Mouse NGF Research Articles

Overproduction of biologically-active human nerve growth factor in Escherichia coli.

A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the beta-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the beta-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells.

The use of genetically altered astrocytes to provide nerve growth factor to adrenal chromaffin cells grafted into the striatum

Transplantation of adrenal chromaffin cells into the striatum of Parkinson's disease patients is unlikely to become a reliable therapy unless techniques are devised to improve cell survival. To address this issue, we investigated the use of genetically altered astrocytes that constitutively secrete β-nerve growth factor (NGF) to provide trophic support for adrenal chromaffin cells grafted into the dopamine-denervated striatum of the rat. Primary rat astrocytes were altered genetically in vitro by infection with a retroviral vector harboring a mouse β-NGF transgene under constitutive long terminal repeat transcriptional control. Confluent cultures of these genetically altered astrocytes secrete NGF into their culture medium at a rate of approximately 9 pg/10 5 cells/h. This rate of NGF secretion is at least 10-fold higher than that of confluent sister cultures of uninfected astrocytes. The effects of the NGF-secreting astrocytes on the survival and neuronal transformation of dissociated adrenal chromaffin cells were assessed in vitro and following transplantation into the dopamine-denervated striatum of the adult rat. In vitro experiments demonstrated that neuritic outgrowth is stimulated when postnatal day 12 chromaffin cells are grown on a monolayer of the genetically altered astrocytes. When co-grafted with genetically altered astrocytes, young postnatal chromaffin cells displayed extensive neuritic outgrowth within the host brain 2 weeks postimplantation, whereas chromaffin cells grafted alone or with normal astrocytes retain an endocrine-like morphology. Survival of the chromaffin cells is also enhanced 3–6-fold when co-grafted with the genetically altered astrocytes. In addition, the neuronally transformed chromaffin cells appear to lose adrenergic properties as assessed by diminished immunoreactivity to the adrenergic marker, phenylethanolamine-N-methyltransferase. Although their survival is also enhanced approximately 4-fold relative to controls, adult chromaffin cells do not convert to a neuronal morphology when co-grafted with the genetically altered astrocytes. These studies demonstrate that rat astrocytes carrying a mouse NGF transgene provide trophic support for intrastriatal chromaffin cell grafts.

Two conserved domains in the NGF propeptide are necessary and sufficient for the biosynthesis of correctly processed and biologically active NGF.

The three members of the neurotrophin family (NGF, BDNF and NT-3) are synthesized as large precursor proteins which undergo proteolytic processing to yield biologically active, mature neurotrophic factors. We have used in vitro mutagenesis to examine the pro-region in the NGF precursor protein as a first step towards a general understanding of the role of propeptides in the biosynthesis of neurotrophins. Our results demonstrate that only two small domains within the NGF propeptide are required for the expression and secretion of properly processed and biologically active, recombinant mouse NGF in COS-7 cells. Domain I plays an important role in the expression of active NGF while domain II is involved in proteolytic processing. Both domains are partially conserved between the propeptides of NGF proteins isolated from different species as well as BDNF and NT-3.

Regulation of axonal caliber, neurofilament content, and nuclear localization in mature sensory neurons by nerve growth factor

