and plasticity of mossy fibers in transgenic mice with developmental overexpression of brain-derived neurotrophic factor. Despite 2–3-fold elevated BDNF levels in the hippocampus sufficient to increase the intensity of neuropeptide Y immunoreactivity in interneurons, no visible changes in mossy fiber Timm staining patterns were observed in the inner molecular layer of adult mutant hippocampus compared to wild-type mice. In addition, no changes of the mRNA expression of two growth-associated proteins, GAP-43 and SCG-10 were found. These data suggest that early and persistent elevations of brain-derived neurotrophic factor in granule cells are not sufficient to elicit this pattern of axonal plasticity in the hippocampus. J. Neurosci. Res. 64:268–276, 2001. © 2001 Wiley-Liss, Inc.

Chronic exposure of brain neurons to nerve growth factor in vitro and in vivo results in increased levels of the nerve growth factor receptor TrkA. In contrast, in the present study, we have found that chronic exposure of rat embryonic cortical neurons to brain-derived neurotrophic factor (BDNF) leads to a pronounced reduction of the levels of protein and messenger RNA for the full-length but not the truncated BDNF receptor TrkB. Similar effects were observed with the other TrkB ligands neurotrophin-3 and neurotrophin-4/5. After pretreatment with BDNF, neurotrophin-3 or neurotrophin-4/5, subsequent tyrosine phosphorylation responses of the remaining Trks to the same factors were greatly reduced. Three days exposure of rat embryonic cortical neurons to BDNF induced an absolute refractory period of several hours, with no subsequent response to the same factor. Similar but less pronounced refractory effects were observed with neurotrophin-3 and neurotrophin-4/5. Our results suggest a negative regulatory effect of BDNF and other TrkB ligands on TrkB receptors. Down-regulation of the TrkB response by its ligands might play a role in the control of BDNF action during early development, when BDNF levels significantly increase. Our findings are also of potential clinical relevance, since the possibility of ligand-induced down-regulation of the receptor response needs to be addressed when considering BDNF or other neurotrophins for the therapy of neurodegeneration.

Glutathione (GSH) is considered the primary molecule responsible for peroxide removal from the brain. Inhibition of its rate-limiting synthetic enzyme, glutamylcysteine synthetase (GCS), results in morphological damage to both cortical and nigral neurons in rodents. Here, we report cloning of the catalytic heavy chain GCS mRNA from mouse and its localization in the murine brain. Heavy chain GCS appears to be localized in glial populations in the hippocampus, cerebellum and olfactory bulb, with lower levels of expression in the cortex and substantia nigra. Variations in GCS levels and subsequent GSH synthesis may explain differences in susceptibility to neuropathology associated with oxidative stress noted in these various brain regions.

Several recent studies have demonstrated that expression of the tumour-suppressor gene p53 increases within the nervous system after injury. In various cell lines wild-type-p53, induced by DNA damage, has been shown to function to halt cell-cycle progression and under certain circumstances to induce programmed-cell death or apoptosis. Since wild type-p53 can act as a transcription factor to regulate the expression of p53-responsive genes it is possible that either, or both, functions of p53 are mediated by down-stream effector genes. However wild-type-p53 only weakly activates transcription and it remains to be determined whether p53-responsive genes are expressed in lesioned brain. Here we report that excitotoxic lesion of rat brain with the N-methyl-D-aspartate receptor agonist, quinolinic acid, induces expression of p53 messenger RNA and protein in brain regions showing delayed DNA fragmentation and that expression of p53 messenger RNA precedes DNA damage detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling. In addition, using in situ hybridization and immunocytochemistry we demonstrate increased expression of the p53-responsive gene Gadd-45 (preceding p53 expression) and re-expression of the p53-responsive gene Bax (following p53 expression), in these same areas. Bax has been shown to promote neuronal death by interacting with Bcl-2 family members while Gadd-45 expression has been associated with suppression of the cell-cycle and DNA repair. These results suggest that p53 protein may function as an active transcription factor in lesioned brain perhaps initiating the re-expression of Bax in injured brain regions. However, since Gadd-45 precedes p53 expression it appears unlikely that p53 is involved in regulating the early expression of Gadd-45. Taken together however, these results suggest that p53, Bax and Gadd-45 may play important roles in the response (damage/recovery) of the brain following excitotoxic injury.

