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Tackling neurodegeneration and neuroinflammation is particularly challenging due to the complexity of central nervous system (CNS) disorders, as well as the limited drug accessibility to the brain. The activation of tropomyosin-related kinase A (TRKA) receptor signaling by the nerve growth factor (NGF) or the neurosteroid dehydroepiandrosterone (DHEA) may combat neurodegeneration and regulate microglial function. In the present study, we synthesized a C-17-spiro-cyclopropyl DHEA derivative (ENT-A010), which was capable of activating TRKA. ENT-A010 protected PC12 cells against serum starvation-induced cell death, dorsal root ganglia (DRG) neurons against NGF deprivation-induced apoptosis and hippocampal neurons against Aβ-induced apoptosis. In addition, ENT-A010 pretreatment partially restored homeostatic features of microglia in the hippocampus of lipopolysaccharide (LPS)-treated mice, enhanced Aβ phagocytosis, and increased Ngf expression in microglia in vitro. In conclusion, the small molecule ENT-A010 elicited neuroprotective effects and modulated microglial function, thereby emerging as an interesting compound, which merits further study in the treatment of CNS disorders.

Mesenchymal stem/stromal cells (MSCs) are promising for the treatment of degenerative diseases and traumatic injuries. However, MSC engraftment is not always successful and requires a strong comprehension of the cytokines and their receptors that mediate the biological behaviors of MSCs. The effects of nerve growth factor (NGF) and its two receptors, TrkA and p75NTR, on neural cells are well studied. Increasing evidence shows that NGF, TrkA, and p75NTR are also involved in various aspects of MSC function, including their survival, growth, differentiation, and angiogenesis. The regulatory effect of NGF on MSCs is thought to be achieved mainly through its binding to TrkA. p75NTR, another receptor of NGF, is regarded as a novel surface marker of MSCs. This review provides an overview of advances in understanding the roles of NGF and its receptors in MSCs as well as the effects of MSC‐derived NGF on other cell types, which will provide new insight for the optimization of MSC‐based therapy., In this study, the modulatory effect of exogenous nerve growth factor (NGF) on mesenchymal stem/stromal cell (MSC) functions and the paracrine effects of MSC‐derived NGF on other cell types are discussed. In particular, the cytological functions and superiority of p75NTR+ MSCs as the basis for utilizing p75NTR as a surface marker to identify MSCs is presented.

Nerve growth factor (NGF), a member of the neurotrophin family, has emerged as an active mediator in different crucial events in the peripheral and central nervous system. At the same time, several studies showed that this neurotrophin can also play a role in non-neuronal tissues (e.g., among gonads). In spite of a large number of studies present in mammals, investigations devoted to NGF and its receptor TrkA in the reproductive system of other animal models, such as teleost fish, are scarce. To increase our knowledge of NGF and its receptor in a vertebrate gonads model, the present report describes the expression patterns of ngf and trka mRNA in the testis and ovary of adult zebrafish. By using chromogenic and fluorescence in situ hybridization, we demonstrate that in the testis of adult zebrafish, ngf and its receptor trka are mainly expressed in spermatogony B and spermatocytes. In the ovary of this fish, ngf and trka are expressed at different stages of oocyte development. Altogether, these results show that this neurotrophin and its receptor have an important role in the reproductive system that is conserved during vertebrate evolution.