The neuronal perikaryal response to axonal injury (axon reaction) includes reduction in axonal caliber beginning in the proximal portion of the nerve (somatofugal axonal atrophy), development of nuclear eccentricity, and chromatolysis. The means by which these events are triggered is unknown, but it has been argued that loss of a neurotrophic signal from the target of injured neurons plays a role. To date, the identity of this substance(s) remains unknown. In the present study, we have asked whether NGF normally functions to control axonal caliber of sensory neurons in the L4 and L5 dorsal root ganglia (DRG) of the adult rat. Two approaches were used: (1) NGF was continuously delivered to the proximal stump of a transected sciatic nerve to determine whether NGF administration would prevent the production of somatofugal axonal atrophy; and (2) NGF antisera were administered to normal animals to determine whether NGF deprivation would produce somatofugal axonal atrophy. In the first experiment, 9-week-old rats underwent a unilateral sciatic nerve transection at midthigh, and the proximal stump was connected to an osmotic pump containing either NGF or cytochrome C (as control). At 11 weeks of age, dorsal root fibers in lumbar DRG from the control group appeared smaller in caliber and less circular in shape than fibers from age-matched normal animals. Although smaller than those in normal animals, fibers from the NGF-treated nerves were larger than in axotomized controls. Mean axonal area and shape factor (an index in circularity) were measured and found to be decreased significantly (22% and 15%, respectively) from the control group. Fibers from the NGF-treated nerves were significantly (p less than 0.05) larger in axonal caliber and more circular in shape; mean values were only reduced by 11% and 10%, respectively. Quantitation of neurofilament (NF) numbers revealed that the larger calibers in the NGF-treated nerves result from a greater NF content. NGF treatment did not prevent the atrophy of motor fibers in the proximal ventral root. In the second experiment, 2 antisera to mouse NGF were given daily into the footpad for 11 or 12 d; control animals were given normal goat serum. Quantitation of axonal calibers in the L5 DRG demonstrated that mean axonal area and shape factor were significantly (p less than 0.05) reduced by 14% and 17% respectively. The axoplasm of atrophic fibers demonstrated a paucity of NFs.(ABSTRACT TRUNCATED AT 400 WORDS)

Mouse nerve growth factor prevents degeneration of axotomized basal forebrain cholinergic neurons in the monkey

NGF, a trophic polypeptide, is necessary for the normal development and survival of certain populations of neurons in the CNS and PNS. In the CNS, cholinergic neurons of the basal forebrain magnocellular complex (BFMC) are prominent targets of NGF. During rat development, NGF increases the activity of ChAT in these neurons. In adult rats with experimental injury of axons in the fimbria-fornix, NGF prevents degenerative changes in axotomized cholinergic BFMC neurons in the medial septal nucleus (MSN). Because the amino acid sequences of NGF and its receptor (NGF-R) are highly conserved across species, we hypothesized that mouse NGF would also prevent degeneration of cholinergic BFMC neurons in nonhuman primates. Therefore, the present study was designed to test whether fimbria-fornix lesions result in retrograde degenerative changes in basal forebrain cholinergic neurons in macaques, whether these changes are prevented by mouse NGF, and whether the protective effect of NGF is selective for cholinergic neurons of the basal forebrain. Following unilateral complete transection of the fornix, animals were allowed to survive for 2 weeks, during which time half of the subjects received intraventricular NGF in vehicle and the other half received vehicle alone. In animals receiving vehicle alone, there was a 55% reduction in the number of ChAT-immunoreactive cell bodies within the MSN ipsilateral to the lesion; loss of immunoreactive somata was more severe in caudal planes of the MSN. Remaining immunoreactive neurons appeared smaller than those in control, unoperated animals. In Nissl stains, there was no apparent loss of basophilic profiles in the MSN, but cells showed reduced size and intensity of basophilia. Treatment with NGF almost completely prevented reductions in the number and size of cholinergic neurons and had a significant general effect in preventing atrophy in basophilic magnocellular neurons of the MSN, though some basophilic neurons in the MSN did not appear to respond to NGF. Adjacent 7-microns-thick sections stained with ChAT and NGF-R immunocytochemistry revealed that these markers are strictly colocalized in individual neurons in the MSN in controls and in both groups of experimental animals. Thus, mouse NGF profoundly influences the process of axotomy-induced retrograde degeneration in cholinergic BFMC neurons in primates. The in vivo effectiveness of mouse NGF on primate BFMC neurons suggests that mouse or human recombinant NGF may be useful in ameliorating the ACh-dependent, age-associated memory impairments that occur in nonhuman primates.(ABSTRACT TRUNCATED AT 400 WORDS)

Nerve growth factor mRNA and protein in the testis and epididymis of mouse and rat.

