Hans R. Widmer

Neurotrophin-4/5 Promotes Survival and Differentiation of Rat Striatal Neurons Developing in Culture

Cultures of dissociated striatal neurons from fetal rats were prepared, and were grown in the presence of neurotrophin‐4/5 (NT‐4/5) as well as the other known neurotrophins, nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3). We found that acute administration of NT‐4/5 to 7‐day‐old cultures stimulates the hydrolysis of phosphatidylinositol, an event involved in neurotrophin signal transduction. Growth of striatal cultures in the presence of NT‐4/5 resulted in increased cell survival, as indicated by elevations in cell number, protein content, and a measure of mitochondrial enzyme activity (MTT assay). NT‐4/5 increased GABA uptake and staining intensity in these cultures, as indicated by GABA immunocytochemistry, indicating a trophic action on GABAergic neurons, the predominant neuron type in the striatum. To further identify responsive cell populations we analysed for calretinin, a calcium‐binding protein known to colocalize with GABA in a number of neuronal cells. In cultures prepared from rats of embryonic day 15, NT‐4/5 strongly increased the number of calretinin‐positive cells as well as calretinin levels, as determined by Western blot analysis. When the cultures were prepared from embryonic day 18 rats, NT‐4/5 very strongly increased the morphological differentiation of calretinin‐positive cells, whereas the increase in cell number was less prominent. All effects produced by NT‐4/5 were mimicked by BDNF with similar potency. NT‐3 was less effective than NT‐4/5 and BDNF, and its effects were limited to cultures prepared from embryonic day 15 rats, suggesting a role in the regulation of cell survival at early developmental stages. NGF did not affect any of the measured parameters. Our findings identify NT‐4/5 as potent neurotrophic factor for striatal neurons, able to promote their survival and differentiation.

Immunohistochemical visualization of brain-derived neurotrophic factor in the rat brain.

A purified polyclonal antibody preparation was made against recombinant human brain‐derived neurotrophic factor (BDNF) in guinea pig and characterized for use in immunoassays and immunohistochemistry. The anti‐BDNF antibodies specifically recognized BDNF in Western blots and immunoprecipitation. There was no cross‐reactivity with the other known mammalian members of the neurotrophin family, nerve growth factor, neurotrophin‐3 and neurotrophin‐4/5. In immunohistochemical analysis, the anti‐BDNF recognized exogenous BDNF injected into the brain of rats, whereas no signal was obtained with the other neurotrophins. Preabsorption with native BDNF abolished the immunoreactivity in brain sections. These studies identify the anti‐BDNF as a tool for immunocytochemistry and the development of an immunoassay. Immunohistochemical analysis revealed widespread neuronal localization of BDNF in many brain areas. BDNF was localized in all subpopulations of hippocampal neurons. The distribution in the hippocampus suggests localization in the cytoplasm of cell bodies and dendrites.

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.

STIMULATION OF GABAERGIC NEURON DIFFERENTIATION BY NT-4/5 IN CULTURES OF RAT CEREBRAL CORTEX

Primary cultures of fetal rat cortical cells were used to investigate trophic effects of neurotrophin-4/5 (NT-4/5) on GABAergic neurons. Chronic administration of NT-4/5 resulted in a significant increase in high-affinity GABA uptake and in a stronger immunohistochemical staining pattern and bigger cells size of neurons visualized by GABA immunohistochemistry. Brain-derived neurotrophic factor (BDNF) exerted similar actions. These effects were observed in cultures prepared from rat fetuses from embryonic day (ED) 15 and ED18. The total number of cells was marginally increased in ED15 but not ED18 cultures. NT-4/5 did not elevate uptake of excitatory amino acids. Administration of NT-4/5 and BDNF resulted in higher levels of the calcium binding protein, calbindin, in these cultures. Our findings suggest that subpopulations of cortical GABAergic and calbindin-expressing neurons respond to NT-4/5 during development.

Response of embryonic rat hippocampal neurons in culture to neurotrophin-3, brain-derived neurotrophic factor and basic fibroblast growth factor

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.

Stimulation of Phosphatidylinositol Hydrolysis by Brain‐Derived Neurotrophic Factor and Neurotrophin‐3 in Rat Cerebral Cortical Neurons Developing in Culture

Abstract: 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.

Neurotrophin-4/5 treatment reduces infarct size in rats with middle cerebral artery occlusion

The aim of this study was to determine whether neurotrophin-4/5 (NT-4/5) treatment alters infarction volume following permanent focal cerebral ischemia in the rat. Permanent focal cerebral ischemia was produced in adult male rats by intraluminal occlusion of the right middle cerebral artery. NT-4/5 was administered intraventricularly one day before and immediately following occlusion. Rats were sacrificed at 1, 4 and 7 days after occlusion. NT-4/5 treatment reduced infarction volume by 34% when compared to control rats 1 day after occlusion. Infarction volume was unaltered by treatment 4 to 7 days after occlusion. Middle cerebral artery occlusion led to a significant reduction in levels of mRNAs coding for catalytic and truncated TrkB receptors. This expression was unaffected by NT-4/5 treatment.

Down-regulation of phosphatidylinositol response to BDNF and NT-3 in cultures of cortical neurons.

The hydrolysis of phosphatidyl 4,5-bisphosphate (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 neutrophins. 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.

Epidermal Growth Factor Induces PC12 Cell Differentiation in the Presence of the Protein Kinase Inhibitor K‐252a

Abstract: 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 or 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‐γ1 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‐γ1. Finally, although K‐252a alone did not induce neurite outgrowth or tyrosine phosphorylation of Erks or phospholipase C‐γ1, 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 for EGF and bFGF. Our results are further compatible with the hypothesis that sustained activation of Erks may be important in PC12 differentiation.

Therapeutic Use of Neurotrophic Factors

Publisher Summary Neurotrophic factor therapy is still at an embryonic stage; however, it appears to be one of the most promising pharmacological approaches toward effective treatment of age-related neurodegenerative diseases. This chapter provides a matrix of established interactions among neurotrophic factors and populations of neurons vulnerable in specific diseases, suggesting clinical use of the proteins. Recent detailed studies indicate that TrkA receptors are predominantly expressed by small cutaneous and visceral sensory neurons and TrkC receptors by large proprioceptive neurons, whereas TrkB receptors are expressed by a fraction of all types of sensory neurons. In line with these findings, small cutaneous sensory neurons are absent in mutant mice that lack functional NGF or TrkA genes. Nerve growth factor (NGF) deprivation caused by autoimmunization exhibits degenerative changes of sensory neurons. Expression studies and analysis of mutant mice suggest that NGF may be useful in the treatment of neuropathies affecting small sensory and visceral sensory neurons whereas NT-3 is expected to be beneficial for large proprioceptive sensory neurons.