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Many routes may lead to the transition from a healthy to a diseased phenotype. However, there are not so many routes to travel in the opposite direction; that is, therapy for different diseases. The following pressing question thus remains: what are the pathogenic routes and how can be they counteracted for therapeutic purposes? Human cells contain >500 protein kinases and nearly 200 protein phosphatases, acting on thousands of proteins, including cell growth factors. We herein discuss neurotrophins with pathogenic or metabotrophic abilities, particularly brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), pro-NGF, neurotrophin-3 (NT-3), and their receptor Trk (tyrosine receptor kinase; pronounced "track"). Indeed, we introduced the word trackins, standing for Trk-targeting drugs, that play an agonistic or antagonistic role in the function of TrkBBDNF, TrkCNT−3, TrkANGF, and TrkApro-NGF receptors. Based on our own published results, supported by those of other authors, we aim to update and enlarge our trackins concept, focusing on (1) agonistic trackins as possible drugs for (1a) neurotrophin-deficiency cardiometabolic disorders (hypertension, atherosclerosis, type 2 diabetes mellitus, metabolic syndrome, obesity, diabetic erectile dysfunction and atrial fibrillation) and (1b) neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, and multiple sclerosis), and (2) antagonistic trackins, particularly TrkANGF inhibitors for prostate and breast cancer, pain, and arrhythmogenic right-ventricular dysplasia. Altogether, the druggability of TrkANGF, TrkApro-NGF, TrkBBDNF, and TrkCNT−3 receptors via trackins requires a further translational pursuit. This could provide rewards for our patients. [ABSTRACT FROM AUTHOR]

The neurotrophin nerve growth factor (NGF) and its precursor proNGF are both bioactive and exert similar or opposite actions depending on the cell target and its milieu. The balance between NGF and proNGF is crucial for cell and tissue homeostasis and it is considered an indicator of pathological conditions. Proteolytical cleavage of proNGF to the mature form results in different fragments, whose function and/or bioactivity is still unclear. The present study was conducted to investigate the distribution of proNGF fragments derived from endogenous cleavage in brain and peripheral tissues of adult rats in the healthy condition and following inflammatory lipopolysaccharide (LPS) challenge. Different anti‐proNGF antibodies were tested and the presence of short peptides corresponding to the prodomain sequence (pdNGFpep) was identified. Processing of proNGF was found to be tissue‐specific and accumulation of pdNGFpeps was found in inflamed tissues, mainly in testis, intestine and heart, suggesting a possible correlation between organ functions and a response to insults and/or injury. The bioactivity of pdNGFpep was also demonstrated in vitro by using primary hippocampal neurons. Our study supports a biological function for the NGF precursor prodomain and indicates that short peptides from residues 1–60, differing from the 70–110 sequence, induce apoptosis, thereby opening the way for identification of new molecular targets to study pathological conditions. [ABSTRACT FROM AUTHOR]

There is increasing evidence that nerve growth factor (NGF) and its receptors, the neurotrophic receptor tyrosine kinase 1 (NTRK1/TrkA), the common neurotrophin receptor (NGFR/p75NTR) and the membrane receptor sortilin, participate in cancer growth. In melanoma, there have been some reports suggesting that NGF, TrkA and p75NTR are dysregulated, but the expression of the NGF precursor (proNGF) and its membrane receptor sortilin is unknown. In this study, we investigated the expression of NGF, proNGF, TrkA, p75NTR and sortilin by immunohistochemistry in a series of human tissue samples (n = 100), including non-cancerous nevi (n = 20), primary melanomas (n = 40), lymph node metastases (n = 20) and distant metastases (n = 20). Immunostaining was digitally quantified and revealed NGF and proNGF were expressed in all nevi and primary melanomas, and that the level of expression decreased from primary tumors to melanoma metastases (p = 0.0179 and p < 0.0001, respectively). Interestingly, TrkA protein expression was high in nevi and thin primary tumors but was strongly downregulated in thick primary tumors (p < 0.0001) and metastases (p < 0.0001). While p75NTR and sortilin were both expressed in most nevi and melanomas, there was no significant difference in expression between them. Together, these results pointed to a downregulation of NGF/ProNGF and TrkA in melanoma, and thus did not provide evidence to support the use of anti-proNGF/NGF or anti-TrkA therapies in advanced and metastatic forms of melanoma. [ABSTRACT FROM AUTHOR]