The influence of NGF in male reproduction in some animal species and humans has already been assessed. Many of these effects are mediated by the distribution and abundance of tropomyosin receptor kinase A (TrKA) and p75 neurotrophin (p75NTR) receptors on sperm cells. The aim of this research was to investigate the role of NGF and its receptors, TrKA and p75NTR, in rabbit sperm outcomes during in vitro storage. Major semen traits (kinetic parameters, apoptotic, necrotic and live sperm) were recorded in rabbit semen samples from 0 to 12 h of storage (every 4 h). Three experimental hypotheses were formulated: i) sperm storage changes NGF receptor abundance in rabbit sperm; ii) TrKA and p75NTR differently modulate NGF signalling (assessed by the neutralisation of receptors); iii) NGF-receptor interactions show different responses during storage (evaluated by the addition of exogenous NGF). The results demonstrate that: (i) the receptor number changed in a time-dependent manner with a significant increase in p75NTR after 8–12 h of storage; ii) the neutralisation of NGF receptors largely affected VCL, apoptotic, necrotic and live cells during sperm storage, i.e. blockade of TrKA significantly increased speed, capacitation, necrosis and apoptosis, whereas blockade of p75NTR improved motility and live cells; iii) the addition of exogenous human NGF (100 ng/mL) at different time points of storage (0, 4, 8 h) differently influenced sperm traits i.e. NGF addition at time 0 positively affected all the pro-vital traits (kinetic, live cells) whereas, after 4–8 h, the effect of NGF was null or negative. In conclusion, NGF affects kinetic and other physiological traits (capacitation, apoptosis and necrosis) of rabbit sperm in a time-dependent manner. Most of these modifications are modulated by the receptors involved (TrKA or p75NTR), which changed considerably during sperm storage (increase of p75NTR).

Purpose: TRK inhibitors achieve marked tumor-agnostic efficacy in TRK fusion–positive cancers and consequently are now an established standard of care. Little is known, however, about the demographics, outcomes, response to alternative standard therapies, or genomic characteristics of TRK fusion–positive cancers. Experimental Design: Utilizing a center-wide screening program involving more than 26,000 prospectively sequenced patients, genomic and clinical data from all cases with TRK fusions were extracted. An integrated analysis was performed of genomic, therapeutic, and phenomic outcomes. Results: We identified 76 cases with confirmed TRK fusions (0.28% overall prevalence) involving 48 unique rearrangements and 17 cancer types. The presence of a TRK fusion was associated with depletion of concurrent oncogenic drivers (P < 0.001) and lower tumor mutation burden (P < 0.001), with the exception of colorectal cancer where TRK fusions cooccur with microsatellite instability (MSI-H). Longitudinal profiling in a subset of patients indicated that TRK fusions were present in all sampled timepoints in 82% (14/17) of cases. Progression-free survival on first-line therapy, excluding TRK inhibitors, administered for advanced disease was 9.6 months [95% confidence interval (CI), 4.8–13.2]. The best overall response rate achieved with chemotherapy containing–regimens across all lines of therapy was 63% (95% CI, 41–81). Among 12 patients treated with checkpoint inhibitors, a patient with MSI-H colorectal cancer had the only observed response. Conclusions: TRK fusion–positive cancers can respond to alternative standards of care, although efficacy of immunotherapy in the absence of other predictive biomarkers (MSI-H) appears limited. TRK fusions are present in tumors with simple genomes lacking in concurrent drivers that may partially explain the tumor-agnostic efficacy of TRK inhibitors.

Background NGF-TrkA is well known to play a key role in propagating and sustaining pruritogenic signals, which form the pathology of chronic pruritus. Inhibition of NGF-TrkA is a known strategy for the treatment of pruritus. In the present paper, we describe the identification, in vitro characterization, structure–activity analysis, and inhibitory evaluation of a novel TrkA inhibitory scaffold exemplified by Cucurbitacins (Cus). Methods Cus were identified as TrkA inhibitors in a large-scale kinase library screen. To obtain structural models of Cus as TrkA inhibitors, AutoDock was used to explore their binding to TrkA. Furthermore, PC12 cell culture systems have been used to study the effects of Cus and traditional Chinese medicinal plants (Tian Gua Di and bitter gourd leaf) extracts on the kinase activity of TrkA. Results Cus block the phosphorylation of TrkA on several tyrosine sites, including Tyr490, Tyr674/675, and Tyr785, and inhibit downstream Akt and MAPK phosphorylation in response to NGF in PC12 cell model systems. Furthermore, traditional Chinese medicinal plants (Tian Gua Di and bitter gourd leaf) containing Cu extracts were shown to inhibit the phosphorylation of TrkA and Akt. These data reveal mechanisms, at least partly, of the anti-pruritus bioactivity of Cus. Conclusion Taken together, with the recent discovery of the important role of TrkA as a therapeutic target, Cus could be the basis for the design of improved TrkA kinase inhibitors, which could someday help treat pruritus.

