Michael D. Coughlin

The Precursor Pro-Nerve Growth Factor Is the Predominant Form of Nerve Growth Factor in Brain and Is Increased in Alzheimer's Disease

Nerve growth factor (NGF) is important for regulation, differentiation, and survival of peripheral and central nervous system neurons, including basal forebrain cholinergic neurons (BFCN) which degenerate in Alzheimers disease (AD). Mature NGF protein is processed from a larger precursor, proNGF. We demonstrate that proNGF is the predominant form of NGF in mouse, rat, and human brain tissue, whereas little or no mature NGF is detected. Previous reports showed NGF protein, measured by ELISA, is increased in AD BFCN target regions such as hippocampus and cortex. Using Western blotting, we demonstrate a twofold increase in proNGF in AD parietal cortex compared to controls, indicating that it is this precursor form, proNGF, that accumulates in AD. This increase may reflect either a role for biologically active proNGF or posttranslational disturbances in NGF biosynthesis that decrease the processing of proNGF to mature NGF in AD.

The nerve growth factor precursor proNGF exhibits neurotrophic activity but is less active than mature nerve growth factor

Nerve growth factor (NGF) promotes neuronal survival and differentiation and stimulates neurite outgrowth. NGF is synthesized as a precursor, proNGF, which undergoes post‐translational processing to generate mature β‐NGF. It has been assumed that, in vivo, NGF is largely processed into the mature form and that mature NGF accounts for the biological activity. However, we recently showed that proNGF is abundant in CNS tissues whereas mature NGF is undetectable, suggesting that proNGF has biological functions beyond its role as a precursor. To determine whether proNGF exhibits biological activity, we mutagenized the precursor‐processing site and expressed unprocessed, cleavage‐resistant proNGF protein in insect cells. Survival and neurite outgrowth assays on murine superior cervical ganglion neurons and PC12 cells indicated that proNGF exhibits neurotrophic activity similar to mature 2.5S NGF, but is approximately fivefold less active. ProNGF binds to the high‐affinity receptor, TrkA, as determined by cross‐linking to PC12 cells, and is also slightly less active than mature NGF in promoting phosphorylation of TrkA and its downstream signaling effectors, Erk1/2, in PC12 and NIH3T3‐TrkA cells. These data, coupled with our previous report that proNGF is the major form of NGF in the CNS, suggest that proNGF could be responsible for much of the biological activity normally attributed to mature NGF in vivo.

Nerve growth factor-independent development of embryonic mouse sympathetic neurons in dissociated cell culture☆

Although ganglia from neonatal mouse sympathetic ganglia require nerve growth factor (NGF) for survival in culture, explanted sympathetic ganglia from early embryonic stages do not require added NGF for survival and growth. To determine whether the change in growth factor requirement is due to changes in the neurons themselves, to variations in neuronal populations, or to changes in nonneuronal cells, we examined the response to growth factors by dissociated sympathetic neurons at various stages of development. Results indicate that neurons from the 14-day gestational (E14) superior cervical ganglion (SCG) do not require NGF for initial survival and neurite extension, but do require the conditioned medium neurite extension factor, CMF. By 2 to 3 days thereafter, whether in vivo or in culture, most neurons have developed a requirement for NGF for survival in culture. During the same period, there is a concomitant increase in responsiveness to NGF alone as a trophic agent. Changes in response to NGF are not due to changes in NGF content of ganglia, to interactions in culture with nonneuronal cells, or to age-related differences in NGF requirements for maximum survival. The changes in growth factor requirements may be related to mechanisms regulating specificity of nerve-target connections.

ProNGF: a neurotrophic or an apoptotic molecule?

Nerve growth factor (NGF) acts on various classes of central and peripheral neurons to promote cell survival, stimulate neurite outgrowth and modulate differentiation. NGF is synthesized as a precursor, proNGF, which undergoes processing to generate mature NGF. It has been assumed, based on studies in the mouse submandibular gland, that NGF in vivo is largely mature NGF, and that mature NGF accounts for the molecules biological activity. However, recently we have shown that proNGF is abundant in central nervous system tissues whereas mature NGF is undetectable, suggesting that proNGF may have a function distinct from its role as a precursor. A recent report that proNGF has apoptotic activity contrasts with other data demonstrating that proNGF has neurotrophic activity. This chapter will review the structure and processing of NGF and what is known about the biological activity of proNGF. Possible reasons for the discrepancies in recent reports are discussed.

Biological Activity of Nerve Growth Factor Precursor Is Dependent upon Relative Levels of Its Receptors

Nerve growth factor (NGF) is produced as a precursor called pro-nerve growth factor (proNGF), which is secreted by many tissues and is the predominant form of NGF in the central nervous system. In Alzheimer disease brain, cholinergic neurons degenerate and can no longer transport NGF as efficiently, leading to an increase in untransported NGF in the target tissue. The protein that accumulates in the target tissue is proNGF, not the mature form. The role of this precursor is controversial, and both neurotrophic and apoptotic activities have been reported for recombinant proNGFs. Differences in the protein structures, protein expression systems, methods used for protein purification, and methods used for bioassay may affect the activity of these proteins. Here, we show that proNGF is neurotrophic regardless of mutations or tags, and no matter how it is purified or in which system it is expressed. However, although proNGF is neurotrophic under our assay conditions for primary sympathetic neurons and for pheochromocytoma (PC12) cells, it is apoptotic for unprimed PC12 cells when they are deprived of serum. The ratio of tropomyosin-related kinase A to p75 neurotrophin receptor is low in unprimed PC12 cells compared with primed PC12 cells and sympathetic neurons, altering the balance of proNGF-induced signaling to favor apoptosis. We conclude that the relative level of proNGF receptors determines whether this precursor exhibits neurotrophic or apoptotic activity.