Radical prostatectomy for prostate cancer can not only induce cavernous nerve injury (CNI), but also causes cavernous hypoxia and cavernous structural changes, which lead to a poor response to phosphodiesterase 5 inhibitors. To investigate the therapeutic effect of oral administration of LM11A-31, a small molecule p75 neurotrophin receptor (p75NTR) ligand and proNGF antagonist, in a mouse model of bilateral CNI, which mimics nerve injury–induced erectile dysfunction after radical prostatectomy. 8-week-old male C57BL/6 mice were divided into sham operation and CNI groups. Each group was divided into 2 subgroups: phosphate-buffered saline and LM11A-31 (50 mg/kg/day) being administered once daily starting 3 days before CNI via oral gavage. 2 weeks after CNI, we measured erectile function by electrical stimulation of the bilateral cavernous nerve. The penis was harvested for histologic examination and Western blot analysis. The major pelvic ganglia was harvested and cultured for assays of ex vivo neurite outgrowth. Intracavernous pressure, neurovascular regeneration in the penis, in vivo or ex vivo functional evaluation, and cell survival signaling were measured. Erectile function was decreased in the CNI group (44% of the sham operation group), while administration of LM11A-31 led to a significant improvement of erectile function (70% of the sham operation group) in association with increased neurovascular content, including cavernous endothelial cells, pericytes, and neuronal processes. Immunohistochemical and Western blot analyses showed significantly increased p75NTR expression in the dorsal nerve of CNI mice, which was attenuated by LM11A-31 treatment. Protein expression of active PI3K, AKT, and endothelial nitric oxide synthase was increased, and cell death and c-Jun N-terminal kinase signaling was significantly attenuated after LM11A-31 treatment. Furthermore, LM11A-31 promoted neurite sprouting in cultured major pelvic ganglia after lipopolysaccharide exposure. LM11A-31 may be used as a strategy to treat erectile dysfunction after radical prostatectomy or in men with neurovascular diseases. Unlike biological therapeutics, such as proteins, gene therapies, or stem cells, the clinical application of LM11A-31 would likely be relatively less complex and low cost. Our study has some limitations. Future studies will assess the optimal dosing and duration of the compound. Given its plasma half-life of approximately 1 hour, it is possible that dosing more than once per day will provide added efficacy. Specific inhibition of the proNGF-p75NTR degenerative signaling via oral administration of LM11A-31 represents a novel therapeutic strategy for erectile dysfunction induced by nerve injury. Yin GN, Ock J, Limanjaya A, et al. Oral Administration of the p75 Neurotrophin Receptor Modulator, LM11A-31, Improves Erectile Function in a Mouse Model of Cavernous Nerve Injury. J Sex Med 2021;18:17–28. [ABSTRACT FROM AUTHOR]

Background: Radical prostatectomy induces some degree of cavernous nerve injury (CNI) and causes denervation‐induced pathologic changes in cavernous vasculature, regardless of the advances in surgical techniques and robotic procedures. The precursor for nerve growth factor (proNGF) is known to be involved in neuronal cell apoptosis and microvascular dysfunction through its receptor p75NTR. Objectives: To determine the expression of proNGF/p75NTR and the efficacy of proNGF neutralizing antibody (anti‐proNGF‐Ab) in a mouse model of ED induced by CNI. Materials and Methods: Age‐matched 12‐week‐old C57BL/6 mice were distributed into three groups: sham group and bilateral CNI group treated with intracavernous injections of PBS (20 μL) or of anti‐proNGF‐Ab (20 µg in 20 μL of PBS) on days −3 and 0. Two weeks after treatment, erectile function was measured by electrical stimulation of cavernous nerve. Penis tissues from a separate group of animals were harvested for further analysis. We also determined the efficacy of anti‐proNGF‐Ab on neural preservation in major pelvic ganglion (MPG) ex vivo. Results: We observed increased penile expression of proNGF and p75NTR after CNI. Intracavernous administration of anti‐proNGF‐Ab increased nNOS and neurofilament expression probably by enhancing the production of neurotrophic factors, such as neurotrophin‐3, NGF, and brain‐derived neurotrophic factor. Anti‐proNGF‐Ab preserved the integrity of cavernous sinusoids, such as pericytes, endothelial cells, and endothelial cell‐to‐cell junctions, possibly by controlling angiogenic factors (angiopoietin‐1, angiopoietin‐2, and vascular endothelial growth factor) and induced endogenous eNOS phosphorylation in CNI mice. And finally, treatment with anti‐proNGF‐Ab rescued erectile function in CNI mice. Anti‐proNGF‐Ab also enhanced neurite sprouting from MPG exposed to lipopolysaccharide. Discussion and Conclusion: The preservation of damaged cavernous neurovasculature through inhibition of the proNGF/p75NTR pathway may be a novel strategy to treat radical prostatectomy‐induced erectile dysfunction. [ABSTRACT FROM AUTHOR]

