Production of functional human nerve growth factor from the submandibular glands of mice using a CRISPR/Cas9 genome editing system.

Nerve growth factor (NGF) is an essential trophic factor for the growth and survival of neurons in the central and peripheral nervous systems. For many years, mouse NGF (mNGF) has been used to treat various neuronal and non-neuronal disorders. However, the biological activity of human NGF (hNGF) is significantly higher than that of mNGF in human cells. Using the CRISPR/Cas9 system, we constructed the transgenic mice expressing hNGF specifically in their submandibular glands. As demonstrated by fluorescence immunohistochemical staining, these mice produced hNGF successfully, with 0.8 mg produced per gram of submandibular glands. hNGF with 99% purity was successfully extracted by two-step ion-exchange chromatography and one-step size-exclusion chromatography from the submandibular glands of these transgenic mice. Further, the purified hNGF was verified by LC-MS/MS. We analyzed the NH2-terminus of hNGF using both Edman degradation and LC-MS/MS-based methods. Both results showed that the obtained hNGF lost the NH2-terminal octapeptide (SSSHPIFH). Moreover, the produced hNGF demonstrated a strong promotion in the proliferation of TF1 cells.

Kisspeptin induces LH release and ovulation in an induced ovulator†.

Kisspeptin has been implicated in the ovulatory process of several species of spontaneous ovulators but in only one induced ovulator. In contrast, NGF in semen is the principal trigger of ovulation in other species of induced ovulators-camelids. We tested the hypotheses that kisspeptin induces luteinizing hormone (LH) secretion in llamas through a hypothalamic mechanism, and kisspeptin neurons are the target of NGF in its ovulation-inducing pathway. In Experiment 1, llamas were given either NGF, kisspeptin, or saline intravenously, and LH secretion and ovulation were compared among groups. All llamas treated with NGF (5/5) or kisspeptin (5/5) had an elevation of LH blood concentrations after treatment and ovulated, whereas none of the saline group did (0/5). In Experiment 2, llamas were either pretreated with a gonadotropin-releasing hormone (GnRH) receptor antagonist or saline and treated 2 h later with kisspeptin. Llamas pretreated with saline had elevated plasma LH concentrations and ovulated (6/6) whereas llamas pretreated with cetrorelix did not (0/6). In Experiment 3, we evaluated the hypothalamic kisspeptin-GnRH neuronal network by immunohistochemistry. Kisspeptin neurons were detected in the arcuate nucleus, the preoptic area, and the anterior hypothalamus, establishing synaptic contacts with GnRH neurons. We found no colocalization between kisspeptin and NGF receptors by double immunofluorescence. Functional and morphological findings support the concept that kisspeptin is a mediator of the LH secretory pathway in llamas; however, the role of kisspeptins in the NGF ovulation-inducing pathway in camelids remains unclear since NGF receptors were not detected in kisspeptin neurons in the hypothalamus.

Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors.

The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 µg/mL) was selected. hNGF protein was successfully purified from the saliva of these TG mice and its identity was verified. The purified hNGF was highly functional as it displayed the ability to induce neuronal differentiation of PC12 cells. Furthermore, it strongly promoted proliferation of TF1 cells, above the levels observed with mouse NGF. Additionally, saliva collected from TG mice and containing unpurified hNGF was able to significantly enhance the growth of TF1 cells. This study not only provides a new and efficient approach for the synthesis of therapeutic hNGF but also supports the concept that salivary gland from TG animals is an efficient system for production of valuable foreign proteins.

Optimization of recombinant ß-NGF expression in Escherichia coli using response surface methodology.