In situ hybridization using beta-nerve growth factor (NGF) DNA probes was used to demonstrate NGF mRNA in spermatocytes and early spermatids of adult mouse. NGF mRNA-containing cells were also identified in the epithelium of convoluted ducts in mouse corpus epididymidis. Blot-hybridization analysis of RNA prepared from mouse testis and epididymis as well as from rat epididymis confirmed the presence of a 1.3-kilobase (kb) NGF mRNA in these tissues. In the rat testis, however, only a 1.5-kb NGF mRNA was found, corresponding in size to a minor NGF mRNA detected in the rat brain, heart, and epididymis. By using affinity-purified anti-NGF antibodies, NGF-like immunoreactivity was observed in germ cells of rat and mouse testis and in the lumen of epididymis. Extracts of both mouse epididymis and testis stimulated fiber outgrowth in cultured sympathetic ganglia, and the effect was blocked by antibodies to mouse NGF. A two-site enzyme immunoassay showed the presence of 10 and 70 ng of NGF per g of tissue in the mouse testis and epididymis, respectively. Furthermore, RNA blot analysis showed the presence of mRNA for the NGF receptor in mouse testis. These results suggest a nonneurotrophic role for NGF in the male reproductive system, possibly in survival maturation and/or motility of spermatozoa.

Comparison of the Biochemical and Immunological Properties of Nerve Growth Factors from Various Animals

Nerve growth factors (NGF's) were purified from mouse submaxillary gland, guinea pig prostate gland, bovine seminal plasma, and venom of Naja naja atra, and their biochemical and immunological properties were compared. The NGF's from the four sources stimulated the maximal response of nerve fiber outgrowth from chick dorsal root ganglia at 10ng/ml. Two-site enzyme immunoassay (EIA) systems for these NGF's showed that mouse NGF, guinea pig NGF, and bovine NGF were similar in immunological properties and that the crossreactivity was less than 1% between snake NGF and mammalian NGF's. However, pretreatment with anti-snake NGF antibody inhibited the biological activity of mammalian NGF's even when their immunological activity was not completely inhibited. These results suggest that snake NGF and mammalian NGF's are similar in their biological activity but not in their immunological activity and that anti-snake NGF antibody recognizes the parts of mammalian NGF's essential for the biological activity.

Highly sensitive enzyme immunoassays for β-nerve growth factor

A comparison was made between three different strategies for measuring β-nerve growth factor (NGF) by fluorometric enzyme immunoassay. The substrate used was 4-methylumbelliferyl-β-galactoside and the enzyme reaction was followed in a Microfluor plate reader (Dynatech). After optimizing incubation times, concentrations, buffers, pH, and washings, a primary anti-NGF antibody directly conjugated to β-galactosidase gave the best detection limit (2 × 10 −17 M) of purified mouse NGF ( M r 26 000) in a two-site sandwich assay. Biotinylated secondary antibodies followed by streptavidin conjugated β-galactosidase proved to be 200-fold less sensitive in a similar assay. Finally, blotting NGF onto nitrocellulose membranes for detection with the same biotin-streptavidin steps after incubation with unlabelled primary antibodies resulted in a detection limit of 3 × 10 −12 M. All three methods indicated the same level (4 × 10 −11 M) of endogenous NGF in the rat brain hippocampus.

Localization of nerve growth factor-like molecule in astrocytes in the mammalian brain by monoclonal antibodies to 2.5 s mouse NGF

Localization of nerve growth factor-like molecule in astrocytes in the mammalian brain by monoclonal antibodies to 2.5 s mouse NGF

Localization of nerve growth factor-like molecule in astrocytes in the mammalian brain by monoclonal antibodies to 2.5 S mouse NGF

Localization of nerve growth factor-like molecule in astrocytes in the mammalian brain by monoclonal antibodies to 2.5 S mouse NGF

Neurite-promoting factors from embryonic neurons

Dissociated chick embryo peripheral and central nervous system cultures enriched for neurons by differential adherence, defined medium and cytosine arabinoside release to the culture environment molecular species which enhance the performance of neurons in limiting conditions. Culture medium conditioned by the neurons can be depleted of substrate-attached material by serial passage on poly- d-lysine substrate, leaving in the medium factors which promote neurite outgrowth on poly- d-lysine. The substrate-attached material which enhances neuron survival and neurite extension is heat- and trypsin-labile but is not affected by prior treatment with antisera to mouse NGF, human plasma fibronectin or laminin. The autostimulation phenotype displayed by neurons may play a role in neuronal survival or axonal growth during neuronal development.