• Reactive astrocytes display increased expression of proNGF in mouse model of injury spinal cord. • Astrocytic proNGF appears in both exosome-like vesicles and homogeneous form in the cytoplasm. • Activated astrocytes also increase expression level and release of proNGF following LPS exposure. Astrocytes are major glial cells critically in maintaining stability of the central nervous system and functional activation of astrocytes occurs rapidly in various diseased or traumatic events. We are interested in functional changes of astrocytes during the spinal cord injury, and studied expression of nerve growth factor (NGF) in activated astrocytes by mouse model of contused spinal cord injury and cell culture experiment. It revealed that the spinal cord injury resulted in apparent activation of astrocytes and microglial cells and decreased BMS scores. A larger number of astrocytes showed immunoreactivity to proNGF in the injured spinal cord areas, and proNGF expression increased and remained high level at 7 to 14dpi, which was coincided with upregulation of glial fibrillary acidic protein. The proNGF was clearly localized in both exosome-like vesicles and cytoplasm of astrocytes in culture. Electron microscopy confirmed exosome-like vesicles with proNGF-immunoreactivity in diameter sizes of 50–100 nm. Finally, cell culture with lipopolysaccharide (LPS) experiment indicated increasing expression and release of proNGF in the astrocytes with LPS exposure. This study demonstrated that reactive astrocytes increased proNGF expression after spinal cord injury, also suggesting involvement of exosome-like proNGF transport or release in triggering neuronal apoptosis and aggravating progression of spinal cord injury. [ABSTRACT FROM AUTHOR]

Accumulating data highlight the role of neurotrophins and their receptors in human breast cancer. This family includes nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), both synthetized as proneurotrophins (proNGF and proBDNF). (pro)NGF and (pro)BDNF initiate their biological effects by binding to both their specific receptors TrkA and TrkB, respectively, and the common receptor p75NTR. Currently, no data are available about their expression and potential role in canine mammary tumors. The aim of this study was to investigate expression of proNGF and BDNF as well as their receptors TrkA, TrkB, and p75NTR in canine mammary carcinomas, and to correlate them with clinicopathological parameters (grade, histological type, lymph node status, recurrence, and distant metastasis) and survival. Immunohistochemistry was performed on serial sections of 96 canine mammary carcinomas with antibodies against proNGF, BDNF, TrkA, TrkB, and p75NTR. Of the 96 carcinomas, proNGF expression was detected in 71 (74%), BDNF in 79 (82%), TrkA in 94 (98%), TrkB in 35 (37%), and p75NTR in 44 (46%). No association was observed between proNGF, BDNF, or TrkA expression and either clinicopathological parameters or survival. TrkB and p75NTR expression were associated with favorable clinicopathological parameters as well as better overall survival. [ABSTRACT FROM AUTHOR]

Abstract Background Precursor of nerve growth factor (proNGF) was previously considered biologically inactive; however, it has recently been identified as having important roles in the pathology of cancer development. Aim This study aimed to explore the therapeutic effects of proNGF siRNA on the proliferation, invasion, and anoikis of pancreatic cancer cells and determine the functions of proNGF. Methods Pancreatic ductal adenocarcinoma (PDAC) and paired paracancerous tissue samples were collected from 60 patients for evaluation of proNGF expression by immunohistochemistry staining, qPCR, and western blotting. PDAC cell proliferation, migration, apoptosis, and anoikis following proNGF siRNA knockdown were investigated in two pancreatic cancer cell lines, Panc-1 and Bxpc-3, using BrdU incorporation assays, EdU staining, Ki-67 immunofluorescence (IF) staining, wound-healing assays, transwell invasion assays, and EthD-1 IF staining. Autophagy-related proteins were also measured by western blotting. Results Levels of proNGF protein were higher in pancreatic cancer tissues and cells lines than those in paracancerous tissues and normal pancreatic duct epithelial cells, respectively. In vitro , ProNGF knockdown by siRNA led to significantly reduced cell proliferation, remarkably inhibited wound-healing, and reduced the number of invaded PDAC cells in migration and transwell assays. Treatment with proNGF siRNA also downregulated ATG5 and Beclin 1 protein levels, increased those of P62, and increased EthD-1 staining in PDAC cells. Conclusion ProNGF expression is elevated in PDAC tissues and cell lines, and proNGF siRNA can inhibit cell proliferation, migration, and invasion, and promote anoikis of pancreatic cancer cells, in which decreased proNGF may participate. [ABSTRACT FROM AUTHOR]