One of the aspects of Alzheimer disease is loss of cholinergic neurons in the basal forebrain, which leads to development of cognitive impairment. Here, we used a model of cholinergic deficit caused by immunotoxin 192IgG-saporin to study possible beneficial effects of adeno-associated virus (AAV)–mediated overexpression of nerve growth factor (NGF) in the hippocampus of rats with cholinergic deficit. Suspension of recombinant AAV carrying control cassette or cassette with NGF was injected into both hippocampi of control rats or rats with cholinergic deficit induced by intraseptal injection of 192IgG-saporin. Analysis of choline acetyltransferase (ChAT) immunostaining showed that NGF overexpression in the hippocampus did not prevent strong loss of ChAT-positive neurons in the septal area caused by the immunotoxin. Induction of cholinergic deficit in the hippocampus led to impairments in Y-maze and beam-walking test but did not affect behavioral indices in the T-maze, open field test, and inhibitory avoidance training. NGF overexpression in the rats with cholinergic deficit restored normal animal behavior in Y-maze and beam-walking test. Recording of field excitatory postsynaptic potentials in vivo in the hippocampal CA1 area showed that induction of cholinergic deficit decreased magnitude of long-term potentiation (LTP) and prevented a decrease in paired-pulse ratio after LTP induction, and NGF overexpression reversed these negative changes in hippocampal synaptic characteristics. The beneficial effect of NGF was not associated with compensatory changes in the number of cells that express NGF receptors TrkA and NGFR in the hippocampus and medial septal area. NGF overexpression also did not prevent a 192IgG-saporin–induced decrease in the activity of acetylcholine esterase in the hippocampus. We conclude that NGF overexpression in the hippocampus under conditions of cholinergic deficit induces beneficial effects which are not related to maintenance of cholinergic function.

Neurotrophins (NTs) and their signal-transducing Trk receptors play a crucial role in the development and maintenance of specific neuronal subpopulations in nervous and sensory systems. NTs are supposed to regulate two sensory systems in fish, the inner ear and the lateral line system (LLS). The latter is one of the major mechanosensory systems in fish. Considering that annual fishes of the genus Nothobranchius, with their short life expectancy, have become a suitable model for aging studies and that the occurrence and distribution of neurotrophin Trk receptors have never been investigated in the inner ear and LLS of killifish (Nothobranchius guentheri), our study aimed to investigate the localization of neurotrophin-specific Trk receptors in mechanosensory systems of N. guentheri. For histological and immunohistochemical analysis, adult specimens of N. guentheri were processed using antibodies against Trk receptors and S100 protein. An intense immunoreaction for TrkA and TrkC was found in the sensory cells of the inner ear as well as in the hair cells of LLS. Moreover, also the neurons localized in the acoustic ganglia displayed a specific immunoreaction for all Trk receptors (TrkA, B, and C) analyzed. Taken together, our results demonstrate, for the first time, that neurotrophins and their specific receptors could play a pivotal role in the biology of the sensory cells of the inner ear and LLS of N. guentheri and might also be involved in the hair cells regeneration process in normal and aged conditions.

