Two peptides derived from the nerve growth factor precursor are biologically active.

This report provides evidence that the proregion of the NGF precursor protein contains two novel bioactive peptides. The presence of pairs of basic amino acid (aa) residues in the NGF proregion suggests that two or three peptides other than NGF may be generated by proteolytic cleavage. Synthetic peptides of 29 aa (LIP1) and 38aa (LIP2) corresponding to the sequences -71 to -43 and -40 to -3 of the proNGF, respectively, were used in this study. ELISA specific for these two peptides revealed their presence in the rat intestine. LIP1 was localized by immunohistochemistry in endocrine cells of the intestinal epithelium, and LIP2 was immunoprecipitated from an intestinal extract. We also provide evidence for the presence of specific receptors for LIP2 in several cell lines. Scatchard analysis indicated the presence of a low affinity binding site with a Kd of approximately 10(-7) M and a high affinity binding site of 10(-9) M. Cross-linking studies revealed receptor forms of about 140 kD and 93 kD in a prostatic adenocarcinoma cell line. LIP1 and LIP2 induced rapid F-actin redistribution in PC12 cells within 2 min of incubation, which suggests a role of LIP1 and LIP2 in the process of neurite outgrowth. Furthermore, both propeptides induced rapid tyrosine phosphorylation of the Trk protein in both prostatic adenocarcinoma cells and PC12 cells, thus implicating trk in their mechanism of action. These results support our hypothesis that two peptides within the NGF precursor protein are biologically active.

Peptides other than the neurotrophins that can be cleaved from proneurotrophins: a neglected story.

The members of the family of neurotrophic factors known as neurotrophins, NGF, BDNF, NT-3 and NT4/5 are known to be cleaved intracellularly from immature precursors, the proneurotrophins. NGF and the other neurotrophins regulate neurite outgrowth and neuronal survival during development via binding to Trk receptor tyrosine kinases and the p75 neurotrophin receptor. Surprisingly, the proneurotrophins were shown to be also biologically active ligands. ProNGF and proBDNF induce neuronal apoptosis via binding to a complex of p75 and sortilin. Therefore, life and death seems to be a delicate interplay between 'cleavage' or 'not cleavage' of the proneurotrophins. However, there is a third aspect to this story. In general, peptide-hormone precursors are known to give rise to several biologically active peptides from one precursor molecule. The paradox with the proneurotrophins is that although they have several additional potential cleavage sites that would necessarily give rise to other peptides besides the neurotrophins and thus new members in the neurotrophin family, this aspect has been largely neglected. This article aims to review evidence for biologically active peptides other than the NGF and NT-3 that can be generated from the proNGF and proNT-3.

Multiple forms of an extracellular cyclic-AMP phosphodiesterase from Dictyostelium discoideum.

The extracellular cyclic-AMP phosphodiesterase of a mutant of Dictyostelium discoideum which accumulates this enzyme was found to exist in multiple forms. Using the isoelectric focusing technique the phosphodiesterase activity was distributed into three peaks with isoelectric points of 4.6, 6.5 and 8.3, designated as p4, p6 and p8. Gel filtration and sucrose gradient analysis showed that the p4 activity consisted of two forms of different sedimentation coefficients. At high enzyme concentrations, the heavy form was favored. Dilution of enzyme activity shifted the equilibrium toward the light form. Direct analysis by sucrose gradient sedimentation of all isoelectric forms demonstrated that besides p4, p6 activity also existed as a mixture of the heavy (9.7 S) and the light (5.4 S) components. In contrast, the p8 activity displayed only the light form. The heterogeneity of the p4 and p6 isoelectric forms was also observed by polyacrylamide gel electrophoresis. A procedure for a partial purification of the extracellular enzyme to about 70-fold is presented.

Presence of nerve growth factor and its receptors in an in vitro model of islet cell development: implication in normal islet morphogenesis.