Abstract Introduction Penile neurovascular dysfunction is a major cause of erectile dysfunction (ED) in diabetic patients, which causes poor response to oral phosphodiesterase-5 inhibitors. Nerve growth factor precursor (proNGF) and its p75 neurotrophin receptor (p75NTR) have been known to be involved in microvascular complications and neurodegeneration. Aim To examine the role of proNGF and its receptor p75NTR signaling pathway in diabetic ED, and to determine the effectiveness of proNGF neutralizing antibody (proNGF-Ab) in restoring erectile function in streptozotocin (STZ)-induced diabetic mice. Methods Diabetes mellitus was induced by intraperitoneal injection of STZ (50 mg/kg) into 8-week-old C57BL/6 male mice for 5 consecutive days. At 8 weeks after the induction of diabetes mellitus, the animals were distributed into 3 groups: controls and STZ-induced diabetic mice receiving 2 intracavernous injections of either saline (days −3 and 0; 20 μL) or proNGF-Ab (days −3 and 0; 20 μg in 20 μL of saline). We also examined the effect of proNGF-Ab or p75NTR small interfering RNA in primary cultured mouse cavernous endothelial cells, pericytes, and major pelvic ganglion. Main Outcome Measures Erectile function was measured by electrical stimulation of the cavernous nerve at 2 weeks after treatment, and the penis was then harvested for histologic and biochemical studies. Results The cavernous expression of proNGF and p75NTR was upregulated under diabetic conditions. Intracavernous injection of proNGF-Ab successfully restored erectile function in diabetic mice, which reach 93–96% of control values. ProNGF-Ab significantly restored cavernous endothelial cell, pericyte, and neuronal cell content, and increased endothelial cell-to-cell junction proteins in the diabetic mice. Under the high-glucose condition, proNGF-Ab or p75NTR small interfering RNA promoted tube formation in mouse cavernous endothelial cells and pericytes, decreased apoptosis of endothelial cells and pericytes, and enhanced neurite sprouting from major pelvic ganglion. Clinical Implications The ProNGF/p75NTR pathway will be a new therapeutic target for diabetic ED. Strength & Limitations This is the first study demonstrating the efficacy of the inhibition of proNGF/p75NTR pathway in diabetic ED. Further studies are needed to test whether a different dosing of proNGF-Ab would induce more durable erectile function recovery. Conclusion Our findings suggest that inhibition of the proNGF/p75NTR signaling pathway is a promising therapeutic strategy for diabetic ED. Nguyen NM, Song K-M, Choi M-J, et al. Inhibition of proNGF and p75NTR Pathway Restores Erectile Function Through Dual Angiogenic and Neurotrophic Effects in the Diabetic Mouse. J Sex Med 2019;16:351–364. [ABSTRACT FROM AUTHOR]

Previous studies demonstrated exercise-induced modulation of neurotrophins, such as Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF). Yet, no study that we are aware of has examined their change as a function of different training paradigms. In addition, the understanding of the possible training-induced relationship between NGF and BDNF change is still lacking. Consequently, in the current study we examined the effect of a Walking Training (WT) and of Quadrato Motor Training (QMT) on NGF and BDNF precursors (proNGF and proBDNF). QMT is a specifically structured sensorimotor training that involves sequences of movements based on verbal commands, that was previously reported to improve spatial cognition, reflectivity, creativity as well as emotion regulation and general self-efficacy. In addition, QMT was reported to induce electrophysiological and morphological changes, suggesting stimulation of neuroplasticity processes. In two previous independent studies we reported QMT-induced changes in the salivary proNGF and proBDNF levels. Our present results demonstrate that following 12 weeks of daily QMT practice, proNGF level increases while proBDNF showed no significant change. More importantly, while no correlation between the two neurotrophins prior to training was detectable, there was a significant correlation between change in proNGF and proBDNF levels. Taken together the current results suggest that the two neurotrophins undergo a complex modulation, likely related to the different pathways by which they are produced and regulated. Since variations of these neurotrophins have been previously linked to depression, stress and anxiety, the current study may have practical implications and aid in understanding the possible physiological mechanisms that mediate improved well-being, and the dynamic change of neurotrophins as a result of training.

Previous studies demonstrated exercise-induced modulation of neurotrophins, such as Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF). Yet, no study that we are aware of has examined their change as a function of different training paradigms. In addition, the understanding of the possible training-induced relationship between NGF and BDNF change is still lacking. Consequently, in the current study we examined the effect of a Walking Training (WT) and of Quadrato Motor Training (QMT) on NGF and BDNF precursors (proNGF and proBDNF). QMT is a specifically structured sensorimotor training that involves sequences of movements based on verbal commands, that was previously reported to improve spatial cognition, reflectivity, creativity as well as emotion regulation and general self-efficacy. In addition, QMT was reported to induce electrophysiological and morphological changes, suggesting stimulation of neuroplasticity processes. In two previous independent studies we reported QMT-induced changes in the salivary proNGF and proBDNF levels. Our present results demonstrate that following 12 weeks of daily QMT practice, proNGF level increases while proBDNF showed no significant change. More importantly, while no correlation between the two neurotrophins prior to training was detectable, there was a significant correlation between change in proNGF and proBDNF levels. Taken together the current results suggest that the two neurotrophins undergo a complex modulation, likely related to the different pathways by which they are produced and regulated. Since variations of these neurotrophins have been previously linked to depression, stress and anxiety, the current study may have practical implications and aid in understanding the possible physiological mechanisms that mediate improved well-being, and the dynamic change of neurotrophins as a result of training. [ABSTRACT FROM AUTHOR]