Human nerve growth factor a member of the neurotrophin family can be used to treat neurodegenerative diseases. As it has disulfide bonds in its structure, periplasmic expression of it using appropriate signal sequence is beneficial. Therefore, in this work ß-nerve growth factor (ß-NGF) was expressed in Escherichia coli using pET39b expression vector containing DsbA signal sequence. In an initial step, the effect of isopropyl ß-D-1-thiogalactopyranoside (IPTG) and lactose concentration as inducer on protein production was investigated using response surface methodology. Then the effect of different postinduction time and temperature on protein production was studied. Our results indicated that the highest ß-NGF production was achieved with 1 mM IPTG and low concentrations of lactose (0-2% w/v), low cultivation temperature of 25°C and postinduction time of 2 hr. Also following ß-NGF purification, bioassay test using PC12 cell line was done. The biological activity of the purified ß-NGF showed a similar cell proliferation activity with the standard recombinant human ß-NGF. In conclusion, the results indicated an optimized upstream process to obtain high yields of biologically active ß-NGF.

Large-scale implementation of sequential protein and peptide immunoaffinity enrichment LC/nanoLC-MS/MS for human ß-nerve growth factor.

BACKGROUND: A previously described immunoaffinity (IA)-LC-MS/MS assay for human ß-nerve growth factor (ß-NGF) was implemented to support large-scale sample testing for multiple clinical trials. Methodology & results: The procedure was modified to increase throughput by simultaneous preparation of two 96-well plates and LC duty-cycle reduction. Robustness of the LC method and nano-ESI was ensured during large-scale assay execution by closely monitoring and, if needed, replacing system components prior to failure. Following validation, the assay was used to analyze approximately 19,000 samples from multiple clinical studies over several years. CONCLUSION: Routine implementation of the ß-NGF IA-LC-MS/MS assay supported drug development programs. This optimized assay format now serves as a template for other clinical protein biomarker assays.

Effect of purified murine NGF on isolated photoreceptors of a rodent developing retinitis pigmentosa.

A number of different studies have shown that neurotrophins, including nerve growth factor (NGF) support the survival of retinal ganglion neurons during a variety if insults. Recently, we have reported that that eye NGF administration can protect also photoreceptor degeneration in a mice and rat with inherited retinitis pigmentosa. However, the evidence that NGF acts directly on photoreceptors and that other retinal cells mediate the NGF effect could not be excluded. In the present study we have isolated retinal cells from rats with inherited retinitis pigmentosa (RP) during the post-natal stage of photoreceptor degenerative. In presence of NGF, these cells are characterized by enhanced expression of NGF-receptors and rhodopsin, the specific marker of photoreceptor and better cell survival, as well as neuritis outgrowth. Together these observations support the hypothesis that NGF that NGF acts directly on photoreceptors survival and prevents photoreceptor degeneration as previously suggested by in vivo studies.

Association of p75(NTR) and α9ß1 integrin modulates NGF-dependent cellular responses.

Direct interaction of α9ß1 integrin with nerve growth factor (NGF) has been previously reported to induce pro-proliferative and pro-survival activities of non-neuronal cells. We investigated participation of p75(NTR) in α9ß1 integrin-dependent cellular response to NGF stimulation. Using selective transfection of glioma cell lines with these receptors, we showed a strong, cation-independent association of α9 integrin subunit with p75(NTR) on the cellular membrane by selective immunoprecipitation experiments. The presence of the α9/p75(NTR) complex increases NGF-dependent cell adhesion, proliferation and migration. Other integrin subunits including ß1 were not found in complex with p75(NTR). FRET analysis indicated that p75(NTR) and α9 integrin subunit are not closely associated through their cytoplasmic domains, most probably because of the molecular interference with other cytoplasmic proteins such as paxillin. Interaction of α9ß1 integrin with another ligand, VCAM-1 was not modulated by the p75(NTR). α9/p75(NTR) complex elevated NGF-dependent activation of MAPK Erk1/2 arty for integrin that may create active complexes with other types of receptors belonging to the TNF superfamily.

Expression, purification, and characterization of recombinant mouse nerve growth factor in Chinese hamster ovary cells.