Neurotrophic factors, particularly the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and related molecules are proposed for the experimental treatment of neurodegenerative disease. Earlier observations had suggested down-regulation of the neurotrophin receptor response with chronic stimulation. We therefore tested for effects of acute and chronic NGF treatment in vivo on the tyrosine phosphorylation response of Trk-type neurotrophin receptors in adult and aged rats. Rats were treated for 1 week with daily injections of NGF directly into the striatum. Surprisingly, this chronic neurotrophin treatment induced long-lasting tyrosine phosphorylation of Trk type receptors beyond the last injection. A similar result was obtained with 1 week of daily injections of BDNF into the hippocampus. Persistent Trk tyrosine phosphorylation was also observed after single neurotrophin injections. With 1 μg of NGF injected, Trk-type receptors were maximally stimulated from immediately after the injection until 3 days after the treatment. Maintaining Trk tyrosine phosphorylation required maintained energy levels in the tissue. Incubation of microslices of brain tissue from NGF-injected animals in glucose-free buffer completely abolished all Trk tyrosine phosphorylation signals. Recovery of the tissue in presence of glucose restored the signals in microslices derived from NGF-injected animals, in absence of acute NGF treatment. This result, together with dose–response comparisons after 2-h and 2-day survival times suggest that Trk protein remains tyrosine phosphorylated due to trophic protein which is only slowly being cleared out of the tissue during several days after the injection. Experiments with aged rats indicated similar extent and duration of Trk receptor activation after NGF administration in young adult and in aged brain.

Adrenalectomy (ADX) in rats has been shown to induce apoptosis of hippocampal granule cells. We tested whether neurotrophins are able to protect hippocampal neurons in this neurodegeneration model. Acid fucshsin stain was used to identify pyknotic cells in ADX rats treated for 4 days with NT-3, NT-4-5 or cytochrome-C, as a control protein. Cytochrome-C injections slightly decreased cell death on the ipsilateral side. NT-3 did not further promote this effect. Significantly less cell death was observed bilaterally in hippocampus treated with NT-4/5. TUNEL end labeling also confirmed the results. Our results demonstrated that NT-4/5, but not NT-3, promotes hippocampal neuron survival in adrenalectomized rats. They further show that injections of a control solution can induce a local protective effect.

In search of the possible involvement of neurotrophic factors in inherited neurological disease, we examined brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) mRNA expression patterns in the ataxic mutant mouse stargazer (stg). Using in situ hybridization, we found a selective and near total reduction in BDNF mRNA in the cerebellar granule cell layer. NT-3 or NGF mRNA expression in the cerebellum was normal. Northern blot analysis demonstrated a 70% reduction in BDNF mRNA in the whole cerebellum. BDNF mRNA levels in other mutant brain regions were unchanged. Absence of BDNF mRNA in granule cells was observed at postnatal age (P15), coincident with the onset of ataxia, and expression levels failed to follow the developmental increase found in the wild type at later ages (P20 and P30). Despite the severe BDNF reduction, in situ hybridization patterns for both the full-length and the truncated BDNF TrkB receptor mRNA were unaltered. No major cytoarchitectural abnormalities were apparent in the stg/stg cerebellum. BDNF expression in a related ataxic mutant, tottering, was unaltered. These data show that BDNF can be regulated selectively in distinct brain regions, possibly by differential activation of its multiple promoters. Absence of cerebellar granule cell BDNF mRNA in stg/stg mice demonstrates that sustained expression of this neurotrophin is not required for cell survival in the developing cerebellar cortex. Our data, in contrast, suggest a role of BDNF in maturation of specific cerebellar neurons and pathways. Early failure of cerebellar BDNF expression may be related to the ataxic phenotype in stg mice.

TrkA is a receptor tyrosine kinase whose activation transduces NGF signaling. TrkA expression has been demonstrated in NGF-responsive adult basal forebrain cholinergic neurons (BFCNs). Several lines of evidence have suggested that endogenous NGF plays a role in the development and differentiation of these neurons. We examined TrkA expression during development. TrkA mRNA and protein were present in basal forebrain neurons during the entire postnatal period; the distribution of neurons bearing these markers was identical to that for those containing choline acetyltransferase (ChAT) mRNA, suggesting that, as in the adult, TrkA gene expression is localized to BFCNs. The expression of TrkA and ChAT followed a very similar temporal pattern, suggesting regulation by the same factor(s). We discovered that NGF administration in vivo activated TrkA receptors, and increased both TrkA and ChAT mRNA; conversely, anti-NGF infusions suppressed expression of both genes. These results suggest that endogenous NGF regulates expression of TrkA and ChAT. Finally, while NGF infusion increased the size of developing BFCNs, NGF antibodies inhibited the normal developmental increase. The results are evidence that endogenous NGF acts on developing BFCNs to enhance gene expression and cellular differentiation.

