Hans Thoenen

Neurotrophins and Neuronal Plasticity

There is increasing evidence that neurotrophins (NTs) are involved in processes of neuronal plasticity besides their well-established actions in regulating the survival, differentiation, and maintenance of functions of specific populations of neurons. Nerve growth factor, brain-derived neurotrophic factor, NT-4/5, and corresponding antibodies dramatically modify the development of the visual cortex. Although the neuronal elements involved have not yet been identified, complementary studies of other systems have demonstrated that NT synthesis is rapidly regulated by neuronal activity and that NTs are released in an activity-dependent manner from neuronal dendrites. These data, together with the observation that NTs enhance transmitter release from neurons that express the corresponding signal-transducing Trk receptors, suggest a role for NTs as selective retrograde messengers that regulate synaptic efficacy.

Purification of a new neurotrophic factor from mammalian brain.

We report the purification from pig brain of a factor supporting the survival of, and fibre outgrowth from, cultured embryonic chick sensory neurons. The purified factor migrates as one single band, mol. wt. 12 300, on gel electrophoresis in the presence of sodium dodecylsulphate (SDS) and is a basic molecule (pI greater than or equal to 10.1). Approximately 1 microgram factor was isolated from 1.5 kg brain. The final degree of purification was estimated to be 1.4 X 10(6)‐fold, and the specific activity 0.4 ng/ml/unit, which is similar to that of nerve growth factor (NGF) using the same assay system. This factor is the first neurotrophic factor to be purified since NGF, from which it is clearly distinguished because it has different antigenic and functional properties.

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.

Activity dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by non-NMDA glutamate receptors.

The mRNAs of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) exhibit a similar, though not identical, regional and cellular distribution in the rodent brain. In situ hybridization experiments have shown that BDNF, like NGF, is predominantly expressed by neurons. The neuronal localization of the mRNAs of these two neurotrophic molecules raised the question as to whether neuronal activity might be involved in the regulation of their synthesis. After we had demonstrated that depolarization with high potassium (50 mM) resulted in an increase in the levels of both BDNF and NGF mRNAs in cultures of hippocampal neurons, we investigated the effect of a large number of transmitter substances. Kainic acid, a glutamate receptor agonist, was by far the most effective in increasing BDNF and NGF mRNA levels in the neurons, but neither N‐methyl‐D‐aspartic acid (NMDA) nor inhibitors of the NMDA glutamate receptors had any effect. However, the kainic acid mediated increase was blocked by antagonists of non‐NMDA receptors. Kainic acid also elevated levels of BDNF and NGF mRNAs in rat hippocampus and cortex in vivo. These results suggest that the synthesis of these two neurotrophic factors in the brain is regulated by neuronal activity via non‐NMDA glutamate receptors.

The changing scene of neurotrophic factors.

The purification of brain-derived neurotrophic factor (BDNF), the elucidation of its primary structure, and the subsequent identification of neurotrophin-3 (NT-3) ended the monopoly of NGF as the only well-characterized, target-derived neurotrophic molecule. NGF, BDNF and NT-3 are members of a gene family called neurotrophins. They have strictly conserved domains that determine their basic structure. However, they also have distinctly variable domains that determine their different neuronal specificity mediated by different high affinity receptors, and that share a common low affinity subunit. These similarities and dissimilarities between the members of the neurotrophin gene family are also reflected by their regional distribution, cellular localization and developmental regulation. In this article the neurotrophins are compared with ciliary neurotrophic factor (CNTF), which is a representative of the category of neurotrophic molecules that, according to their regional distribution, developmental expression and cellular localization, do not fulfil the criteria of a target-derived neurotrophic molecule. The physiological and pathophysiological functions of neurotrophins and CNTF are discussed in the context of their potential use for the treatment of traumatic and degenerative diseases of the peripheral and central nervous systems.

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.