In vivo, the differentiation of pancreatic islet stem cells depends on unknown soluble factors produced by the mesenchyme surrounding these cells. We have previously demonstrated that, like some neuronal cells, different beta-cell lines express functional nerve growth factor (NGF) receptors and can respond to NGF by extending neurite-like processes. NGF receptors are also expressed in vivo in mature rat islets and early during development in pancreatic ductular cells, which represent putative beta-stem cells. In this study, we have further characterized an in vitro model of islet development and studied the expression of NGF receptors and its ligand in this model. We have demonstrated the expression of Trk-A messenger RNA coding for the high affinity NGF receptor in islet cells and the localization of Trk protein in both alpha- and beta-islet cells. Moreover, the cells, from which islet cells "bud," also express Trk-A. Furthermore, NGF is produced and secreted by the nonendocrine cells surrounding the islets, suggesting a possible paracrine mode of action of NGF on the adjacent islet cells. Finally, islet morphogenesis is significantly retarded in the presence of K252a, an inhibitor of the tyrosine kinase activity of the family of Trk receptors, suggesting an implication of the neurotrophin-neurotrophin receptor axis in islet development.

Interaction of antibodies to synthetic peptides of proNGF with in vitro synthesized NGF precursors.

Sera raised against three synthetic peptides that reproduce sequences of the pro-nerve growth factor (proNGF) protein were tested in immunoprecipitation experiments using in vitro translation products of SP6-directed NGF mRNA in a rabbit reticulocyte lysate. The interaction of these antibodies with bacterially synthesized chimeric preproNGF was also examined. Digestion of the translation products by the gamma-subunit generated the 22 and 18 kDa intermediates. A predominant 13 kDa intermediate was obtained after digestion of translation products in wheat germ extract. This is shown to be the N-terminal peptide by immunoprecipitation with an anti-peptide serum. These antibodies may be used to detect NGF precursor cleavage products in vivo.

Synthesis and partial maturation of the alpha- and gamma-subunits of the mouse submaxillary gland nerve growth factor in Xenopus laevis oocytes.

Sera raised against the alpha-, beta- and gamma-subunits of the mouse 7 S NGF were used to characterize translation products coded by submaxillary gland mRNAs microinjected into Xenopus oocytes. Anti-beta NGF sera did not cross-react with any material. In contrast, the precursors of the alpha- and gamma-subunits, as well as that of renin were identified. Use of tunicamycin, and a comparison of the translation products obtained in oocytes or in the reticulocyte lysate indicated that oocytes achieved the cleavage of signal sequences, the glycosylation of the alpha- and gamma-precursors, and the subsequent secretion of the 3 proteins. In the submaxillary gland, however, the mature forms of alpha NGF, gamma NGF and renin are composed of peptides of smaller size than those produced by the oocytes. These latter appear to lack specific proteases involved in the terminal processing of the submaxillary gland proteins.

Serum and thyroid hormones T3 and T4 regulate nerve growth factor mRNA levels in mouse L cells.

Mouse L cells synthesize and secrete a neurotrophic factor related to the beta subunit of the submaxillary gland nerve growth factor (NGF) of male mice. Use of a cDNA probe which encodes the beta-NGF mRNA demonstrated that L cells produce a transcript identical in size to that of the submaxillary gland. Moreover, target sites of restriction enzymes EcoRI, PstI and BamHI were not significantly rearranged in the beta-NGF gene locus of these cells. The abundance of the beta-NGF transcript was found to depend on culture conditions. Removal of serum depressed the cellular content of polyadenylated RNA by a factor of 1.7, and decreased specifically the pool of beta-NGF transcript by an additional factor of 4. The presence of 10(-7) M testosterone in the serum-free medium did not modify the level of beta-NGF mRNA, while addition of 10(-7) M T3 (or T4) increased this level by a factor of 1.5. These data provide the first evidence that the beta-NGF mRNA of L cells is subjected to regulation, but in a way apparently different from that described for the submaxillary gland.

RNA involvement in T4 DNA synthesis in toluene-treated cells.

In T4-infected cells made permeable with toluene, pulses with [(alpha-32P deoxyribonucleoside triphosphates demonstrated covalent linkage of RNA to DNA of the Okazaki fragments. Analysis of the transfer of the 32P label to the 2'(3') ribonucleoside monophosphates indicated that the 3'-end of the RNA primer is heterogeneous. The most frequently encountered ribonucleotide was rCMP, but also transfer to rUMP, rAMP and rGMP occurred at different frequencies. In contrast, no heterogeneity was observed for the deoxyribonucleoside at the RNA-DNA junction. Of all the [to-32P] deoxyribonucleoside triphosphates tested, transfer of the 32P label to 2'(3') rNMPs was predominant when [alpha32P] dGTP was the substrate, indicating that the deoxyribonucleoside most frequently encountered at the RNA-DNA linkage is dG. These observations suggest that the starts for the Okazaki fragments may occur at unique sites of the T4 genome.

