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NGF

[nerve growth factor]
SOURCES

NGF was isolated initially from the submaxillary glands of mice. NGF is synthesized in the hypothalamus, pituitary, thyroid gland, testes, and the epididymis. It is produced also by various cell types including vascular smooth muscle cells and fibroblasts.

The expression of NGF in specific neurons of the central nervous system (cortex, hippocampus) can be influenced positively by glutamate mediated or negatively by GABAergic neuronal activity.

Serum, phorbol 12-myristate 13-acetate (see also: Phorbol esters), and vitamin D3 are potent inducers of NGF synthesis. Glucocorticoids inhibit the synthesis of NGF.

The synthesis of NGF in astrocytes is enhanced by IL1, TNF-alpha, PDGF and TGF-beta. These factors do not influence the synthesis of NGF in Schwann cells; NGF synthesis in Schwann cells is decreased by TGF-beta.


PROTEIN CHARACTERISTICS

NGF is synthesized as a preproprotein of 305 amino acids including a secretory signal sequence of 18 amino acids and a prosequence of 103 amino acids. The mature factor is obtained by proteolytic processing at the N- and C-terminus of the precursor protein. NGF sequences have been conserved during evolution and display little sequence deviations in various species. For a pathophysiological role of the unprocessed NGF precursor see: proNGF.


NGFsequence
Sequences of NGF derived from different species.

This sequence comparison shows the strong evolutionary conservation of NGF sequences among species. Identical amino acid positions are shown in blue. Conserved cysteine residues are shown in yellow.


The 7S form of NGF (7S NGF) initially isolated from the submaxillary gland of mice is a complex of three proteins (alpha, beta, and gamma; sometimes also referred to as NGF-alpha, NGF-beta (gene symbol NGFB), and NGF-gamma (gene symbol NGFG). The complex is stabilized by zinc ions (Young et al, 1988). The 26 kDa beta subunit is a homodimer of two disulfide-bonded proteins with a length of 118 amino acids and displays the biological activity of NGF.

The NGF-alpha and NGF-gamma subunits of the NGF complex are closely related members of the Kallikreins protein family that are structurally related to trypsin-like serine proteases (Ronne et al, 1984). NGF-alpha (referred to in databanks also as kallikrein Klk1b4 or Klk1b4, is closely related to NGF-gamma but appears to be enzymatically inactive (Young et al, 1988; Evans and Richards, 1985; Isackson et al, 1984). The gamma subunit in the mouse is an enzyme involved in processing NGF (also referred to as kallikrein Klk1b3 or Klk1b3) (Mason et al, 1983). The gamma subunit of NGF is related to a binding protein for EGF (EGF-PB and has been shown also to cleave and activate the precursor of a macrophage stimulating protein (MSP).


GENE STRUCTURE

The murine gene encoding Beta-NGF has a length of at least 45 kb and contains four exons. The 3' end of the fourth exons contains the coding sequence for NGF-beta. The mouse genes for NGF-alpha and NGF-gamma, located on mouse chromosome 7, are contiguous, transcribed from the same DNA strand, and separated by 5.3 kb of intergenic DNA. The human NGF gene maps to chromosome 1p13 and is organized in a similar way.

Differential splicing of the primary transcripts yields two different mRNAs that are expressed differentially also in different tissues. NGF-alpha and NGF-gamma are encoded by separate genes. They show a marked sequence homology and are probably derived from a common ancestor.

The first exon of the NGF gene contains a response element for phorbol esters (see: TRE), which functions as a binding site for transcription factors (see also: gene expression) that enhance the expression of the NGF gene.


RELATED FACTORS

Two proteins, BDNF (brain-derived neurotrophic factor) and NT-3 (neurotrophin-3) are related to NGF. They show some overlapping but not identical biological activities. The three proteins form the NGF family of proteins. They are also referred to collectively as Neurotrophins.

NGF, TGF-beta-2 (see: TGF-beta), and PDGF-BB (see: PDGF) share a similar overall topology (see also: Cystine knot growth factor family). For another factor related to NGF see also: NGF-2.


RECEPTORS

One receptor that is responsible for mediating most of the activities of NGF is expressed preferentially in neuronal tissues. This glycoprotein of 140 kDa (gp140trk) is the product of the trk gene. It possesses an intrinsic tyrosine-specific protein kinase in its intracellular domain. The expression of trk in monocytes as well as other findings suggest that NGF, in addition to its neurotrophic function, is an immunoregulatory cytokine acting on monocytes.