12 páginas, 6 figuras. CSs fromTrkA-TMD and p75-TMD are deposited in the Biological Magnetic Resonance Data Bank with accession number 25872for TrkA-TMD and 19673 for p75-TMD., The neurotrophin receptors p75 and tyrosine protein kinase receptor A (TrkA) play important roles in the development and survival of the nervous system. Biochemical data suggest that p75 and TrkA reciprocally regulate the activities of each other. For instance, p75 is able to regulate the response of TrkA to lower concentrations of nerve growth factor (NGF), and TrkA promotes shedding of the extracellular domain of p75 by α-secretases in a ligand-dependent manner. The current model suggests that p75 and TrkA are regulated by means of a direct physical interaction; however, the nature of such interaction has been elusive thus far. Here, using NMR in micelles, multiscale molecular dynamics, FRET, and functional studies, we identified and characterized the direct interaction between TrkA and p75 through their respective transmembrane domains (TMDs). Molecular dynamics of p75-TMD mutants suggests that although the interaction between TrkA and p75 TMDs is maintained upon mutation, a specific protein interface is required to facilitate TrkA active homodimerization in the presence of NGF. The same mutations in the TMD protein interface of p75 reduced the activation of TrkA by NGF as well as reducing cell differentiation. In summary, we provide a structural model of the p75-TrkA receptor complex necessary for neuronal development stabilized by TMD interactions., This study was supported bythe Spanish Ministry of Economy and Competitiveness (project BFU2013-42746-P and SAF2017-84096-R), the GeneralitatValenciana Prometeo grant 2018/055 (to M. V.), and the National Institutes of Health GM068619 (to K. H.). NMR studies of TRKA-TM and p75-TM were supported by the Russian Science Founda-tion (grant no. 19-74-30014 to A. S. A.)

Alzheimer’s disease (AD) is the most common neurodegenerative disorder and results in severe neurodegeneration and progressive cognitive decline. Neurotrophins are growth factors involved in the development and survival of neurons, but also in underlying mechanisms for memory formation such as hippocampal long-term potentiation. Our aim was to identify small molecules with stimulatory effects on the signaling of two neurotrophins, the nerve growth factor (NGF) and the brain derived neurotrophic factor (BDNF). To identify molecules that could potentiate neurotrophin signaling, 25,000 molecules were screened, which led to the identification of the triazinetrione derivatives ACD855 (Ponazuril) and later on ACD856, as positive allosteric modulators of tropomyosin related kinase (Trk) receptors. ACD855 or ACD856 potentiated the cellular signaling of the neurotrophin receptors with EC50 values of 1.9 and 3.2 or 0.38 and 0.30 µM, respectively, for TrkA or TrkB. ACD855 increased acetylcholine levels in the hippocampus by 40% and facilitated long term potentiation in rat brain slices. The compounds acted as cognitive enhancers in a TrkB-dependent manner in several different behavioral models. Finally, the age-induced cognitive dysfunction in 18-month-old mice could be restored to the same level as found in 2-month-old mice after a single treatment of ACD856. We have identified a novel mechanism to modulate the activity of the Trk-receptors. The identification of the positive allosteric modulators of the Trk-receptors might have implications for the treatment of Alzheimer’s diseases and other diseases characterized by cognitive impairment.

JAK2–STAT3 and TrkA signaling pathways have been separately implicated in aggressive breast cancers, however, whether they are co-activated or undergo functional interaction has not been thoroughly investigated. Herein we report, for the first time that STAT3 and TrkA are significantly co-overexpressed and co-activated in triple-negative breast cancer (TNBC) and HER2-enriched breast cancer, as shown by immunohistochemical staining and data mining. Through immunofluorescence staining–confocal microscopy and immunoprecipitation–Western blotting, we found that TrkA and STAT3 co-localize and physically interact in the cytoplasm, and the interaction is dependent on STAT3-Y705 phosphorylation. TrkA–STAT3 interaction leads to STAT3 phosphorylation at Y705 by TrkA in breast cancer cells and cell-free kinase assays, indicating that STAT3 is a novel substrate of TrkA. β-NGF-mediated TrkA activation induces TrkA–STAT3 interaction, STAT3 nuclear transport and transcriptional activity, and the expression of STAT3 target genes, SOX2 and MYC. The co-activation of both pathways promotes breast cancer stem cells. Finally, we found that TNBC and HER2-enriched breast cancer with JAK2–STAT3 and TrkA co-activation are positively associated with poor overall metastasis-free and organ-specific metastasis-free survival. Collectively, our study uncovered that TrkA is a novel activating kinase of STAT3, and their co-activation enhances gene transcription and promotes breast cancer stem cells in TNBC and HER2-enriched breast cancer.