Evidence that natural autoantibodies against the nerve growth factor (NGF) may be potential carriers of NGF.

Nerve growth factor (NGF) was detected by enzyme-linked immunosorbent assay using the monoclonal anti-NGF antibody 27/21 in natural NGF autoantibodies (NGF NA) purified from sera of control human subjects as well as from sera of patients with rheumatoid arthritis, autoimmune thyroiditis and to a lesser degree in patients with systemic lupus erythematosus as well as in NGF NA from the synovial fluid of patients with spondylarthropathies. Our results suggest that NGF NA may be potential carriers of NGF in the circulation.

Naturally occurring antibodies against nerve growth factor in human and rabbit sera: comparison between control and herpes simplex virus-infected patients.

Antibodies against nerve growth factor (NGF) in sera were detected by enzyme-linked immunosorbent assays (ELISA), by their isolation after passage of sera through NGF immunoadsorbent columns and by their specificity to bind and immunoprecipitate mouse NGF as well as to stain by immunohistochemical methods cellular sites of NGF synthesis. Increased levels of anti-NGF antibodies were found in sera of herpes simplex virus (HSV)-infected patients but not in HSV-inoculated rabbits. As HSV latency is known to be promoted by NGF in vitro, these results may suggest that anti-NGF antibodies modulate the cytokine function of NGF and thus might play a role in HSV infection. The biological function of circulating antibodies against NGF, in general, is now open to future investigation.

Natural autoantibodies against the nerve growth factor in autoimmune diseases.

High titers of natural autoantibodies against the nerve growth factor (NGF) were detected in the sera of patients with systemic lupus erythematosus, autoimmune thyroiditis and rheumatoid arthritis. Autoantibodies to NGF from these pathological cases displayed higher avidity for NGF and a higher polyreactivity with certain cytoskeletal proteins and with DNA as compared to those from control human subjects. The biological activity, immunoglobulin composition and physiological relevance of these autoantibodies are discussed.

Characterization and visualization of [125I] stromal cell-derived factor-1alpha binding to CXCR4 receptors in rat brain and human neuroblastoma cells.

Stromal cell-Derived Factor-1 (SDF-1alpha), binds to the seven-transmembrane G protein-coupled CXCR4 receptor and modulates cell migration, differentiation, and proliferation. CXCR4 has been reported to be expressed in various tissues including brain. Moreover, CXCR4 has recently been shown to be one of the coreceptors for HIV-1 infection which could be implicated in HIV encephalitis. In the present study, the binding properties and autoradiographic distribution of [125I]SDF-1alpha binding to CXCR4 were characterized in the adult rat brain. SDF-1alpha binding and CXCR4 coupling system were also studied in human neuroblastoma cell line SK-N-SH. The binding of [125I]SDF-1alpha on rat brain sections was specific, time-dependent and reversible. The highest densities of CXCR4 were detected in the choroid plexus of the lateral and the dorsal third ventricle. Lower densities of [125I]SDF-1alpha binding sites were observed in various brain regions including cerebral cortex, anterior olfactory nuclei, hippocampal formation, thalamic nuclei, blood vessels and pituitary gland. In the choroid plexus, the IC(50) and K(d) of [125I]SDF-1alpha binding were respectively 0.6 nM and 0. 36 nM. Similar IC(50) values were obtained in other brain structures. A CXCR4 antagonist, bicyclam, competed with SDF-1alpha binding (30% inhibition at 10(-6) M). In SK-N-SH cells, [125I]SDF-1alpha bound to CXCR4 with a K(d) of 5.0 nM and a maximal binding capacity of 460 fmol/mg of protein. SDF-1alpha induced a rapid and transient intracellular calcium increase in SK-N-SH cells. These findings suggest that CXCR4 is highly expressed in some brain structures and have a regulatory role in the nervous system. The significance of this expression in the brain parenchyma and more specifically in the choroid plexus remains to be clarified in the normal as well as in the infected brain.

Characterization of nerve growth factor precursor protein expression in rat round spermatids and the trophic effects of nerve growth factor in the maintenance of Sertoli cell viability.

