Rolf Heumann

Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress

Erythropoietin (EPO) promotes neuronal survival after hypoxia and other metabolic insults by largely unknown mechanisms. Apoptosis and necrosis have been proposed as mechanisms of cellular demise, and either could be the target of actions of EPO. This study evaluates whether antiapoptotic mechanisms can account for the neuroprotective actions of EPO. Systemic administration of EPO (5,000 units/kg of body weight, i.p.) after middle-cerebral artery occlusion in rats dramatically reduces the volume of infarction 24 h later, in concert with an almost complete reduction in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling of neurons within the ischemic penumbra. In both pure and mixed neuronal cultures, EPO (0.1–10 units/ml) also inhibits apoptosis induced by serum deprivation or kainic acid exposure. Protection requires pretreatment, consistent with the induction of a gene expression program, and is sustained for 3 days without the continued presence of EPO. EPO (0.3 units/ml) also protects hippocampal neurons against hypoxia-induced neuronal death through activation of extracellular signal-regulated kinases and protein kinase Akt-1/protein kinase B. The action of EPO is not limited to directly promoting cell survival, as EPO is trophic but not mitogenic in cultured neuronal cells. These data suggest that inhibition of neuronal apoptosis underlies short latency protective effects of EPO after cerebral ischemia and other brain injuries. The neurotrophic actions suggest there may be longer-latency effects as well. Evaluation of EPO, a compound established as clinically safe, as neuroprotective therapy in acute brain injury is further supported.

Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation.

The levels of nerve growth factor (NGF) and its mRNA in the rat central nervous system were determined by two‐site enzyme immunoassay and quantitative Northern blots, respectively. Relatively high NGF levels (0.4‐1.4 ng NGF/g wet weight) were found both in the regions innervated by the magnocellular cholinergic neurons of the basal forebrain (hippocampus, olfactory bulb, neocortex) and in the regions containing the cell bodies of these neurons (septum, nucleus of the diagonal band of Broca, nucleus basalis of Meynert). Comparatively low, but significant NGF levels (0.07‐0.21 ng NGF/g wet weight) were found in various other brain regions. mRNANGF was found in the hippocampus and cortex but not in the septum. This suggests that magnocellular cholinergic neurons of the basal forebrain are supplied with NGF via retrograde axonal transport from their fields of innervation. These results, taken together with those of previous studies showing that these neurons are responsive to NGF, support the concept that NGF acts as trophic factor for magnocellular cholinergic neurons.

NGF-Mediated increase of choline acetyltransferase (ChAT) in the neonatal rat forebrain: Evidence for a physiological role of NGF in the brain?

Abstract Neonatal rats received intraventricular injections of mouse submandibular gland nerve growth factor (NGF) on days 1, 3, 5 and 7 postpartum. After killing the animals at day 8, activities of choline acetyltransferase (ChAT), acetylcholine esterase (AChE) and tyrosine hydroxylase (TH) were measured in different brain areas. NGF treatment increased ChAT activity in the septal area by 78%, in the hippocampus by 30%, and in the cortex by 73% relative to control animals. No increase was observed in other brain areas. The elevation of ChAT activity was not accompanied by an increased activity of AChE, and the concomitant 30–40% increase of TH activity observed in the cortex and brainstem was abolished after immunosympathectomy, reflecting the ingrowth of peripheral sympathetic peripheral fibers into the central nervous system (CNS) in response to centrally administered NGF23. In adult rats, repeated injections of NGF over 4 weeks caused a small but statistically significant increase of ChAT activity (15%) in the forebrain. In contrast, repeated intraventricular or intracortical injections into neonatal rats of large amounts of purified antibodies against mouse NGF (anit-NGF) failed to reduce ChAT activity in the same forebrain areas. Moreover, the offspring of rats autoimmunized against mouse NGF showed no reduction of ChAT activity in the brain, even though the TH activity was reduced by 76% in the superior cervical ganglia (SCG) of these animals. Antibodies against mouse NGF were also without effect on ChAT activity in cultures of dissociated septal neurons, though these cells also responded to NGF with an increase in ChAT activity. Anti-NGF blocked the effect of exogenous NGF but failed to reduce basal ChAT activity in these cultures. It is concluded that exogenous NGF can affect forebrain cholinergic neurons during their development. NGF does not seem to be identical with an endogenous neurotrophic factor produced by hippocampus or neocortex acting on cholinergic neurons of the forebrain.