A low affinity receptor (kdis = 10**-9 - 10**-8 M) of unknown physiological role is encoded by the LNGFR gene (low affinity nerve growth factor receptor). LNGFR is a transmembrane cysteine-rich glycoprotein of 399 amino acids (gp80LNGFR) with a very short intracellular domain.

The low affinity receptor is expressed by many neurons that do not respond to NGF. It is expressed also in non-neuronal tissues such as testes, muscles, lymphoid tissues, and lymphocytes. LNGFR is also a receptor for BDNF (brain-derived neurotrophic factor) and NT-3.

It has been suggested that the combination of trk and LNGFR yields a high-affinity NGF receptor (kdis = 10**-11 M) but this issue is still unclear as trk alone has been shown to allow cell growth in the absence of LNGFR in heterologous systems. NRH-2, a protein related to the NGF receptor p75NTR, has been shown to interact with trkA and to form high affinity binding sites for NGF. trkA has been shown to form high affinity binding receptors after interaction with NRH-2 [neurotrophin receptor homolog-2].

The intracellular signal transduction mechanisms initiated by binding of NGF to its receptor are largely unknown. The participation of cAMP as second messenger has been ruled out. Signal transduction may involve a G-protein. The expression of the ras oncogene also appears to be necessary for signaling since an inhibition of ras expression also inhibits NGF mediated differentiation of certain cells (see: PC12).


BIOLOGICAL ACTIVITIES

NGF is not a mitogen so that its name is actually a misnomer. It is mainly responsible for the survival and the differentiation and the functional activities of sensory and sympathetic neurons in the peripheral nervous system. It also plays an important role in the development and functional activities of cholinergic neurons in the central nervous system.

The continuous infusion of NGF prevents the death of neurons in rat, following experimental transsection of the fimbria hippocampi in the fore brain. The treatment of newborn rats and mice with anti-NGF antibodies leads to the almost complete degeneration of neurons of the sympathetic nervous system and causes a variety of neuroendocrine deficits.

Immortalized rat fibroblasts, genetically altered to secrete NGF into the conditioned medium, have been shown to decrease excitotoxic lesion size by 80 % after implantation in rat brain near the striatum 7 days before striatal infusion of excitotoxic quantities of an NMDA-receptor agonist.

NGF induces the synthesis of a number of specific transmitter-like peptides in sensory neurons, including SP (substance P; see also: Tachykinins), Somatostatin, and VIP (vasoactive intestinal peptide). NGF inhibits the release of noradrenaline in sympathetic nerve ends and also acts as an inhibitory neuromodulator for adrenergic processes, possibly building an inhibitory feedback loop for synthesis of NGF stimulated by catecholamine.

Since NGF is synthesized also in non-neuronal tissues it may have a much wider spectrum of biological activities than thought previously. NGF stimulates chemotactic migration of human polymorphonuclear leukocytes in vitro (see also: Chemotaxis; Motogenic cytokines). Subdermal injection of NGF in mice also stimulates rapid and marked chemotactic recruitment of leukocytes at nanomolar concentrations. NGF also promotes the proliferation of mast cells. NGF enhances histamine release and strongly modulates the formation of lipid mediators by basophils in response to various stimuli.

NGF stimulates the growth and differentiation of B-cells and the growth of T-cells and of some tumor cell types. NGF inhibits immunoglobulin production by various human plasma cell. NGF induced inhibition of Ig production is restored by IL6. NGF also influences the differentiation of eosinophils and basophils. Since these cells are involved in immune and inflammatory reactions NGF may play an accessory role in the regulation of these processes. Torcia et al (1996) have shown that NGF is an autocrine survival factor for memory B-lymphocytes.

The cytokines IL1, IL6, and bFGF are potent inducers of NGF. NGF induces the synthesis of IL1 in pheochromocytoma cells (see: PC12) which in turn acts as a growth factor for glial cells and induces the synthesis of NGF following nerve injuries.

In thymic stromal cells (see also: Stromal cell line) NGF induces the synthesis of IL6. NGF induces the synthesis of the fos oncogene and the myc oncogene and also influences the expression of EGF.


TRANSGENIC ANIMALS, KNOCK-OUT, AND ANTISENSE STUDIES

Experiments with transgenic mice reveal that NGF may act as a growth factor under certain circumstances. The ectopic expression in the pituitary of NGF driven by a Prolactin promoter causes a pronounced hyperplasia of this gland with size enlargements of up to 10-100-fold. The overexpression of NGF in the pancreas of transgenic mice induces the enhanced innervation of this tissue.