Nerve growth factor (NGF) is expressed by rat round spermatids and is thought to participate in the paracrine regulation of spermatogenesis. In order to elucidate the role of NGF in the rat testis, we further characterized the NGF immunoreactive protein secreted by round spermatids and examined the effect of NGF beta and related neurotrophin family members on the maintenance of Sertoli cell viability. Round spermatids were isolated from rat testes by centrifugal elutriation and the conditioned medium dialyzed/concentrated for the preparation of round spermatid protein (RSP). Immunoblot analysis of RSP with anti-NGF beta antibody identified two immunoreactive bands of 31 and 22 kD, whereas the 13 kD mature form of NGF beta was not observed. Similarly, immunoblot analysis of RSP with an antibody raised against a synthetic peptide (L38) corresponding to the -3 to -40 sequence of proNGF also recognized two immunoreactive bands of 31 and 22 kD. These results are consistent with the identification of two NGF precursors. Interestingly, immunoblot analysis of RSP with an antibody raised against a synthetic peptide (N4) corresponding to the -71 to -46 sequence of proNGF only recognized one immunoreactive band of 31 kD, consistent with the larger NGF precursor observed with the L38 antibody. In a bioactivity test of PC-12 neurite outgrowth, the 31 kD NGF precursor induced flattening and neurite outgrowth of PC-12 cells, consistent with NGF bioactivity. The 22 kD NGF precursor induced modest and inconsistent neurite outgrowth. These results suggest that round spermatids express the 31 and 22 kD precursor forms of the NGF gene product, and that processing of this gene product is incomplete such that the 13 kD mature form of NGF beta is not observed. In view of the role of round spermatids in the paracrine regulation of spermatogenesis, we examined the effect of RSP on the rescue of the viability of Sertoli cells cultured under serum deprived conditions. In the absence of serum, RSP was able to extend the viability of Sertoli cells, and the elimination of this activity by anti-NGF antibody immunoprecipitation of RSP suggests that the NGF precursors in RSP support the maintenance of Sertoli cell viability. In addition, treatment with exogenous NGF beta was able to rescue Sertoli cell viability, whereas L38 peptide, N4 peptide, or the neurotrophins, brain derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 were unable to rescue Sertoli cell viability above control levels. Hence, it appears that round spermatids express the precursor forms of the NGF gene product, but not the mature form of NGF beta, and that the NGF beta moiety of the NGF precursor proteins exhibits trophic activity in the rescue of Sertoli cell viability, consistent with the paracrine regulation of spermatogenesis.

Regulation of NGF, BDNF and LNGFR gene expression in ROS 17/2.8 cells.

The secosteroid hormone 1.25-dihydroxyvitamin D3 (1,25(OH)2D3) has been recently shown to enhance the synthesis of NGF to mouse L929 fibroblasts. In view of the critical role of 1,25(OH)2D3 on bone metabolism, it has been investigated if ROS 17/2.8 osteoblastic cells were able to express the nerve growth factor (NGF) gene and if this process was responsive to 1,25(OH)2D3. Results indicate that these cells respond in a dose-dependent manner to the presence of 1,25(OH)2D3 by an increase in NGF mRNA levels. However, the phorbol ester PMA, previously reported to augment the synthesis of NGF via the recruitment of AP-1 complexes, depressed the expression of the NGF gene in ROS cells. In contrast, the mRNA levels of an NGF-related trophic factor, brain-derived neurotrophic factor (BDNF), was increased by PMA but not following 1,25(OH)2D3 treatment. Binding of 125I-NGF to ROS cells displayed the properties of a low affinity NGF receptor (dissociation constant Kd approximately 10(-9) M). In agreement with this result, the mRNA encoding the low affinity NGF receptor (LNGFR) was detected in ROS 17/2.8 cells, unlike trkA transcripts which encode the high affinity receptor. These data suggest that neurotrophins and their low affinity receptor could play an unsuspected role in bone tissue.

Nerve growth factor (NGF) autoantibodies and NGF in the synovial fluid: implications in spondylarthropathies.