Nerve growth factor increases choline acetyl-transferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons

Neurons dissociated from the septal area of fetal rat brains were grown in culture. Cholinergic neurons were identified by immunocytochemical visualization of choline acetyltransferase and cytochemical demonstration of acetyl cholinesterase. Choline acetyltransferase immunocytochemistry stained cell bodies and proximal processes while acetylcholinesterase cytochemistry visualized the entire neuron. Choline acetyltransferase-positive neurons could only be identified in cultures grown under conditions that produced the maximal choline acetyltransferase activity, measured biochemically. All of the choline acetyltransferase-positive neurons were double stained for acetylcholinesterase while only 6% of the acetylcholinesterase-positive cells were choline acetyltransferase negative in these cultures. These results indicate that acetylcholinesterase is a reliable marker for cholinergic cells in cultures of dissociated septal neurons. Being the more sensitive method, acetylcholinesterase staining was therefore used to identify cholinergic cells in cultures with choline acetyltransferase levels insufficient for immunocytochemical visualization of this enzyme. Addition of nerve growth factor or antibodies to nerve growth factor to the medium did not affect the number of cholinergic neurons surviving in culture. Furthermore, nerve growth factor and anti-nerve growth factor failed to influence the general morphological appearance and the number of processes of these neurons. However, nerve growth factor elevated the biochemically measured activity of choline acetyltransferase up to two-fold. The nerve growth factor-mediated increase in choline acetyltransferase activity was dose dependent with an ED50 of 10 ng/ml (4 X 10(-10) M). The increase was highly specific for nerve growth factor. It was blocked by anti-nerve growth factor, and epidermal growth factor, insulin and other control proteins failed to exert a similar effect. Nerve growth factor had to be present for at least 3 days in the culture medium to increase choline acetyltransferase activity, suggesting that the increase was due to an elevated choline acetyltransferase synthesis rather than to an activation of the enzyme.

Synaptic secretion of BDNF after high-frequency stimulation of glutamatergic synapses.

The protein brain‐derived neurotrophic factor (BDNF) has been postulated to be a retrograde or paracrine synaptic messenger in long‐term potentiation and other forms of activity‐dependent synaptic plasticity. Although crucial for this concept, direct evidence for the activity‐dependent synaptic release of BDNF is lacking. Here we investigate secretion of BDNF labelled with green fluorescent protein (BDNF–GFP) by monitoring the changes in fluorescence intensity of dendritic BDNF–GFP vesicles at glutamatergic synaptic junctions of living hippocampal neurons. We show that high‐frequency activation of glutamatergic synapses triggers the release of BDNF–GFP from synaptically localized secretory granules. This release depends on activation of postsynaptic ionotropic glutamate receptors and on postsynaptic Ca2+ influx. Release of BDNF–GFP is also observed from extrasynaptic dendritic vesicle clusters, suggesting that a possible spatial restriction of BDNF release to specific synaptic sites can only occur if the postsynaptic depolarization remains local. These results support the concept of BDNF being a synaptic messenger of activity‐dependent synaptic plasticity, which is released from postsynaptic neurons.

BDNF and NT-4/5 enhance glutamatergic synaptic transmission in cultured hippocampal neurones

THE effects of BDNF, and NT-4/5 on AMPA receptor-mediated synaptic transmission were investigated with the patch clamp technique applied to embryonic, and postnatal rat hippocampal neurones, cultured in serum-free medium. Spatially restricted application of neurotrophin-containing solution on to the recorded cells was performed, and evoked as well as miniature excitatory postsynaptic currents (mepscs) were monitored. In approximately 25% of neurones tested a transient augmentation of evoked synaptic currents, and a transient increase in the frequency of mepscs occurred with a delay of 0.5–5 min after the onset of BDNF or NT-4/5 application. The amplitudes of the AMPA receptor mediated mepscs were unaffected, suggesting a presynaptic action of BDNF, and NT-4/5.

