Beat Knusel

K‐252 Compounds: Modulators of Neurotrophin Signal Transduction

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.

The nature of the trophic action of brain-derived neurotrophic factor, des(1-3)-insulin-like growth FACTOR-1, and basic fibroblast growth factor on mesencephalic dopaminergic neurons developing in culture

Brain-derived neurotrophic factor, basic fibroblast growth factor and des(1-3)-insulin-like growth factor-1, a brain specific form of insulin-like growth factor-1, were analysed, in the rat, for their influence on survival, morphological growth, and transmitter-specific differentiation of dopaminergic neurons in vitro. Brain-derived neurotrophic factor, des-insulin-like growth factor-1, and basic fibroblast growth factor were found to differentially regulate development of dopaminergic cells. Brain-derived neurotrophic factor stimulated survival, the formation of primary neurites and dopamine uptake activity. des-Insulin-like growth factor-1 was most effective in promoting survival, stimulated dopamine uptake less effectively than brain-derived neurotrophic factor and did not alter the morphology of dopaminergic cells. Basic fibroblast growth factor produced comparatively mild increases in survival and dopamine uptake, and slightly reduced neurite growth of the cells. None of the factors stimulated the expression of the tyrosine hydroxylase gene. These findings suggest that (i) effective growth factors may stimulate different, but partially overlapping, molecular pathways during developmental differentiation, (ii) none of the factors stimulates dopaminergic cell differentiation comparable to the pronounced trophic action of nerve growth factor on peripheral sympathetic or basal forebrain cholinergic neurons, and (iii) localization and effects of none of the factors are compatible with a role as target-derived survival-regulating neurotrophic factor.

Ligand-induced down-regulation of Trk messenger RNA, protein and tyrosine phosphorylation in rat cortical neurons.

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.

Excitotoxic lesion of rat brain with quinolinic acid induces expression of p53 messenger RNA and protein and p53-inducible genes Bax and Gadd-45 in brain areas showing DNA fragmentation

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.

Recombinant human nerve growth factor prevents retrograde degeneration of axotomized basal forebrain cholinergic neurons in the rat

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 Alzheimers disease.

K‐252b Selectively Potentiates Cellular Actions and trk Tyrosine Phosphorylation Mediated by Neurotrophin‐3

Abstract: 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 PC 12 pheochromocytoma cells, when used at >2 μM 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 PC 12 cells. In PC 12 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.

Expression of neurotrophin and trk receptor genes in adult rats with fimbria transections : effect of intraventricular nerve growth factor and brain-derived neurotrophic factor administration

The expression of the specific trk receptors for nerve growth factor and brain-derived neurotrophic factor (trkA and trkB) 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 trkA messenger RNA showed an identical pattern to that of choline acetyltransferase messenger RNA, supporting the view that trkA 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 trkA 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 trkA 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 trkA 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 trkA 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 trkA messenger RNA in the striatum. The contrasting findings between septum and striatum suggest different regulatory mechanisms for trkA messenger RNA expression in the two cholinergic populations. Since nerve growth factor was found to upregulate the expression of its trkA receptor, we tested whether brain-derived neurotrophic factor administration had similar effects on the regulation of its trkB receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Rapid Phosphorylation of Phospholipase Cγ 1 by Brain‐Derived Neurotrophic Factor and Neurotrophin‐3 in Cultures of Embryonic Rat Cortical Neurons

Abstract: Phospholipase Cγ1 (PLC‐γ1) 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‐γ1 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‐γ1 in primary cultures of embryonic rat brain cells. BDNF and NT‐3 stimulated the phosphorylation of PLC‐γ1, followed by hydrolysis of PI. The stimulation of PLC‐γ1 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 τ25 ng/ml. Phosphorylation of PLC‐γ1 by BDNF and NT‐3 was found in cultures from all major brain areas. K‐252b, a compound known to inhibit selectively neurotrophin actions by interfering with the phosphorylation of trk‐type neurotrophin receptors, prevented the BDNF‐ and NT‐3‐stimulated phosphorylation of PLC‐γ1. Receptors of the trk type were coprecipitated with anti‐PLC‐γ1 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‐γ1 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‐γ1 followed by PI hydrolysis.

Cerebellar Brain-Derived Neurotrophic Factor-TrkB Defect Associated with Impairment of Eyeblink Conditioning in Stargazer Mutant Mice

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-Cγ1, 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 produces striatal apoptosis in Sprague-Dawley rats but not in BALB/c byJ mice

Metabolic compromise with systemic 3-nitropropionic acid (3-NP) results in the degeneration of striatal cells, mimicking the pathology of Huntingtons 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.