Mouse NGF (mNGF) extracted from mouse submaxillary gland has been approved on the market in China for treating nerve damage caused by N-hexane poisoning for over a decade, and many researches showed the clinical effectiveness of mNGF for the treatment of other nerve system diseases. The extracted mNGF have risks of potential viral contamination due to the animal origin. Here, we report the successful expression, purification, and characterization of recombinant mNGF (rmNGF). An expression plasmid of mouse nerve growth factor (mNGF) was constructed and transfected into CHO-S cells. Stable transfectants were obtained using a two-phase selection scheme with the addition of different concentrations of methotrexate and puromycin. Recombinant mNGF (rmNGF) was purified from cell culture medium by a two-step procedure: cation exchange followed by size-exclusion chromatography. The purity of rmNGF was 98.6% determined by size exclusion high performance liquid chromatography (SEC-HPLC). The molecular weight, isoelectric point and N-terminal sequence of rmNGF were identical to the theoretical values entirely. In TF-1/MTS, the specific activity of the protein was approximately 1.7×10(6)U/mg against rhNGF (the reference standard). In DRGs, the specific activity was approximately 7.3×10(5)AU/mg against mNGF (the reference standard). Our results showed that a high quality of rmNGF with marked biological activity comparable with mNGF was produced, and laid the basis for further research and development of rmNGF.

Ovulation-inducing factor (OIF/NGF) from seminal plasma origin enhances Corpus Luteum function in llamas regardless the preovulatory follicle diameter.

Ovulation-inducing factor (OIF) is a protein present in llama seminal plasma that has recently been identified as ß-Nerve Growth Factor (NGF) and it induces not only a high rate of ovulation but also appears to have luteotrophic properties in this species. A 2-by-2 experimental design was used to determine the effect of treatments (OIF/NGF vs GnRH) and categories of preovulatory follicle diameter (7-10 vs >10mm) on ovulation rate, CL diameter and function in llamas. Llamas (n=32 llamas per group) were randomly assigned to receive an intramuscular dose of: (a) 1mg purified OIF/NGF in the presence of a follicle of 7-10mm in diameter; (b) 50 µg of GnRH in the presence of a follicle of 7-10mm in diameter; (c) 1mg purified OIF/NGF in the presence of a follicle >10mm in diameter; (d) 50 µg of GnRH in the presence of a follicle >10mm in diameter. Llamas were examined by ultrasonography every 12h from treatment to Day 2 (Day 0=treatment) to detect ovulation, and again on Day 8 to determine CL diameter. Ovulation rates did not differ among groups. There was an effect of preovulatory follicle size on Corpus Luteum diameter at Day 8 (P<0.001), however plasma progesterone concentration (n=15/per group) was higher (P<0.05) in the OIF/NGF - than that of the GnRH - treated group by the same day. We conclude that OIF/NGF treatment enhances CL function regardless preovulatory follicle size at the time of treatment.

Development of a method that eliminates false-positive results due to nerve growth factor interference in the assessment of fulranumab immunogenicity.

Fulranumab, a human IgG2 monoclonal antibody that neutralizes nerve growth factor (NGF), is currently in development for the treatment of pain. Our initial immunogenicity test method was found to be prone to NGF interference, leading to a high apparent incidence of anti-drug antibody (ADA) in phase 1 studies. The ADA immunoassay comprised a homogeneous bridging electrochemiluminescence (ECL) format with biotin and ruthenium-labeled fulranumab bound together ("bridged") by ADA in test samples for detection. In this assay, NGF produced a false-positive signal due to its ability to bridge fulranumab molecules. Thus, we developed a specificity assay to eliminate the NGF false-positive results. We encountered the challenge of eliminating drug interference as well as drug target interference, and discovered that the acid-dissociation-based pretreatment of samples used for mitigating drug interference dramatically increased drug target interference. Several strategies were investigated to eliminate the NGF interference; yet only one strategy specifically removed NGF and produced true fulranumab-specific ADA results by using competitive inhibition with fulranumab and utilizing an alternative NGF binding antibody to eliminate NGF interference. Using this new method, we confirmed that the high apparent anti-fulranumab antibody incidence (>60%) in clinical study samples was in fact due to fulranumab-bound NGF released during the acid-dissociation step of the ADA testing method. We conclude that our revised method accurately identifies anti-fulranumab antibodies by incorporating steps to eliminate fulranumab and NGF interference. We advise that acid-dissociation pretreatment must not be universally applied to improve ADA assays without investigating its bioanalytical risks versus benefits.