We investigated the presence of nerve growth factor (NGF) autoantibodies and NGF in the synovial fluid (SF) of patients with different forms of chronic arthritis such as spondylarthropathy (SpA), rheumatoid arthritis (RA), calcium pyrophosphate dihydrate crystal deposition disease (CPPD) and osteoarthritis (OA) and compared them to their levels in serum. NGF autoantibodies were detected by ELISA and by their capacity to immunoprecipitate NGF and to inhibit its biological activity. NGF was measured with a two-site enzyme-linked immunosorbent assay. Significantly high NGF autoantibody levels (p < 10(-4)) and high frequency of detectable NGF (p < 0.01) were observed in the SF of SpA patients and to a lesser degree in RA patients as compared to CPPD and OA patients. In the serum high frequency of detectable NGF was observed only in RA patients. These results suggest a role of NGF autoantibodies and NGF in joint inflammation especially in spondylarthropathies.

Anti-NGF autoantibodies and NGF in sera of Alzheimer patients and in normal subjects in relation to age.

It has been suggested that inflammation may be a possible cause of Alzheimer's disease (AD). Increased anti-NGF autoantibody levels and increased NGF frequency in serum have previously been associated with inflammatory responses. In this study no changes in anti-NGF autoantibody titers or in NGF frequency were detected in sera of AD patients, suggesting that they are not involved in the neuroimmunological mechanisms underlying AD. There were neither age-associated changes in NGF frequency in sera of four groups of normal subjects between 18-91 years of age. In contrast, anti-NGF autoantibodies were significantly increased in sera of the 31-45 yr age group.

Nerve growth factor precursors in the rat thyroid and hippocampus.

A nerve growth factor (NGF) precursor form of about 24 kDa was identified in homogenates of rat thyroid and hippocampus by immunoprecipitation using three sera raised against a synthetic peptide that reproduces the sequence -71 to -46 of the proNGF molecule. Besides this species, a 31 kDa protein, as well as a cleavage product of 12 kDa were also immunoprecipitated in both tissues by one of these sera.

Expression of recombinant human nerve growth factor in Escherichia coli.

Nerve growth factor (NGF) is a neurotrophic factor for basal forebrain cholinergic neurons and may be of benefit in neurodegenerative diseases of humans. A method is described to obtain significant amounts of biologically active recombinant human NGF (rhNGF) in one step. RhNGF was expressed in E. coli and the majority of the protein accumulated in inclusion bodies. It was immunoprecipitated by a serum against mouse NGF. Solubilization of the inclusion bodies was done in 3M guanidine HCl and renaturation was effected by dilution and air oxidation in the presence of 6 microM CuSO4. Recoveries were 10-12 micrograms of rhNGF per ml of bacterial suspension. Its biological activity was tested in a bioassay system employing sympathetic chick embryo ganglia and was inhibited by the monoclonal antibody 27/21 against mouse NGF.

Neuroprotective effects of leptin in vivo and in vitro.

The present study explored the efficacy of leptin in protecting against in vivo induction of excitotoxic lesions by the glutamatergic analogue ibotenate injected into the developing mouse brain and against in vitro NMDA-induced cell death in primary neuronal cultures. Ibotenate injected intracerebrally (i.c.) to mice on postnatal day 5 produced transcortical necrosis and white matter cysts. Co-treatment with leptin administered i.c. or i.p. reduced ibotenate-induced cortical lesions and white matter cysts by 50%. in vitro, leptin afforded significant neuroprotection of mouse cortical neurons against NMDA cytotoxicity. The neuroprotective effect of leptin was antagonized both in vivo and in vitro by the Jak2 inhibitor AG490, indicating that it was mediated via the leptin receptor and Jak2 activation. These findings are the first evidence for a role of leptin in neuroprotection.

Increased frequency of NGF in sera of rheumatoid arthritis and systemic lupus erythematosus patients.

Nerve growth factor (NGF) levels were measured by a two-site enzyme-linked immunosorbent assay (ELISA) in sera of patients with three autoimmune diseases, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and thyroiditis. Serum NGF levels were variable (15 pg ml(-1)-1.6 ng ml-1) but not significantly different among these groups compared with control subjects. However the frequency of detectable circulating NGF was significantly higher in RA and SLE patients but not in thyroiditis patients compared with controls. The present data provides evidence for NGF involvement in two autoimmune rheumatic diseases and suggests a possible differential role of NGF as immunomodulatory agent in systemic versus certain organ-specific autoimmune diseases.