William C. Mobley

p140trk mRNA marks NGF-responsive forebrain neurons: Evidence that trk gene expression is induced by NGF

Nerve growth factor (NGF) appears to act as a neurotrophic factor for basal forebrain and caudate-putamen cholinergic neurons. The mechanism by which NGF transduces its signal in these neurons is yet to be defined. Recent data indicate that the product of the trk gene, p140trk, is a critical component of the NGF receptor. Herein, we show that p140trk mRNA is highly restricted in its distribution in the adult rat forebrain, that it is present in cholinergic neurons, and that most if not all cholinergic neurons contain p140trk mRNA. Furthermore, induction of trk expression by NGF suggests that neurotrophin-mediated up-regulation of their receptor tyrosine kinases is an important feature of their actions and that neurotrophins may regulate the activity of responsive neurons through increasing the level of their receptors.

EGF Receptor Signaling Stimulates SRC Kinase Phosphorylation of Clathrin, Influencing Clathrin Redistribution and EGF Uptake

Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor (EGFR) signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling.

Nerve Growth Factor Signaling in Caveolae-like Domains at the Plasma Membrane

Nerve growth factor (NGF) binding to its receptors TrkA and p75NTR enhances the survival, differentiation, and maintenance of neurons. Recent studies have suggested that NGF receptor activation may occur in caveolae or caveolae-like membranes (CLM). This is an intriguing possibility because caveolae have been shown to contain many of the signaling intermediates in the TrkA signaling cascade. To examine the membrane localization of TrkA and p75NTR, we isolated caveolae from 3T3-TrkA-p75 cells and CLM from PC12 cells. Immunoblot analysis showed that TrkA and p75NTR were enriched about 13- and 25-fold, respectively, in caveolae and CLM. Binding and cross-linking studies demonstrated that the NGF binding to both TrkA and p75NTRwas considerably enriched in CLM and that about 90% of high affinity binding to TrkA was present in CLM. When PC12 cells were treated with NGF, virtually all activated (i.e. tyrosine phosphorylated) TrkA was found in the CLM. Remarkably, in NGF-treated cells, it was only in CLM that activated TrkA was coimmunoprecipitated with phosphorylated Shc and PLCγ. These results document a signaling role for TrkA in CLM and suggest that both TrkA and p75NTRsignaling are initiated from these membranes.

Evidence for normal aging of the septo-hippocampal cholinergic system in apoE (-/-) mice but impaired clearance of axonal degeneration products following injury.

The association of the epsilon4 allele of apoE with increased risk for Alzheimers disease (AD) and with poor clinical outcome after certain acute brain injuries has sparked interest in the neurobiology of apoE. ApoE (-/-) mice provide a tool to investigate the role of apoE in the nervous system in vivo. Since integrity of the basal forebrain cholinergic system is severely compromised in AD, with severity of dysfunction correlating with apoE4 gene dosage, the present study tested the hypothesis that apoE is required to maintain the normal integrity of basal forebrain cholinergic neurons (BFCNs). Histological and biochemical analyses of the septo-hippocampal cholinergic system were performed in apoE (-/-) mice during aging and following injury. Using unbiased quantitative methods, there was little or no evidence for defects in the septo-hippocampal cholinergic system, as assessed by p75(NTR)-immunoreactive neuron number and size in the medial septum, cholinergic fiber density in the hippocampus, and choline acetyltransferase activity in the hippocampus, cortex, and striatum in aged apoE (-/-) mice (up to 24 months of age) as compared to age-matched wild-type mice of the same strain. In addition, cholinergic neuronal survival and size following fimbria-fornix transection in apoE (-/-) mice did not differ from controls. However, following entorhinal cortex lesion, there was persistence of degeneration products in the deafferented hippocampus in apoE (-/-) mice. These data suggest that although apoE is not required for the maintenance of BFCNs in vivo, it may play a role in the clearance of cholesterol-laden neurodegeneration products following brain injury.

NGF effects on developing forebrain cholinergic neurons are regionally specific.