The effect of culture on the neuronotrophic activity of avian smooth muscle

The expansor secundariorum is a smooth muscle of the chicken wing which receives a dense noradrenergic innervation and contains high concentrations of trophic activity for sympathetic neurons. Expansor cells were dissociated in trypsin and grown to confluency in standard nutrient medium. Medium that had been conditioned by expansor cells contained trophic activity for chick sympathetic nerves which differed from the activity which is found within the muscle prior to culture, in that it was effective on a different neuronal subpopulation. Furthermore, unlike the factor(s) found in vivo, the trophic activity of the conditioned medium was not enhanced by NGF and could be partially inhibited by mouse NGF antibodies. Both the production of total trophic activity in the conditioned medium and the activity which differed from that in the muscle extract, increased rapidly and to the same extent between 24 and 48 h culture. Greater concentrations of trophic activity were present in medium conditioned by smooth muscle cells than by skin cells. The conditioned medium was able to support more neurons from 11 and 18 day incubated embryos than a saturating dose of NGF. Neither this effect nor the additive effect of conditioned medium with expansor muscle extract could be attributed to the presence of neurite promoting factors. We conclude that the cultured muscle produces neuronotrophic activity that is similar to the activity found in denervated but not innervated muscle.

Effects of autoimmune NGF deprivation in the adult rabbit and offspring

An experimental autoimmune approach to the production of nerve growth factor deprivation, which we have previously described in the rat and guinea pig, has been applied to the rabbit. This species was chosen for study because of several potential advantages. The rabbit produces large litters and has a relatively short gestation period. More importantly, rabbits generate high titers of antibody against mouse NGF and large amounts of maternal antibody are passively transferred to the developing rabbit fetus compared to most other species, particularly the rat. The sympathetic nervous system of adult rabbit immunized against mouse NGF underwent degeneration with up to an 85% decrease in neuronal numbers in the superior cervical ganglion after 10 months of immunization, thus providing further evidence that NGF is required for the survival of mature sympathetic neurons. Despite the fact that newborn rabbits born to anti-NGF producing mothers had much higher titers of anti-NGF than did rats, the effects on the developing sympathetic and sensory nervous systems were not found to be any greater than in rats. Reductions in norepinephrine levels in the heart and spleen of adult rabbits born to anti-NGF producing mothers were greater than in small intestine. Prenatal exposure to maternal anti-NGF caused reductions (up to 70%) in the number of neurons in the dorsal root ganglia. Substance-P immunoreactivity was reduced in the substantia gelatinosa of the spinal cord of rabbit exposed to maternal anti-NGF. These changes, however, were not greater than seen in the rat. We conclide that although the rabbits offers some advantage in the study of the effects of NGF deprivation in the adult animal, it appears less well suited than the rat or guinea pig to the study of the effects of NGF deprivation on development.

Neurite growth promoting factors of embryonic chick--ontogeny, regional distribution, and characteristics.

A single neuronal cell biological assay for NGF has been used to quantitate factors in soluble extract of chick embryo that promote neurite outgrowth. The neurite growth promoting activity is detected as early as day 1.5 of embryonic life. Specific activity increases to maximum over the first four days and then decreases from day 5 to 6 during a growth spurt of the embryo. The tissue distribution of neurite growth promoting activity at eight embryonic days shows higher specific activity in peripheral structures that are richly innervated. Extract from all embryonic stages and from all tissues studied induces neurite outgrowth that is inhibited only partially by antibody to mouse NGF. Furthermore, the kinetics of neurite outgrowth and the neuronal populations responsive to extract differ from that of NGF. These observations indicate heterogeneity of the neurite growth promoting factors in soluble extracts of embryonic chick and demonstrate the presence of these factors before the responsive neurons leave the cell cycle and begin their differentiation processes.