Neurotrophins have been intensively studied and have multiple roles in the brain. Neurotrophins are first synthetized as proneurotrophins and then cleaved intracellularly and extracellularly. Increasing evidences demonstrate that proneurotrophins and mature neurotrophins exerts opposing role in the central nervous system. In the present review, we explore the role of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4) and their respective proform in cellular processes related to learning and memory. We focused on their roles in synaptic activity and plasticity in the brain with an emphasis on long-term potentiation, long-term depression, and basal synaptic transmission in the hippocampus and the temporal lobe area. We also discuss new findings on the role of the Val66Met polymorphism on the BDNF propeptide on synaptic activity. [ABSTRACT FROM AUTHOR]

Evidence from human neuropathological studies indicates that the levels of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are compromised in Alzheimer's disease. However, the causes and temporal (pathology-dependent) evolution of these alterations are not completely understood. To elucidate these issues, we investigated the McGill-R-Thy1-APP transgenic rat, which exhibits progressive intracellular and extracellular amyloid-beta (Aβ) pathology and ensuing cognitive deficits. Neurochemical analyses revealed a differential dysregulation of NGF and BDNF transcripts and protein expression. While BDNF mRNA levels were significantly reduced at very early stages of amyloid pathology, before plaques appeared, there were no changes in NGF mRNA expression even at advanced stages. Paradoxically, the protein levels of the NGF precursor were increased. These changes in neurotrophin expression are identical to those seen during the progression of Alzheimer's disease. At advanced pathological stages, deficits in the protease cascade controlling the maturation and degradation of NGF were evident in McGill transgenic rats, in line with the paradoxical upregulation of proNGF, as seen in Alzheimer's disease, in the absence of changes in NGF mRNA. The compromise in NGF metabolism and BDNF levels was accompanied by downregulation of cortical cholinergic synapses; strengthening the evidence that neurotrophin dysregulation affects cholinergic synapses and synaptic plasticity. Our findings suggest a differential temporal deregulation of NGF and BDNF neurotrophins, whereby deficits in BDNF mRNA appear at early stages of intraneuronal Aβ pathology, before alterations in NGF metabolism and cholinergic synapse loss manifest. [ABSTRACT FROM AUTHOR]

This review summarizes the present knowledge concerning the retinal localization of the nerve growth factor (NGF), its precursor proNGF, and the receptors TrkA and p75 NTR in the developing and mature rodent retina. We further discuss the changes in the expression of NGF and the receptors in experimental models of retinal disorders and diseases like inherited retinitis pigmentosa, retinal detachment, glaucoma, and diabetic retinopathy. Since proNGF is now recognized as a bioactive signaling molecule which induces cell death through p75 NTR activation, the role of proNGF in the induction of retinal cell loss under neurodegenerative conditions is also highlighted. In addition, we present the evidences for a potential therapeutic intervention with NGF for the treatment of retinal neurodegenerative diseases. Different strategies have been developed and experimentally tested in mice and rats in order to reduce cell loss and Müller cell gliosis, e.g., increasing the availability of endogenous NGF, administration of exogenous NGF, activation of TrkA, and inhibition of p75 NTR . Here, we discuss the several lines of evidence supporting a protective effect of NGF on retinal cell loss, with specific emphasis on photoreceptor and retinal ganglion cell degeneration. A better understanding of the mechanisms underlying the effects of NGF and proNGF in the modulation of neurodegeneration and gliosis in the retina will help to develop efficient therapeutic strategies for various retinal diseases. [ABSTRACT FROM AUTHOR]

The homeostasis between mature neurotrophin NGF and its precursor proNGF is thought to be crucial in physiology and in pathological states. Therefore, the measurement of the relative amounts of NGF and proNGF could serve as a footprint for the identification of disease states, for diagnostic purposes. Since NGF is part of proNGF, their selective identification with anti-NGF antibodies is not straightforward. Currently, many immunoassays for NGF measurement are available, while the proNGF assays are few and not validated by published information. The question arises, as to whether the commercially available assays are able to distinguish between the two forms. Also, since in biological samples the two forms coexist, are the measurements of one species affected by the presence of the other? We describe experiments addressing these questions. For the first time, NGF and proNGF were measured together and tested in different immunoassays. Unexpectedly, NGF and proNGF were found to reciprocally interfere with the experimental outcome. The interference also calls into question the widely used NGF ELISA methods, applied to biological samples where NGF and proNGF coexist. Therefore, an immunoassay, able to distinguish between the two forms is needed. We propose possible ways forward, toward the development of a selective assay. In particular, the use of the well validated anti-NGF aD11 antibody in an alphaLISA assay with optimized incubation times would be a solution to avoid the interference in the measurement of a mixed sample containing NGF and proNGF. Furthermore, we explored the possibility of measuring proNGF in a biological sample. But the available commercial kit for the detection of proNGF does not allow the measurement of proNGF in mouse brain tissues. Therefore, we validated an SPR approach for the measurement of proNGF in a biological sample. Our experiments help in understanding the technical limits in the measurement of the NGF/proNGF ratio in biological samples, and propose concrete solutions toward the solution of this problem. [ABSTRACT FROM AUTHOR]