Nerve growth factor (NGF) has been shown to have an effect on neurons in the central nervous system (CNS). A number of observations suggest that NGF acts as a trophic factor for cholinergic neurons of the basal forebrain and the caudate-putamen. We sought to further characterize the CNS actions of NGF by examining its effect on choline acetyltransferase (ChAT) activity in the cell bodies and fibers of developing neurons of the septum and caudate-putamen. ChAT activity was increased after even a single NGF injection. Interestingly, the magnitude of the effect of multiple NGF injections suggested that repeated treatments may augment NGF actions on these neurons. The time-course of the response to NGF was followed after a single injection on postnatal day (PD) 2. NGF treatment produced long-lasting increases in ChAT activity in septum, hippocampus and caudate-putamen. The response in cell body regions (septum, caudate-putamen) was characterized by an initial lag period of approximately 24 hr, a rapid rise to maximum values, a plateau phase and a return to baseline. The response in hippocampus was delayed by 48 hr relative to that in septum, indicating that NGF actions on ChAT were first registered in septal cell bodies. Finally, developmental events were shown to have a regionally specific influence on the response of neurons to NGF. For though the septal response to a single NGF injection was undiminished well into the third postnatal week, little or no response was detected in caudate-putamen at that time. In highlighting the potency and regional specificity of NGF effects, these observations provide additional, support for the hypothesis that NGF is a trophic factor for CNS cholinergic neurons.

Developmental regulation of nerve growth factor and its receptor in the rat caudate-putamen

In prior studies, nerve growth factor (NGF) administration induced a robust, selective increase in the neurochemical differentiation of caudate-putamen cholinergic neurons. In this study, expression of NGF and its receptor was examined to determine whether endogenous NGF might serve as a neurotrophic factor for these neurons. The temporal pattern of NGF gene expression and the levels of NGF mRNA and protein were distinct from those found in other brain regions. NGF and high-affinity NGF binding were present during cholinergic neurochemical differentiation and persisted into adult-hood. An increase in NGF binding during the third postnatal week was correlated with increasing choline acetyltransferase activity. The data are consistent with a role for endogenous NGF in the development and, possibly, the maintenance of caudate-putamen cholinergic neurons.

Nerve growth factor and the neurotrophic factor hypothesis

The discovery of nerve growth factor (NGF) over 40 years ago led to the formulation of the Neurotrophic Factor Hypothesis. This hypothesis states that developing neurons compete with each other for a limited supply of a neurotrophic factor (NTF) provided by the target tissue. Successful competitors survive; unsuccessful ones die. Subsequent research on NTFs has shown that NTF expression and actions are considerably more complex and diverse than initially predicted. Even for NGF, different regulatory patterns are seen for different neuronal populations. As would be predicted by the Neurotrophic Factor Hypothesis, NGF levels critically regulate basal forebrain cholinergic neuron size and neurochemical differentiation. In contrast, the level of trkA, the NGF receptor, regulates these properties in caudate-putamen cholinergic neurons. Understanding NTF regulation and actions on neurons has led to their use in clinical trials of human neurological diseases. NTFs may emerge as important therapies to prevent neuronal dysfunction and death.

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.

Induction of the c-fos gene product in rat forebrain following cortical lesions and NGF injections.

Neocortical lesions and NGF injections into neocortex induce the immunostaining of Fos, the c-fos gene product, in neuronal nuclei in ipsilateral cortex, and amygdala. Adjacent structures including hippocampus, septal nuclei, globus pallidus, and thalamus were unaffected. It is hypothesized that trophic molecules or other chemicals are released at the injury site and these induce the c-fos gene in cells throughout the ipsilateral hemisphere. Fos induction might mediate metabolic or plasticity responses to the focal injury.

Multiple Levels for Regulation of TrkA in PC12 Cells by Nerve Growth Factor

Abstract: TrkA is a receptor tyrosine kinase for nerve growth factor (NGF). Recent studies indicate that NGF regulates not only activation of trkA kinase but also expression of the trkA gene. To further define NGF actions on trkA, we examined binding and signaling through trkA after both short and long intervals of NGF treatment. Induction of tyrosine phosphorylation on gp140trkA was rapidly followed by down‐regulation of cell surface and total cellular gp140trkA. At later intervals, increased expression of trkA was evident in increased mRNA and protein levels. At 7 days, there was increased binding to gp140trkA and increased signaling through this receptor. NGF appears to regulate trkA at several levels. In neurons persistently exposed to NGF, maintenance of NGF signaling may require increased trkA gene expression.