The protein kinase inhibitors K-252a and K-252b have been shown earlier to block the actions of nerve growth factor and other neurotrophins and, at lower concentrations, to selectively potentiate neurotrophin-3 actions. In the present study we show that K-252a, but not K-252b, enhances epidermal growth factor (EGF)-and basic fibroblast growth factor (BFGF)-induced neurite outgrowth of PC12 cells at higher concentrations than required for neurotrophin inhibition. In parallel, tyrosine phosphorylation of extracellular signal-regulated kinases (Erks) elicited by EGF of bFGF was also increased in the presence of K-252a, and this signal was prolonged for 6 h. EGF- and bFGF-induced phosphorylation of phospholipase C-gamma 1 were not changed. The effect of K-252a on Erks was resistant to chronic treatment with phorbol ester, indicating that protein kinase C is not involved in this potentiation. In partial contrast to the actions of K-252a, the neurotrophin-3-potentiating effect of K-252b was accompanied by an increase in tyrosine phosphorylation of the Erks and of phospholipase C-gamma 1. Finally, although K-252a alone did not induce neurite outgrowth or tyrosine phosphorylation of Erks or phospholipase C-gamma 1, this compound alone stimulated phosphatidylinositol hydrolysis. Our findings identify activities of K-252a besides the direct interaction with neurotrophin receptors and suggest that a K-252a-sensitive protein kinase or phosphatase might be involved in signal transduction of EGF and bFGF. Our results are further compatible with the hypothesis that sustained activation of Erks may be important in PC12 differentiation.

Primary cultures of rat hippocampal cells have been used to evaluate trophic effects of neurotrophin-3, brain-derived neurotrophic factor, nerve growth factor, and basic fibroblast growth factor. There was little survival in cultures prepared from embryonic day 17 embryos and grown in defined medium without growth factors. Addition of basic fibroblast growth factor produced a massive increase in the number of neurons present in the cultures seven days after plating. This action reflected proliferation of neuronal precursor cells rather than increased survival of initially plated neurons. Brain-derived neurotrophic factor was ineffective under these conditions, whereas neurotrophin-3 produced a very small, but statistically significant increase in neuronal survival in the range of 20%. However, hippocampal neurons were responsive to brain-derived neurotrophic factor and neurotrophin-3 as demonstrated under culture conditions, resulting in survival in absence of the neurotrophins. Acute administration of brain-derived neurotrophic factor and neurotrophin-3 to hippocampal cultures grown at high density stimulated the hydrolysis of phosphatidylinositol, a response earlier shown to be mediated by tyrosine receptor kinase neurotrophin receptors. Furthermore, when such cultures were grown in presence of neurotrophin-3 rates of glutamate and GABA uptake were increased. In contrast to the findings obtained in cultures of embryonic day 17, cultures prepared from embryonic day 14 or 15 animals were viable in absence of exogenous growth factors. The specific neurotrophin receptor inhibitor, K-252b reduced survival in these cultures and this effect was partly overcome by exogenous neurotrophin-3. Our findings suggest that hippocampal neuron survival at early embryonic stages may involve paracrine neurotrophin mechanisms, whereas the survival of hippocampal neurons of embryonic day 17 is not markedly enhanced by brain-derived neurotrophic factor or neurotrophin-3. However, at this embryonic stage there is a functional response to both neurotrophins as made evident by the activation of tyrosine kinase receptor-linked signal transduction mechanisms and by the stimulation of transmitter-specific differentiation.

The hydrolysis of phosphatidyl 4,5-biphosphate (PI), which is involved in the transduction mechanism of neurotransmitters and growth factors, is stimulated by brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in primary cultures of fetal brain neurons. In the present study we sought to examine the effect of pretreatment with these factors on their acute stimulation capabilities and, furthermore, to substantiate that the effects of BDNF and NT-3 reflect actions on neurons rather than glial cells. Pretreatment with BNDF and NT-3 for 4 days followed by 1 day without growth factor abolished the effect of an acute stimulation with these factors. The growth factors were mutually effective so that BDNF pretreatment abolished the acute response to NT-3 and vice versa. In contrast, the effects of bFGF (basic fibroblast growth factor, a non-neurotrophin growth factor) also stimulating PI hydrolysis in these culture systems, were not reduced by neurotrophin pretreatment. Pretreatment with K-252b, at concentrations known to inhibit trk receptors, did not alter the acute stimulation of PI hydrolysis induced by the neurotrophins. PI hydrolysis stimulated by BDNF and NT-3 in cultures grown in presence of cytosine arabinoside C, containing > 95% neurons, was higher than in cultures containing non-neuronal cells, indicating that the neurotrophin stimulation occurs in neuronal cells. No stimulatory effect was detected in bFGF treated pure neuronal cultures. The findings suggest that prolonged exposure of responsive neurons to BDNF and NT-3 down-regulates their stimulatory effects on PI hydrolysis.