Cucurbitacin B (CuB) isolated from Cucumis melo by using a PC12 cell bioassay system exhibited significant nerve growth factor (NGF)‐mimic or NGF‐enhancing activity in PC12 and primary neuron cells. It was also demonstrated pro‐neurogenesis effects in ICR and APP/PS1 mice and improved memory deficit of APP/PS1 mice. Its possible mechanism includes significant induction of the phosphorylation of glucocorticoid receptor (GR), protein kinase C (PKC), phospholipase C (PLC) and inhibition of cofilin. ChemProteoBase profiling, binding assay and cellular thermal shift assay (CETSA) were used to determine the target protein. Results revealed that CuB could affect actin dynamics as an actin inhibitor but did not bind with GR. The protein level of cofilin in PC12 cells after treating 0.3 μM and different temperatures was significantly higher than that of control group. Other neurotrophic signalling pathways, such as TrkA/TrkB, were analysed with specific inhibitors and Western blot. The inhibitors of TrkA, PLC, PKC, Ras, Raf and ERK1/2 significantly decreased the percentage of PC12 cells with neurite outgrowth and shortened the length of neurite outgrowth induced by CuB. CuB significantly induced the phosphorylation of TrkA, ERK and CREB. The phosphorylation of these proteins was obviously decreased by their specific inhibitors. These results suggest that cofilin is a candidate target protein of CuB in PC12 cells and that the GR/PLC/PKC and TrkA/Ras/Raf/ERK signalling pathways play important roles in the neuroprotective effect of CuB.

During the development of the sympathetic nervous system, signals from tropomyosin-related kinase receptors (Trks) and p75 neurotrophin receptors (p75) compete to regulate survival and connectivity. During this process, nerve growth factor (NGF)- TrkA signaling in axons communicates NGF-mediated trophic responses in signaling endosomes. Whether axonal p75 signaling contributes to neuronal death and how signaling endosomes contribute to p75 signaling has not been established. Using compartmentalized sympathetic neuronal cultures (CSCGs) as a model, we observed that the addition of BDNF to axons increased the transport of p75 and induced death of sympathetic neurons in a dynein-dependent manner. In cell bodies, internalization of p75 required the activity of JNK, a downstream kinase mediating p75 death signaling in neurons. Additionally, the activity of Rab5, the key GTPase regulating early endosomes, was required for p75 death signaling. In axons, JNK and Rab5 were required for retrograde transport and death signaling mediated by axonal BDNF-p75 in CSCGs. JNK was also required for the proper axonal transport of p75-positive endosomes. Thus, our findings provide evidence that the activation of JNK by p75 in cell bodies and axons is required for internalization to a Rab5-positive signaling endosome and the further propagation of p75-dependent neuronal death signals.

Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools., Optical control over receptor tyrosine kinase (RTK) activity can find application in both basic biology and synthetic biology. Here the authors combine the photosensory module of DrBphP bacterial phytochrome with the Trk family of RTKs to obtain Trks that are reversibly switchable with near-infrared and far-red light.