Access to medical records for research purposes: varying perceptions across research ethics boards

Introduction: Variation across research ethics boards (REBs) in conditions placed on access to medical records for research purposes raises concerns around negative impacts on research quality and on human subject protection, including privacy. Aim: To study variation in REB consent requirements for retrospective chart review and who may have access to the medical record for data abstraction. Methods: Thirty 90-min face-to-face interviews were conducted with REB chairs and administrators affiliated with faculties of medicine in Canadian universities, using structured questions around a case study with open-ended responses. Interviews were recorded, transcribed and coded manually. Results: Fourteen sites (47%) required individual patient consent for the study to proceed as proposed. Three (10%) indicated that their response would depend on how potentially identifying variables would be managed. Eleven sites (38%) did not require consent. Two (7%) suggested a notification and opt-out process. Most stated that consent would be required if identifiable information was being abstracted from the record. Among those not requiring consent, there was substantial variation in recognising that the abstracted information could potentially indirectly re-identify individuals. Concern over access to medical records by an outside individual was also associated with requirement for consent. Eighteen sites (60%) required full committee review. Sixteen (53%) allowed an external research assistant to abstract information from the health record. Conclusions: Large variation was found across sites in the requirement for consent for research involving access to medical records. REBs need training in best practices for protecting privacy and confidentiality in health research. A forum for REB chairs to confidentially share concerns and decisions about specific studies could also reduce variation in decisions.

Human Basophilic Cell Differentiation Promoted by 2.5S Nerve Growth Factor

In liquid cultures of human cord blood mononuclear cells, the activities of the 2.5S nerve growth factor (NGF) inducing basophil and eosinophil differentiation were investigated. Various concentrations of immunopurified 2.5S NGF derived from murine submaxillary glands were added to cultures with or without conditioned medium from a human T cell line (Mo-CM), which has previously been shown to produce activities stimulating granulocyte-macrophage colonies. Addition of NGF led to significant increases in differentiation of basophilic cells accompanied by histamine synthesis at 2 weeks in vitro; eosinophil differentiation was not increased in these cultures. In addition, NGF could be shown to amplify basophil differentiation induced by Mo-CM, and the activity of NGF inducing basophil differentiation was dependent on the presence of T lymphocytes. These results indicate that NGF stimulates T-lymphocyte-dependent basophilic cell differentiation from human cord blood progenitors and may in this way support differentiation of basophils or mast cells in vivo at sites of allergic tissue inflammation.

Nerve growth factor enhances antigen and other secretagogue-induced histamine release from rat peritoneal mast cells in the absence of phosphatidylserine.

The effects of 2.5S nerve growth factor (NGF) and epidermal growth factor (EGF), isolated from mouse submaxillary glands, on histamine release from rat peritoneal mast cells (PMCs) were studied. In the absence of phosphatidylserine, NGF (1 ng/ml to 1 microgram/ml) did not cause histamine release from PMCs isolated from normal rats and those infected with the nematode Nippostrongylus brasiliensis. However, when PMCs (greater than 97% pure) were preincubated with NGF and then challenged with worm antigen (Ag), there was a marked enhancement of histamine release (approximately twofold with a maximum effect at 10 ng/ml of NGF [3.8 X 10(-10) mol/L]) compared with the release induced by Ag alone. EGF (1 ng/ml to 1 microgram/ml) neither produced histamine release from PMCs in the presence of phosphatidylserine nor enhanced Ag-induced histamine release. This suggests that NGF acts directly on PMCs by activation of cell-surface receptors. The early kinetics of Ag-induced histamine release were altered by NGF that increased the initial rate at 15 seconds but did not prolong the overall duration of histamine release. Simultaneous addition of Ag and NGF did not cause enhanced histamine release; thus, some preincubation time with NGF (5 minutes or less) was required for the activation of PMCs. Moreover, after PMCs were activated by NGF, that state persisted for 1 hour, even when unbound NGF was removed by washing, and thereafter subsided gradually. Further studies revealed that NGF enhanced histamine release induced by concanavalin A, compound 48/80, and ionophore A23187. These results suggest that NGF might be an important molecule in inflammatory responses through the regulation of mediator release from mast cells.

Exploring a model role description for ethicists

This paper provides a description of the role of the clinical ethicist as it is generally experienced in Canada. It examines the activities of Canadian ethicists working in healthcare institutions and the way in which their work incorporates more than ethics case consultation. The Canadian Bioethics Society established a “Taskforce on Working Conditions for Bioethics” (hereafter referred to as the Taskforce), to make recommendations on a number of issues affecting ethicists and to develop a model role description. This essay carefully assesses this model role description.

Growth Factors Regulating Autonomic Nerve Development

Publisher Summary During ontogenesis, sympathetic and parasympathetic neurons pass through a common series of developmental stages. The cellular interactions that mold the autonomic neuron from the beginning of its migration out of the neural crest to the mature stage of functional activity are mediated by a number of regulatory molecules or growth factors. This chapter provides an overview of these growth factors regulating autonomic nerve development. Although such factors are normally capable of affecting neurons throughout their life history, there appear to be specific stages of development at which different growth factors assume a primary role. Thus, responsiveness to different growth factors change during ontogeny. Specification of neurotransmitter phenotype is normally associated with the initial migratory phase and the stage of ganglion consolidation. Following ganglion formation, neurons extend processes to their peripheral targets. During this period of neurite extension, neurons survive and grow independently of trophic factors. After the period of normal neuronal death, resulting from competition for target-derived factors, neurons appear to regulate the release or activity of trophic factors from targets, thus maintaining their own territory of innervation by inhibiting ingrowth from neighboring axons.