This study examined how possible nerve growth factor (NGF)-induced behaviour changes after septal damage might be modulated by the lesion extent, the dose of NGF administered and the delay between surgery and the onset of testing. In a first experiment, young rats which received electrolytic septal lesions of high or low intensity (inducing respectively large and mild lesions) were treated with 10 or 30 micrograms NGF administered intrahippocampally in a single injection. They were tested 4 months postoperatively for open field ambulation, spontaneous alternation and radial maze performance. It was observed that irrespective of the severity of the lesions rats were impaired in the spontaneous alternation and radial maze tests; however, no obvious changes appeared in the open field test. While an NGF injection did not affect behavioural performances in rats with large lesions, it was capable of ameliorating behavioural deficits in the spontaneous alternation and radial maze tests of rats with mild lesions in both NGF dosage groups. It was also seen that lesions produced a general decrease in hippocampal choline acetyltransferase (ChAT) activity, which was not significantly affected by an NGF administration. There was no significant correlation between ChAT activity and behavioural performance of NGF-treated rats. In a second experiment, young rats received mild septal lesions and were treated with 10 micrograms NGF. These rats were tested 2 weeks postoperatively for radial maze performance. NGF rats exhibited similar behaviour to controls with regard to all of the variables measured. The present results suggest that a single NGF administration spares some abilities to use spatial information efficiently providing lesions are partial.

To elucidate the role of brain derived neurotrophic factor (BDNF) in the protection of cholinergic neurons in the basal forebrain, recombinant human BDNF and as a positive control, human recombinant nerve growth factor (NGF), were infused for 20 days into the lateral ventricle of adult rats with fimbrial transections. BDNF and NGF administration protected cholinergic basal forebrain cells from degenerative changes after axotomy, as assessed with immunohistochemical analysis of the two cholinergic marker proteins ChAT and p75NGFR. Both BDNF and NGF treatment completely prevent the lesion induced loss of p75NGFR-positive cells in the septal area of animals with fimbrial transections. This finding contrasts with the result obtained with ChAT immunohistochemistry, where BDNF treatment protects only part of the population of neurons which disappear following the transections. These findings are compatible with the view that there is a cascade of events induced in cholinergic neurons by the transections, so that ChAT expression is lost before p75NGFR expression, and that BDNF reduces degenerative events in the entire population of cholinergic neurons, maintaining some of them as p75NGFR-positive but ChAT-negative cells.

Phosphatidylinositol (PI) breakdown represents a powerful system participating in the transduction mechanism of some neurotransmitters and growth factors and producing two second messengers, diacylglycerol and inositol trisphosphate. The transformation of PC12 neuroblastoma cells into neuron-like cells induced by nerve growth factor (NGF) is preceded by a rapid stimulation of PI breakdown; however, it was not known whether PI breakdown mediates actions of other members of the neurotrophin family. The present study analyzed the effects of NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) on PI breakdown in primary cultures of embryonic rat brain cells. Cultures were grown for 7 days; PI was then labeled by incubating cultures with myo-[3H]inositol, which then were exposed acutely to growth factors. BDNF and NT-3, but not NGF, elevated the levels of labeled inositol phosphates within 10-15 min after addition to the cultures in a dose-dependent manner. ED50 values for BDNF and NT-3 were 12.4 and 64.5 ng/ml, respectively. Comparable effects were found in cultures of cortical, striatal, and septal cells. The actions of BDNF and NT-3 probably reflect actions on neurons, because no effects were seen in cultures of nonneuronal cells. In contrast, basic fibroblast growth factor induced a marked stimulation of PI breakdown in cultures of nonneuronal cells. K252b, which selectively blocks neurotrophin actions by inhibiting trk-type receptor proteins, prevented the PI breakdown mediated by BDNF and NT-3. The findings suggest that rapid and specific induction of PI breakdown is involved in the signal transduction of BDNF and NT-3, and they provide evidence that cortical neurons are functionally responsive to BDNF and NT-3 during development.