The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival

The survival of cultured chick sympathetic neurons and the outgrowth of neurites were stimulated by the basement membrane protein laminin coated onto polyornithine culture substrates. The survival‐potentiating activity was dependent on the presence of nerve growth factor. Both effects of laminin could be completely inhibited by affinity‐purified antibodies against laminin fragment 3, the product of a limited proteolysis that corresponds to the heparin‐binding globular domain at the end of the long arm of the laminin molecule. Antibodies against other laminin fragments were inactive, including those against previously determined cell‐binding domains. A large laminin fragment, E8, was produced by brief elastase digestion and shown to consist of fragment 3 and an adjacent rod‐like structure. Although lacking the cell binding domains, fragment E8 potentiated both neuronal survival and neurite outgrowth, and these effects could be blocked by antibodies against fragment 3. Weak survival and neurite potentiating activity was also detected in another fragment corresponding to the short arms of laminin, but as these effects were not inhibited by any of the antibodies tested they probably arose de novo during proteolysis. The heparin‐binding domain of laminin is therefore responsible for its effects on neurons.

Neurotrophin-evoked rapid excitation through TrkB receptors

Neurotrophins are a family of structurally related proteins that regulate the survival, differentiation and maintenance of function of different populations of peripheral and central neurons. They are also essential for modulating activity-dependent neuronal plasticity. Here we show that neurotrophins elicit action potentials in central neurons. Even at low concentrations, brain-derived neurotrophic factor (BDNF) excited neurons in the hippocampus, cortex and cerebellum. We found that BDNF and neurotrophin-4/5 depolarized neurons just as rapidly as the neurotransmitter glutamate, even at a more than thousand-fold lower concentration. Neurotrophin-3 produced much smaller responses, and nerve growth factor was ineffective. The neurotrophin-induced depolarization resulted from the activation of a sodium ion conductance which was reversibly blocked by K-252a, a protein kinase blocker which prefers tyrosine kinase Trk receptors. Our results demonstrate a very rapid excitatory action of neurotrophins, placing them among the most potent endogenous neuro-excitants in the mammalian central nervous system described so far.

Brain-derived neurotrophic factor supports the survival of cultured rat retinal ganglion cells

Brain-derived neurotrophic factor (BDNF) is a small, basic protein purified from the mammalian brain that has been shown previously to support the survival of cultured spinal sensory neurons (Barde et al., 1982). In current studies, BDNF was tested for its ability to support the survival of cultured CNS cells isolated from the perinatal rat retina. Both immunofluorescent labeling of Thy-1 and prior retrograde labeling with HRP were used as retinal ganglion cell markers in vitro. With embryonic day (E) 17 retinas, it was found that BDNF allowed the survival of a small subpopulation of neurons (about 7% of the cells plated at this age) identified by the immunofluorescent labeling of Thy- 1. No detectable effects were seen when either the total number of cells or the number of tetanus toxin-positive neurons was measured. BDNF also had an effect on cultured neurons retrogradely labeled after HRP injections in the superior colliculi of neonatal rats. The BDNF- responsive population was therefore detected only in retinal cultures with specific markers and identified as consisting of retinal ganglion cells. These cells could be enriched about 80-fold by density gradient centrifugation, and purified ganglion cell cultures were shown to be responsive to BDNF. Whereas with E17 retinas, the number of surviving Thy-1 positive neurons could be kept constant for at least 4 d, the survival of postnatal neurons was only transiently increased by BDNF. We conclude that in the retina, BDNF affects only the survival of ganglion cells in vitro by a direct action on these cells. The results are discussed in terms of target-derived neurotrophic support during development.

Nerve growth factor (NGF) in the rat CNS: Absence of specific retrograde axonal transport and tyrosine hydroxylase induction in locus coeruleus and substantia nigra

Selective, highly efficient uptake of [125I]NGF by nerve terminals followed by retrograde axonal transport, and specific induction of tyrosine hydroxylase by NGF are well known phenomena in peripheral adrenergic neurons of adult rats. In the present study these parameters were used in order to detect possible interactions of NGF with central catecholaminergic neurons. No selective retrograde transport of [125I]NGF could be detected by light microscopic autoradiography from the caudate nucleus to the dopaminergic neurons in the substantia nigra or from the hippocampus to the noradrenergic nerve cells of the locus coeruleus. Biochemically, no change in tyrosine hydroxylase activity could be observed for up to 3 days after injection of either NGF, anti-NGF antibodies, or control proteins close to the nerve cell bodies in the substantia nigra or the locus coeruleus. These data suggest a fundamental difference between central and peripheral adrenergic neurons with regard to their responsiveness of NGF.