NGF promotes hemodynamic recovery in a rabbit hindlimb ischemic model through trkA- and VEGFR2-dependent pathways.

Nerve growth factor (NGF) has been reported to play an important role in physiological and pathological angiogenesis. Based on these observations, we hypothesized that NGF may induce the formation of functional blood vessels in a hindlimb ischemic rabbit model. Hindlimb ischemia was induced in 34 rabbits bilaterally by endovascular embolization of femoral arteries. On the 7th, 14th, and 20th postembolization days, NGF was injected intramuscularly, in 1 ischemic limb, and vehicle was injected in the contralateral control limb. On the 40th day, newly developed collateral vessels (diameter >500 µm) were quantified by transauricular intraarterial subtraction angiography. Perfusion analysis of an in vivo dynamic computed tomography study was performed to the limbs to investigate the hemodynamic recovery of the distal ischemic tissues. Functional estimation of limb perfusion showed a statistically significant increase of blood flow and blood volume for NGF. However, the increase of the collateral vessels was not detectable angiographically, providing evidence for the existence of a NGF-stimulated capillary angiogenic network but not increase of arteriogenesis. The combination of NGF with either tropomyosin-related kinase type A or vascular endothelial growth factor receptor 2 antagonists abolished the NGF-induced hemodynamic recovery. These findings provide new insights into understanding the involvement of NGF in vascular formation and its applications in therapeutic angiogenesis.

Proteomic identification of camel seminal plasma: purification of ß-nerve growth factor.

The camel seminal plasma contains a diverse array of components including lipids, carbohydrates, peptides, ions and proteins. These are essential for maintaining normal physiology of spermatozoa and are secreted mainly from the prostrate, epidydimis and bulbo-urethral glands of reproductive system. The protein profiles of camel seminal plasma were resolved by two-dimensional gel electrophoresis (2D-PAGE). The majority of the protein was found in acidic regions below pI 7.0 and the 19 brightly stained proteins were identified by MALDI-TOF/MS analysis. On the basis of proteomic profiles, ß-nerve growth factor (ß-NGF) was purified by ion-exchange and gel filtration chromatography and identified by SDS-PAGE and MALDI-TOF/MS analysis. It was further confirmed by western blotting experiments using rabbit anti-ß-NGF primary antibody.

[A neuritogenic compound from Tremella fuciformis].

OBJECTIVE: To search for low-molecular-weight neuritogenic compounds from the traditional Chinese medicine (TCM). METHOD: An extract library of TCM was prepared. Targeted isolation guided by biological screening led to the discovery of compound 1, and its structure was elucidated by analysis of spectroscopic methods and comparison of spectroscopic data with these reported from the literature. RESULT: A neuritogenic compound 1, 3-O-beta-D-glucopyranosyl-22E, 24R-5alpha, 8alpha-epidioxyergosta-6, 22-diene, was isolated and identified from the methanol extract of T. fuciformis. This compound showed a significant neuritogenic activity against PC12 cells at 3 micromol x L(-1)). CONCLUSION: Methonal extract of T. fuciformis and targeted compound 1 both showed significant neuritogenic activity against PC12 cells. These results suggested that the extract and compound 1 might be used to prevent and treat neurodegenerative disease such as Alzheimer's disease.

Conformational plasticity of proNGF.

Nerve Growth Factor is an essential protein that supports neuronal survival during development and influences neuronal function throughout adulthood, both in the central and peripheral nervous system. The unprocessed precursor of NGF, proNGF, seems to be endowed with biological functions distinct from those of the mature protein, such as chaperone-like activities and apoptotic and/or neurotrophic properties. We have previously suggested, based on Small Angle X-ray Scattering data, that recombinant murine proNGF has features typical of an intrinsically unfolded protein. Using complementary biophysical techniques, we show here new evidence that clarifies and widens this hypothesis through a detailed comparison of the structural properties of NGF and proNGF. Our data provide direct information about the dynamic properties of the pro-peptide and indicate that proNGF assumes in solution a compact globular conformation. The N-terminal pro-peptide extension influences the chemical environment of the mature protein and protects the protein from proteolytic digestion. Accordingly, we observe that unfolding of proNGF involves a two-steps mechanism. The distinct structural properties of proNGF as compared to NGF agree with and rationalise a different functional role of the precursor.