Tyrosine phosphorylation of trk type neurotrophin receptors in primary cultures of embryonic rat brain cells was studied by immunoprecipitation and immunoblotting. In cultures containing basal forebrain cholinergic neurons, but not in cultures of cerebral cortex, nerve growth factor (NGF) treatment for 4 min induced tyrosine phosphorylation of trk family proteins. Stimulation with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3), resulted in a very robust phosphorylation signal in basal forebrain and cortical cultures, suggesting actions of these neurotrophins not only on cholinergic cells but probably on most embryonic brain neurons. Trk tyrosine phosphorylation was completely abolished by 5 microM K-252b. Inhibition was rapid, being evident by 30 s following addition of the drug. Corresponding stimulatory and inhibitory effects were seen for phospholipase-C gamma 1 (PLC gamma 1) and extracellular signal-regulated kinase 1 (Erk1), two enzymes involved in second messenger mechanisms. Our findings indicate involvement of trk receptor activation in the NGF response of basal forebrain cholinergic cells and provide evidence for widespread presence of BDNF and NT-3 responsive neurons in the embryonic brain.

Nerve growth factor (NGF) increases the activity of choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine, in rat basal forebrain neurons both in vivo and in vitro. In poly(A) + RNA isolated from cultures prepared from the embryonic (E15) rat basal forebrain, radiolabeled probes from the human ChAT gene detected a 3,700 nt and a less abundant 2,300 nt transcript. After growth in the presence of NGF, the abundance of both mRNAs was increased approximately twofold, parallelling the increase in ChAT enzyme activity. In vivo, the human ChAT probes detected a single 3,700 nt form of ChAT mRNA in both embryonic and adult rat basal forebrain. These results suggest that the NGF-mediated increase in ChAT activity in basal forebrain cultures is regulated at the transcriptional level.

K-252b is a kinase inhibitor structurally related to K-252a, which is known to abolish selectively the effects of nerve growth factor (NGF) on PC12 cells and PNS neurons. We tested whether K-252b, K-252a, and staurosporine, another related compound, are effective and selective inhibitors of NGF actions on CNS neurons. All three compounds, at appropriate concentrations, completely and selectively prevented the NGF-mediated activity increase of the cholinergic marker enzyme choline acetyltransferase in cultures of rat basal forebrain cells. The stimulatory effects of basic fibroblast growth factor and insulin on choline acetyltransferase in these cultures and on dopamine uptake in cultures of dissociated ventral mesencephalon were not affected. No signs of toxicity were observed in cultures treated with K-252b. In contrast, K-252a and staurosporine, at concentrations required to block the NGF actions on cholinergic cells, were cytotoxic and produced cell loss. In addition, K-252a, at higher concentrations and in the absence of growth factors, increased cell numbers. Our study suggests that K-252b is a selective and nontoxic inhibitor of NGF actions in the brain and may become a useful tool to study these actions in vivo.

To study the selectivity of neurotrophic actions in the brain, we analyzed the actions of several known growth factors on septal cholinergic, pontine cholinergic, and mesencephalic dopaminergic neurons in culture. Similar to nerve growth factor (NGF), basic fibroblast growth factor (bFGF) stimulated choline acetyltransferase activity in septal cultures. In contrast to NGF, bFGF also enhanced dopamine uptake in mesencephalic cultures and stimulated cell proliferation in all 3 culture types. Insulin and the insulin-like growth factors I and II stimulated transmitter-specific differentiation and cell proliferation in all culture types. Epidermal growth factor (EGF) produced a small increase in dopamine uptake by mesencephalic cells and stimulated cell proliferation in all culture types. In septal cultures, bFGF was most effective when given at early culture times, NGF at later times. The stimulatory actions of bFGF and insulin did not require the presence of glial cells and were not mediated by NGF. In mesencephalic cultures, the stimulation of dopamine uptake by bFGF and EGF was dependent on glial proliferation. The results suggest different degrees of selectivity of the neurotrophic molecules. NGF and, very similarly, bFGF seem to influence septal cholinergic neurons directly and rather selectively, whereas the neurotrophic actions of insulin and the insulin-like growth factors appear to be more general.

Intracerebral chronic administration of nerve growth factor (NGF) is able to counteract age-related cholinergic dysfunction. This finding suggests that intracerebral administration of neurotrophic factors may become a generally valuable approach when attempting to ameliorate age-related neuronal deficits in experimental animals and humans. This commentary briefly discusses possible strategies for the development of such "neurotrophic factor treatments."