Several reports have shown that a sciatic nerve conditioned media (CM) causes neuronal-like differentiation in PC12 cells. This differentiation is featured by neurite outgrowth, which are exclusively dendrites, without axon or sodium current induction. In previous studies, our group reported that the CM supplemented with a generic inhibitor for tyrosine kinase receptors (k252a) enhanced the CM-induced morphological differentiation upregulating neurite outgrowth, axonal formation and sodium current elicitation. Sodium currents were also induced by depletion of endogenous precursor of nerve growth factorr (proNGF) from the CM (pNGFd-CM). Given that sodium currents, neurite outgrowth and axon specification are important features of neuronal differentiation, in the current manuscript, first we investigated if proNGF was hindering the full PC12 cell neuronal-like differentiation. Second, we studied the effects of exogenous wild type (pNGFwt) and mutated (pNGFmut) proNGF isoforms over sodium currents and whether or not their addition to the pNGFd-CM would prevent sodium current elicitation. Third, we investigated if proNGF was exerting its negative regulation through the sortilin receptor, and for this, the proNGF action was blocked with neurotensin (NT), a factor known to compete with proNGF for sortilin. Thereby, here we show that pNGFd-CM enhanced cell differentiation, cell proportion with long neurites, total neurite length, induced axonal formation and sodium current elicitation. Interestingly, treatment of PC12 cells with wild type or mutated proNGF isoforms elicited sodium currents. Supplementing pNGFd-CM with pNGFmut reduced 35% the sodium currents. On the other hand, pNGFd- CM+pNGFwt induced larger sodium currents than pNGFd-CM. Finally, treatments with CM supplemented with NT showed that sortilin was mediating proNGF negative regulation, since its blocking induced similar effects than the pNGFd-CM treatment. Altogether, our results suggest that proNGF within the CM, is one of the main inhibitors of full neuronal differentiation, acting through sortilin receptor. [ABSTRACT FROM AUTHOR]

The p75 neurotrophin receptor (p75NTR) is a multifunctional receptor that participates in many critical processes in the nervous system, ranging from apoptosis to synaptic plasticity and morphological events. It is a member of the tumor necrosis factor receptor (TNFR) superfamily, whose members undergo trimeric oligomerization. Interestingly, p75NTR interacts with dimeric ligands (i.e., proneurotrophins or mature neurotrophins), but several of the intracellular adaptors that mediate p75NTR signaling are trimeric (i.e., TNFRassociated factor 6 or TRAF6). Consequently, the active receptor signaling unit remains uncertain. To identify the functional receptor complex, we evaluated its oligomerization in vitro and in mice brain tissues using a combination of biochemical techniques. We found that the most abundant homotypic arrangement for p75NTR is a trimer and that monomers and trimers coexist at the cell surface. Interestingly, trimers are not required for ligand-independent or ligand-dependent p75NTR activation in a growth cone retraction functional assay. However, monomersare capable of inducing acute morphological effects in neurons. We propose that p75NTR activation is regulated by its oligomerization status and its levels of expression. These results indicate that the oligomeric state of p75NTR confers differential responses and offers an explanation for the diverse and contradictory actions of this receptor in the nervous system. [ABSTRACT FROM AUTHOR]

The discovery of cancer stem cells (CSCs) fundamentally advanced our understanding of the mechanisms governing breast cancer development. However, the stimuli that control breast CSC self-renewal and differentiation have still not been fully detailed. We previously showed that nerve growth factor (NGF) and its precursor proNGF can stimulate breast cancer cell growth and invasion in an autocrine manner. In this study, we investigated the effects of NGF and proNGF on the breast CSC compartment and found that NGF or proNGF enrich for CSCs in several breast cancer cell lines. This enrichment appeared to be achieved by increasing the number of symmetric divisions of quiescent/slow-proliferating CSCs. Interestingly, in vitro NGF pretreatment of MCF-7 luminal breast cancer cells promoted epithelial to mesenchymal transition in tumors of severe combined immunodeficient mice. Furthermore, p75NTR, the common receptor for both neurotrophins and proneurotrophins, mediated breast CSC self-renewal by regulating the expression of pluripotency transcription factors. Our data indicate, for the first time, that the NGF/proNGF/p75NTR axis plays a critical role in regulating breast CSC self-renewal and plasticity. S tem C ells 2015;33:342-353 [ABSTRACT FROM AUTHOR]