Nerve growth factor inhibits metalloproteinase-disintegrins and blocks ectodomain shedding of platelet glycoprotein VI.

Nerve growth factor (NGF) plays an important role in regulating mammalian neuronal/embryonic development, angiogenesis, and other physiological processes and has recently been investigated as a potential treatment for the neurodegenerative disorder, Alzheimer disease. In this study, we provide evidence that human NGF may also function as a metalloproteinase inhibitor, based on studies of NGF from snake venom. Originally, our aim was to isolate snake venom metalloproteinases targeting platelet receptors and/or ligands relevant to hemostasis and thrombosis, using Ni(2+)-agarose as a purification step based on the conserved metal ion-coordination motif in venom metalloproteinases. However, subsequent analysis of cobra (Naja kaouthia) venom led to the unexpected discovery that cobra venom NGF bound to Ni(2+)-agarose, eluting at approximately 15 mm imidazole, enabling a one-step purification. The identity of the purified protein was confirmed by mass spectrometry and N-terminal sequence analysis. Partial co-purification of NGF within metalloproteinase-enriched venom fractions led us to test whether NGF affected metalloproteinase activity. Venom NGF potently inhibited metalloproteinases isolated from the same or different venom and specifically bound to purified Nk metalloproteinase immobilized on agarose beads. Human NGF also interacted with human metalloproteinases because it blocked metalloproteinase-mediated shedding of the platelet collagen receptor, glycoprotein (GP)VI, and associated with recombinant ADAM10 by surface plasmon resonance. Together, these results suggest that NGF can function as a metalloproteinase inhibitor.

Neurite outgrowth-promoting active constituents of the Japanese cypress (Chamaecyparis obtusa).

In the screening of biologically active constituents from woody plants, the methanol extract of leaves of Chamaecyparis obtusa showed potent neurite outgrowth-promoting activity in neuronal PC12 cells. The ethyl acetate-soluble fraction of the methanol extract showed potent activity and was separated by means of various chromatographic methods to give the two new compounds 1 and 2, as well as 11 known lignan and sesquiterpene derivatives. The structures of the new compounds were determined to be 9-O-acetyldihydrosesamin (1) and 9-O-(11-hydroxyeudesman-4-yl)dihydrosesamin (2), respectively, in NMR studies including 2D-NMR experiments. Of the 13 compounds, the known compound hinokinin (5) and the new compound 2 showed potent neurite outgrowth-promoting activity in PC 12 cells.

[Effect of NGF, which is isolated and purified from the venom of Naja naja atra, on expression of GAP-43 in dorsal root ganglia removed partially from cat].

OBJECTIVE: To investigate the effect of the nerve growth factor (NGF), which is isolated and purified from the venom of Naja naja atra, on expression of GAP-43 in dorsal root ganglia (DRG) removed partially from cat. METHODS: A total of 45 adult male cats (rhizotomy of unilateral L1-Ls, L7-S2 dorsal root, leaving L6 as a spared dorsal root in 45 cats allowed to survive 3 days, 6 days, 12 days respectively) were equally divided into 3 groups based on the dose of using the drug: low-dosage group (1 microg/kg), high-dosage group (2 microg/ kg), normal saline control group (0. 1 mL/kg), with using immunohistochemical reaction to detect the GAP-43 of L6 DRG, count the GAP-43-positive large, medium-small neurons. RESULTS: The results were as follows: After the operation, GAP-43 positive products were mainly distributed in nucleus of large neurons and cytoplasm of medium-small neurons. The expressions of GAP-43 positive products in treatment group and control group were detected at day 3, reached the highest level on the 6th day and began to decrease on the 12th day. The number of GAP-43 positive medium-small neurons of the operated side DRG in treatment group increased more apparently than that of control group, the high-dosage group was significantly (P

Construction of a mutated pro-nerve growth factor resistant to degradation and suitable for biophysical and cellular utilization.