In transgenic mice expressing the beta subunit of NGF in sympathetic neurons using the human dopamine beta-hydroxylase promoter the sympathetic trunk and nerves growing to peripheral tissues are enlarged and contain an increased number of sympathetic fibers. Although sympathetic axons reach peripheral tissues, terminal sympathetic innervation within tissues is decreased. This effect can be reversed in the pancreas by overexpression of NGF in pancreatic islets. It has been suggested that NGF gradients are not required to guide sympathetic axons to their targets, but are required for the establishment of the normal density and pattern of sympathetic innervation within target tissues.

A targeted mutation of the gene encoding the low affinity NGF receptor (LNGFR, p75) has been generated by homologous recombination in ES cells. Knock-out mice homozygous for the mutation are viable and fertile. Mutant mice show markedly decreased sensory innervation by calcitonin gene-related peptide- and substance P-immunoreactive fibers and this is associated with loss of heat sensitivity and the development of ulcers in the distal extremities. Dysfunctions of sympathetic neurons have not been observed in these mice. Embryonic cranial sensory and sympathetic neurons from p75 deficient embryos respond normally to NGF, BDNF, NT-3, and NT-4/5 at saturating concentrations. Dose responses of sympathetic and visceral sensory neurons from mutant embryos are also normal. However, embryonic cutaneous sensory trigeminal neurons isolated from mutant embryos require more NGF for half-maximal survival, indicating that p75 enhances the sensitivity of NGF dependent cutaneous sensory neurons to NGF.

The biological consequences of an NGF gene disruption have been studied in mice generated from ES cells carrying a targeted deletion of the gene. Mice homozygous for a targeted disruption of the NGF gene survive for some days but all animals die by the age of 4 weeks. NGF deficient homozygous animals show a profound cell loss in both sensory and sympathetic ganglia. The effects within the dorsal root ganglia appears to be restricted to small and medium peptidergic neurons. Examination of the central nervous system reveals that basal forebrain cholinergic neurons differentiate and continue to express phenotypic markers for the life span of the mutant mice, showing that differentiation and initial survival of central NGF-responsive neurons can occur in the absence of NGF.

An epidermal-specific gene promoter has been used to produce transgenic mice that overexpress the mouse NGF cDNA in the epidermis and associated hair follicles of the skin. The increase in NGF mRNA correlates with a hypertrophy of peripheral sensory and sympathetic nerves. Increased numbers of nerve processes in the transgenic skin display immunoreactivity for calcitonin gene-related peptide and tyrosine hydroxylase, indicating that both the sensory and sympathetic systems are hypertrophied. The trigeminal and superior cervical ganglia are greatly enlarged. Cell counts of trigeminal ganglia of control and transgenic mice show a 26-117 % increase in the number of neurons in the transgenic s, indicating a reduction or total prevention of the program of naturally occurring apoptotic cell death. These results demonstrate that NGF production by the epidermal target tissue controls neuronal survival, and in so doing, establishes the level of innervation.

Mice overexpressing NGF in skin have been found to display a profound hyperalgesia to noxious mechanical stimulation. Mice overexpressing an NGF antisense message display a profound hypoalgesia to the same stimuli.


DETECTION AND ASSAY METHODS

NGF and NGF-like activities are assayed in bioassays, measuring the outgrowth of neurites in the pheochromocytoma cell line PC12 or its derivatives. NGF is also assayed by its activity on embryonic sensory neurons. Zettler et al (1996) have described an improved extraction procedure that results in the detection of higher NGF concentrations in immunoassays using either polyclonal or commercially available monoclonal antibodies. Sensitive enzyme immunoassays are also available. NGF can be detected also by a modification of the Cell blot assay. An alternative and entirely different detection method is RT-PCR quantitation of cytokines.


CLINICAL USE AND SIGNIFICANCE

Vitamin D3 and some metabolically active precursors are potent inducers of the synthesis of NGF at concentrations of 100 picoM. This may have implications for the treatment of neurodegenerative diseases with NGF. The infusion of human NGF into brains of primates has been shown to prevent the degeneration of cholinergic neurons. It may be possible, therefore, to use NGF to protect cholinergic neurons in Alzheimer's disease which is characterized by a selective degeneration of these cells. Another possible application might be the treatment of diabetes-associated polyneuropathies. In experimental animals NGF prevents chemotherapy induced neuropathies.

Rat hippocampal and human cortical neurons have been shown to be protected by NGF against induced damage induced by iron, which is believed to contribute to the process of cell damage and death resulting from ischemic and traumatic insults (see also: inflammation, wound healing) by catalyzing the oxidation of protein and lipids.

Increased levels of anti-NGF antibodies have been found in the sera of HSV-infected patients. Since NGF promotes the latency of HSV in vitro it is thought that these antibodies may play a role in the course of these viral infections.



 

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