Background Based on our previous findings on the efficacy of ocular applied nerve growth factor as eye drops (o NGF) to act in brain and counteract neuronal damage, we hypothesized that o NGF treatment might revert neuronal atrophy occurring in diabetic brain also by controlling neurotrophin system changes. The major NGF brain target areas, such as the septum and the hippocampus, were used as an experimental paradigma to test this hypothesis. Methods Bilateral o NGF treatment was performed twice a day for 2 weeks in full-blown streptozotocin-treated adult male rats. The forebrain distribution of cholinergic and endothelial cell markers and NGF receptors were studied by confocal microscopy. The septo-hippocampal content of NGF mature and precursor form and NGF receptors expression were also analyzed by Elisa and Western blot. Results o NGF treatment recovers the morphological alterations and the neuronal atrophy in septum and normalized the expression of mature and pro- NGF, as well as NGF receptors in the septum and hippocampus of diabetic rats. In addition, o NGF stimulated brain vascularization and up-regulated the TRKA receptor in vessel endothelium. Conclusions Our findings confirm that reduced availability of mature NGF and NGF signaling impairment favors vascular and neuronal alterations in diabetic septo-hippocampal areas and corroborate the ability of o NGF to act as a neuroprotective agent in brain. [ABSTRACT FROM AUTHOR]

Abstract: Sortilin is a member of the family of vacuolar protein sorting 10 protein domain receptors which has emerged as a co-receptor in cell death and neurodegeneration processes mediated by proneurotrophins. Here we tested the possibility that sortilin deficiency interferes with behavioral and neuropathological endpoints in a chronic Nerve Growth factor (NGF)-deprivation model of Alzheimer’s disease (AD), the AD10 anti-NGF mouse. AD10 mice show cholinergic deficit, increased APP processing and tau hyper-phosphorylation, resulting in behavioral deficits in learning and memory paradigms assessed by novel object recognition and Morris water maze tests. Sort1 −/− mice were crossed with AD10 anti-NGF mice and the neurodegenerative phenotype was studied. We found that the loss of sortilin partially protected AD10 anti-NGF mice from neurodegeneration. A protective effect was observed on non-spatial memory as assessed by novel object recognition, and histopathologically at the level of Aβ and BFCNs, while the phosphotau increase was unaltered by knocking out sortilin. We suggest that sortilin might be involved in different aspects of neurodegeneration in a complex way, supporting the view that sortilin functions in the CNS are broader than being a co-receptor in proneurotrophin and neurotrophin signaling. [Copyright &y& Elsevier]

The complex network of neurotrophic factors is supposed to play a role in neurodegeneration, but the effect of variations in their coding genes on susceptibility to sporadic Alzheimer's disease is not established. The mature form of nerve growth factor (NGF) derives from a precursor, proNGF, which was recently discovered to exert crucial functions in brain. We designed a case-control association study to test the hypothesis as to whether polymorphisms located in the proNGF genomic region influence the liability to Alzheimer's disease and its prodromal form, mild cognitive impairment. Three independent case-control samples, with individuals aged >60 years, were collected in Italian Alzheimer Units. One polymorphism located in the proNGF region, rs6330, demonstrated a minor allele frequency >5% and was used in the association study. The minor allele of rs6330 was more frequent in patients from the three sample series as compared to respective normal controls. Multivariate logistic regression showed a significant association under the dominant model in one cohort (OR 1.83, 95% CI 1.00-3.54) and in the pooled case-control sample (OR 1.47, 95% CI 1.03-2.08). These findings further suggest that proNGF may play a role in Alzheimer-type neurodegeneration and that genetic variations in the NGF locus may influence the occurrence of sporadic, late-onset Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Abstract: proNGF, the precursor of the neurotrophin NGF, is widely expressed in central and peripheral nervous system. Its physiological functions are still largely unknown, although it emerged from studies in the last decade that proNGF has additional and distinct functions with respect to NGF, besides acting chaperone-like for NGF folding during its biogenesis. The regulation of proNGF/NGF ratio represents a crucial process for homeostasis of brain and other tissues, and understanding the molecular aspects of these differences is important. We report the selection and characterization of a recombinant monoclonal anti-proNGF antibody in single chain Fv fragment (scFv) format. The selection exploited the Intracellular Antibody Capture Technology (IACT), starting from a naïve mouse SPLINT (Single Pot Library of INTracellular antibodies) library. This antibody (scFv FPro10) was expressed recombinantly in Escherichia coli, was proven to be highly soluble and stable, and thoroughly characterized from the biochemical–biophysical point of view. scFv FPro10 displays high affinity and specificity for proNGF, showing no cross-reactivity with other pro-neurotrophins. A structural model was obtained by SAXS. scFv FPro10 represents a new tool to be exploited for the selective immunoanalysis of proNGF, both in vitro and in vivo, and might help in understanding the molecular function of proNGF in neurodegeneration. [Copyright &y& Elsevier]