Precursor of nerve growth factor (proNGF) has been found to be proapoptotic in several cell types and mediates its effects by binding to p75 neurotrophin receptor (p75NTR) and sortilin. The proNGF molecule is processed by proteases at three dibasic sites found in the pro domain to form mature NGF (termed herein as sites 1, 2, and 3 from the proNGF N terminus). Of these processing sites, site 3, adjacent to the N terminus of mature NGF, was thought to be the major site responsible for processing of proNGF to mature NGF. We found that mutating this major processing site (site 3) resulted in a form of proNGF that was only partially stable. On introducing additional mutations in the pro domain at the other two dibasic sites, we found the stability of proNGF to increase significantly. Here we describe the construction, expression, and purification of this more stable proNGF molecule. The two consecutive basic residues at each of the three sites were mutated to neutral alanine residues. Expression was performed in stably transfected Sf21 insect cells. Purification involved strong cation-exchange chromatography and N60 immunoaffinity column chromatography. The construct with all three sites mutated (termed proNGF123) gave all proNGF with no mature NGF and was not cleaved by three proconvertases (furin, PACE-4, and PC-2) known to proteolyze proneurotrophins in vivo. This stable proNGF molecule demonstrated proapoptotic activity on rat pheocytochroma PC12 cells, PC12nnr cells, C6 glioblastoma cells, and RN22 schwannoma cells.

Sargachromenol, a novel nerve growth factor-potentiating substance isolated from Sargassum macrocarpum, promotes neurite outgrowth and survival via distinct signaling pathways in PC12D cells.

We previously found that the methanol extract of a marine brown alga, Sargassum macrocarpum showed marked nerve growth factor (NGF)-dependent neurite outgrowth promoting activity to PC12D cells. The active substance purified was elucidated to be sargachromenol. The median effective dose (ED50) was 9 microM against PC12D cells in the presence of 10 ng/ml NGF, although it showed no neurotrophic effect on its own. Pretreatment of cells with protein kinase A (PKA) inhibitor or U0126 substantially suppressed the sargachromenol-enhanced neurite outgrowth from PC12D cells, suggesting that the activation of cyclic AMP-mediated protein kinase and mitogen-activated protein (MAP) kinase 1/2 was apparently required for the action of sargachromenol. On the other hand, sargachromenol significantly promoted the survival of neuronal PC12D cells at 0-50 ng/ml NGF in serum-free medium. Neither PKA inhibitor nor U0126 could inhibit the survival supporting effect of sargachromenol, whereas wortmannin significantly blocked the sargachromenol-induced survival supporting effect on neuronal PC12D cells, suggesting that sargachromenol rescued neuronal PC12D cells by activating phosphatidylinositol-3 kinase. These results demonstrate that sargachromenol promotes neuronal differentiation of PC12D cells and supports the survival of neuronal PC12D cells via two distinct signaling pathways.

Sputa nerve growth factor forms a preferable substitute to mouse 7S-beta nerve growth factor.

The NGF (nerve growth factor) from Naja sputatrix has been purified by gel filtration followed by reversed-phase HPLC. The protein showed a very high ability to induce neurite formation in PC12 cells relative to the mouse NGF. Two cDNAs encoding isoforms of NGF have been cloned and an active recombinant NGF, sputa NGF, has been produced in Escherichia coli as a His-tagged fusion protein. Sputa NGF has been found to be non-toxic under both in vivo and in vitro conditions. The induction of neurite outgrowth by this NGF has been found to involve the high-affinity trkA-p75NTR complex of receptors. The pro-survival mechanism of p75NTR has been mediated by the activation of nuclear factor kappaB gene by a corresponding down-regulation of inhibitory kappaB gene. Real-time PCR and protein profiling (by surface-enhanced laser-desorption-ionization time-of-flight) have confirmed that sputa NGF up-regulates the expression of the endogenous NGF in PC12 cells. Preliminary microarray analysis has also shown that sputa NGF is capable of promoting additional beneficial effects such as the up-regulation of arginine vasopressin receptor 1A, voltage-dependent T-type calcium channel. Hence, sputa NGF forms a new and useful NGF.