Release of nerve growth factor by human glial cells in culture

Non-transformed human glial cells obtained from brain biopsies (lines U-787 CG, U-1169 CG and U-1508 CG) release to their culture medium a factor which, in bioassay, induces neurite outgrowth in spinal and sympathetic embryonic chick ganglia. The neurite-stimulating activity, which was enhanced after pressure dialysis of glial-conditioned medium, is inhibited by specific antiserum prepared to mouse βnerve growth factor (NGF). The glial factor was partially purified by gel filtration on Sephadex G-200 of concentrated, serum-containing, conditioned medium. The activity eluted close to a molecular weight of 30000, as did mouse NGF run under identical conditions. Ion-exchange chromatography on DEAE-Sepharose CL-6B and flat-bed electrofocusing of conditioned medium showed the activity to be associated with a heat-labile entity having an isoelectric point of about 4.1. All purified preparations were blocked by anti(mouse)-βNGF. The results demonstrate the existence of a human glial NGF which in several respects resembles the mouse submandibular gland NGF.

Human nerve growth factor: Lack of immunocrossreactivity with mouse nerve growth factor

Human β-nerve growth factor (hNGF) was purified from term human placenta. The biological potency of hNGF in the chick dorsal root ganglion assay did not differ significantly from that of mouse NGF (mNGF). Molecular weight determinations of mNGF and hMGF were also similar. No immunological crossreactivity was noted between hNGF, at a concentration of 100 μg/ml, and mNGF in a radioimmunoassay for mNGF using 6 different antisera to mNGF. hNGF shares several properties with mNGF but is immunological distinct. The results of studies in man using antisera to mNGF should be interpreted with caution.

Comparison of the nerve growth factor proteins from cobra venom (Naja naja) and mouse submaxillary gland.

The nerve growth factors (NGF's) isolated from cobra venom (Naja naja) and mouse submaxillary gland are closely related proteins. They are structurally similar in that about 60% of their amino acid residues are identical (Hogue-Angeletti, R. A., Frazier, W. A., Jacobs, J. W., Niall, H.D., and Bradshaw, R. A. (1976), Biochemistry, preceding paper in this issue). They are functionally similar in that they both elicit maximum neurite outgrowth from chick embryonic dorsal root ganglia at the same protein concentration. However, the extent of the response is not as great with Naja naja NGF. The cobra NGF has an affinity close to that of mouse NGF for a major proportion of the specific NGF receptors on cells from dissociated embryonic dorsal root ganglia. Despite these similarities there are differences which can be detected between the two proteins. High concentrations of Naja naja NGF will not displace approximately 20% of the binding of mouse NGF to specific NGF receptors. Moreover, Naja naja NGF shows limited cross-reactivity with antiserum to mouse NGF in a competition radioimmunoassay, consistent with the extent of its amino acid sequence homology with mouse NGF. Naja naja NGF does not interact with the alpha- and gamma-subunits of 7S NGF to form a high molecular weight complex. In this behavior it resembles a modified form of mouse NGF which, like Naja naja NGF, lacks COOH-terminal arginine residues.

Effect of mouse submaxillary gland and NGF preparations on competent chick ectodermin vitro, studied by the immunofluorescence method.

The submaxillary gland of the male mouse, but not the female, was shown to act strongly neuralizing on competent chick ectoderm, recorded as frequency of neural antigen producing cultures. NGF preparations from mouse submaxillary gland were shown to cause a high frequency of neural antigen producing cultures. The effect of the NGF preparations diminished with time and ceased after storage at -90° C for 17 days. The possible role of NGF in the present system is discussed; but the possibility could not be excluded that factors other than NGF present in the submaxillary gland and the NGF preparations cause the competent ectoderm to neuralize.