NGF, the principal neurotrophic factor for basal forebrain cholinergic neurons (BFCNs), has been correlated to Alzheimer's disease (AD) because of the selective vulnerability of BFCNs in AD. These correlative links do not substantiate a comprehensive cause-effect mechanism connecting NGF deficit to overall AD neurodegeneration. A demonstration that neutralizing NGF activity could have consequences beyond a direct interference with the cholinergic system came from studies in the AD11 mouse model, in which the expression of a highly specific anti-NGF antibody determines a neurodegeneration that encompasses several features of human AD. Because the transgenic antibody binds to mature NGF much more strongly than to proNGF and prevents binding of mature NGF to the tropomyosin-related kinase A (TrkA) receptor and to p75 neurotrophin receptor (p75NTR), we postulated that neurodegeneration in AD11 mice is provoked by an imbalance of proNGF/NGF signaling and, consequently, of TrkA/p75NTR signaling. To test this hypothesis, in this study we characterize the phenotype of two lines of transgenic mice, one in which TrkA signaling is inhibited by neutralizing anti-TrkA antibodies and a second one in which anti-NGF mice were crossed to p75NTRexonIII(-/-) mice to abrogate p75NTR signaling. TrkA neutralization determines a strong cholinergic deficit and the appearance of β-amyloid peptide (Aβ) but no tau-related pathology. In contrast, abrogating p75NTR signaling determines a full rescue of the cholinergic and Aβ phenotype of anti-NGF mice, but tau hyperphosphorylation is exacerbated. Thus, we demonstrate that inhibiting TrkA signaling activates Aβ accumulation and that different streams of AD neurodegeneration are related in complex ways to TrkA versus p75NTR signaling. [ABSTRACT FROM AUTHOR]

As the population ages, obesity and metabolic complications as well as neurological disorders are becoming more prevalent, with huge economic burdens on both societies and families. New therapeutics are urgently needed. Nerve growth factor (NGF), first discovered in 1950s, is a neurotrophic factor involved in regulating cell proliferation, growth, survival, and apoptosis in both central and peripheral nervous systems. NGF and its precursor, proNGF, bind to TrkA and p75 receptors and initiate protein phosphorylation cascades, resulting in changes of cellular functions, and are associated with obesity, diabetes and its complications, and Alzheimer's disease. In this article, we summarize changes in NGF levels in metabolic and neuronal disorders, the signal transduction initiated by NGF and proNGF, the physiological and pathophysiological relevance, and therapeutic potential in treating chronic metabolic diseases and cognitive decline. • NGF is a response protein associated with changes of adipokines in obesity. • Administration and eyedrops of NGF alleviate degradation of diabetic retinal cell. • Duration of Ras/Raf activation induced by NGF determine the signal flow direction. • GSK-3 activation at different locations results in different neural functions. • NGF can be delivered into human brain via viral vector and encapsulated cell. [ABSTRACT FROM AUTHOR]

Metastases in thyroid cancer are associated with aggressive disease and increased patient morbidity, but the factors driving metastatic progression are unclear. The precursor for nerve growth factor (proNGF) is increased in primary thyroid cancers, but its expression or significance in metastases is not known. In this study, we analysed the expression of proNGF in a retrospective cohort of thyroid cancer lymph node metastases (n = 56), linked with corresponding primary tumours, by automated immunohistochemistry and digital quantification. Potential associations of proNGF immunostaining with clinical and pathological parameters were investigated. ProNGF staining intensity (defined by the median h-score) was significantly higher in lymph node metastases (h-score 94, interquartile range (IQR) 50–147) than in corresponding primary tumours (57, IQR 42–84) (p = 0.002). There was a correlation between proNGF expression in primary tumours and corresponding metastases, where there was a 0.68 (95% CI 0 to 1.2) increase in metastatic tumour h-score for each unit increase in the primary tumour h-score. However, larger tumours (both primary and metastatic) had lower proNGF expression. In a multivariate model, proNGF expression in nodal metastases was negatively correlated with lateral neck disease and being male. In conclusion, ProNGF is expressed in locoregional metastases of thyroid cancer and is higher in lymph node metastases than in primary tumours, but is not associated with high-risk clinical features. [ABSTRACT FROM AUTHOR]

We have previously shown that the expression of pro-nerve growth factor (proNGF) was significantly increased, nerve growth factor (NGF) level was decreased, and the expression of p75NTR was enhanced in Alzheimer's disease (AD) hippocampal samples. NGF regulates cell survival and differentiation by binding TrkA and p75NTR receptors. ProNGF is the precursor form of NGF, binds to p75NTR, and induces cell apoptosis. The objective of this study is to determine whether the increased p75NTR expression in AD is due to the accumulation of proNGF and Rho kinase activation. PC12 cells were stimulated with either proNGF or NGF. Pull-down assay was carried out to determine the RhoA kinase activity. We found the expression of p75NTR was enhanced by proNGF compared to NGF. The proNGF stimulation also increased the RhoA kinase activity leading to apoptosis. The expression of active RhoA kinase was found to be increased in human AD hippocampus compared to control. The addition of RhoA kinase inhibitor Y27632 not only blocked the RhoA kinase activity but also reduced the expression of p75NTR receptor and inhibited the activation of JNK and MAPK induced by proNGF. This suggests that overexpression of proNGF in AD enhances p75NTR expression and activation of RhoA, leading to neuronal cell death. [ABSTRACT FROM AUTHOR]