Regulation of Nerve Growth Factor (NGF) Synthesis in the Rat Central Nervous System: Comparison between the Effects of Interleukin-1 and Various Growth Factors in Astrocyte Cultures and in vivo

In order to obtain information on the physiological regulation of NGF‐synthesis in the central nervous system (CNS) we investigated the effects of a series of growth factors (known to be present in the CNS) in cultures of purified rat astrocytes and compared these effects with those observed after intraventricular injection of the same molecules. After preliminary experiments had shown that 10% fetal calf serum (FCS) produced a marked increase in NGF‐mRNA levels in astrocytes (but neither in microglia nor oligodendrocytes) as demonstrated by Northern blot analysis and in situ hybridization the experiments were performed at low (0.5%) FCS concentrations. Supramaximal concentrations of IL‐1 and various growth factors caused a 5‐ to 7‐fold increase in NGF‐mRNA after 6 h. By 24 h the NGF‐mRNA levels approached control values again, most probably due to inactivation of the added factors since after readdition after 24 h the response was about the same as the initial one. Norepinephrine and 8‐bromo‐cAMP did not change NGF‐mRNA levels. The growth factor‐mediated changes in NGF‐mRNA levels in astrocyte cultures were not consistently reflected by the changes observed after intraventricular injection. IL‐1 produced by far the largest increase in hippocampal NGF‐mRNA after intraventricular injection. This large response to IL‐1 could result from a positive feedback mechanism, since IL‐1β injection not only increases NGF‐mRNA but also IL‐1β‐mRNA in the hippocampus. The understanding of the physiological regulation of NGF synthesis in the CNS is the basis for a rational approach to its pharmacological modification. This, in turn, is an attractive alternative to the (long‐term) infusion of NGF or the transplantation of NGF‐secreting cells with the goal of providing trophic support to the cholinergic neurons of the basal forebrain nuclei. These neurons are consistently affected in the early stages of Alzheimers disease, their impaired function being essentially responsible for the cognitive deficits.

Cellular localization of nerve growth factor synthesis by in situ hybridization.

A very sensitive and specific method for in situ hybridization has been developed. This method detects low copy numbers of mRNA(NGF) transcripts in both tissue sections and cultured cells using 35S‐labelled cRNA and oligonucleotide probes. In order to reduce the high nonspecific background occurring with 35S‐labelled probes, prehybridization in the presence of non‐labelled thio alpha UTP at pH 5.5 proved to be essential, together with a series of additional changes in the standard procedures for in situ hybridization. With this improved method it was possible to demonstrate that in tissues densely innervated by sensory (whisker pad) or both sympathetic and sensory (iris) fibers, NGF is synthesized not only by Schwann cells ensheathing these fibers, but also‐‐and even to a much larger extent‐‐by the target cells of the sensory and sympathetic neurons, i.e. epithelial cells, smooth muscle cells and fibroblasts. Moreover, in the sciatic nerve of newborn rats (where the mRNA(NGF) levels are 15 X higher than in adults) it was demonstrated that all Schwann cells have the capacity to express mRNA(NGF), not just those ensheathing the axons of NGF‐responsive neurons.

Relationship between levels of nerve growth factor (NGF) and its messenger RNA in sympathetic ganglia and peripheral target tissues

We have developed a sensitive assay for the quantification of nerve growth factor mRNA (mRNANGF) in various tissues of the mouse using in vitro transcribed RNANGF. Probes of both polarities were used to determine the specificity of the hybridization signals obtained. Comparison of NGF levels with its mRNA revealed that both were correlated with the density of sympathetic innervation. Thus, vas deferens contained high levels of both NGF and mRNANGF, whereas skeletal muscle levels were barely detectable, indicating that in peripheral tissues NGF levels are primarily regulated by the quantity of mRNANGF and not by the rate of processing of NGF precursor to NGF. However, although superior cervical ganglia contained the highest levels of NGF, its mRNA was barely detectable. Thus, the high levels of NGF in sympathetic ganglia result from retrograde axonal transport rather than local synthesis. The quantity of NGF found in the submandibular glands of female animals was three orders of magnitude higher than expected from their mRNA levels. This observation is discussed in the context of the difference between the mechanism of storage and exocytosis of exocrine glands versus the constitutive release from other tissues.

Macrophage dependence of peripheral sensory nerve regeneration: Possible involvement of nerve growth factor

The levels of NGF and NGF receptor mRNA, the degree of macrophage recruitment, and the ability of sensory and motor axons to regenerate were measured in C57BL/Ola mice, in which Wallerian degeneration following a nerve lesion is very slow. Results were compared with those from C57BL/6J and BALB/c mice, in which degeneration is normal. We found that in C57BL/Ola mice, apart from the actual lesion site, recruitment of macrophages was much lower, levels of mRNA for both NGF and its receptor were raised only slightly above normal, and sensory axon regeneration was much impaired. Motor axons regenerated quite well. These results provide in vivo evidence that macrophage recruitment is an important component of NGF synthesis and of sensory (but not motor) axon maintenance and regrowth.