Objective To evaluate the potential association of nerve growth factor (NGF) and its receptors TrkA and p75NRT with perineural invasion(PNI) and other clinicopathological features of early-stage cervical cancer. Methods We retrospectively analyzed the clinicopathological data of 60 patients with stage Ⅰb-Ⅱa2 cervical cancer treated with radical hysterectomy plus pelvic lymphadenectomy in the Affiliated Tumor Hospital, Guangxi Medical University from January 2010 to June 2014. Immunohistochemistry (IHC) was performed on surgical samples to analyze the expression of NGF, TrkA and p75; the correlation of their expression with perineural invasion and other clinicopathological features was analyzed. Results Perineural invasion was correlated strongly with the high expression of NGF (P=0.025) and TrkA (P=0.011). The rate of NGF-positive area and TrkA-positive area were significantly larger in PNI-positive patients than those in PNI-negative patients (P=0.016, 0.026, respectively). The expressions of NGF and TrkA were significantly associated with lymph-vascular space invasion(LVSI) (P=0.029, 0.019, respectively), but not associated with other clinicopathological features (P > 0.05). While p75 expression was not related to either perineural invasion or other clinicopathological features(P > 0.05). Conclusion High expressions of NGF and TrkA are associated with perineural invasion, which suggests that NGF and TrkA may participate in the related molecular mechanism of PNI.

Nerve growth factor (NGF), by binding to TrkA and p75(NTR) receptors, regulates the survival and differentiation of sensory neurons during development and mediates pain transmission and perception during adulthood, by acting at different levels of the nervous system. Key to understanding the role of NGF as a pain mediator is the finding that mutations (namely, R121W, V232fs and R221W) in the NGF gene cause painlessness disease Hereditary Sensory and Autonomic Neuropathy type V (HSAN V). Here we shall review the consequences of these NGF mutations, each of which results in specific clinical signs: R221W determines congenital pain insensitivity with no overt cognitive disabilities, whereas V232fs and R121W also result in intellectual disability, thus showing similarities to HSAN IV, which is caused by mutations in TrkA, rather than to HSAN V. Comparing the cellular, biochemical and clinical findings of these mutations could help in better understanding not only the possible mechanisms underlying HSAN V, but also mechanisms of NGF signalling and roles. These mutations alter the balance between NGF and proNGF in favour of an accumulation of the latter, suggesting a possible role of proNGF as a molecule with an analgesic role. Furthermore, the neurotrophic and pronociceptive functions of NGF are split by the R221W mutation, making NGF variants based on this mutation interesting for designing therapeutic applications for many diseases. This review emphasizes the possibility of using the mutations involved in "painlessness" clinical disorders as an innovative approach to identify new proteins and pathways involved in pain transmission and perception.Outstanding questions: Why do homozygous HSAN V die postnatally? What is the cause of this early postnatal lethality?Is the development of a mouse or a human feeling less pain affecting higher cognitive and perceptual functions?What is the consequence of the HSAN V mutation on the development of joints and bones? Are the multiple fractures observed in HSAN V patients due exclusively to the carelessness consequent to not feeling pain, or also to an intrinsic frailty of their bones?Are heterodimers of NGF(WT) and NGF(R221W) in the heterozygote state formed? And if so, what are the properties of these heterodimeric proteins?How is the processing of proNGF(R221W) to NGF(R221W) affected by the mutation?

Traumatic brain injury (TBI) remains the leading cause of long-term disability, which annually involves millions of individuals. Several studies on mammals reported that neurotrophins could play a significant role in both protection and recovery of function following neurodegenerative diseases such as stroke and TBI. This protective role of neurotrophins after an event of TBI has also been reported in the zebrafish model. Nevertheless, reparative mechanisms in mammalian brain are limited, and newly formed neurons do not survive for a long time. In contrast, the brain of adult fish has high regenerative properties after brain injury. The evident differences in regenerative properties between mammalian and fish brain have been ascribed to remarkable different adult neurogenesis processes. However, it is not clear if the specific role and time point contribution of each neurotrophin and receptor after TBI is conserved during vertebrate evolution. Therefore, in this review, I reported the specific role and time point of intervention for each neurotrophic factor and receptor after an event of TBI in zebrafish and mammals.

The patterning and ossification of the mammalian skeleton requires the coordinated actions of both intrinsic bone morphogens and extrinsic neurovascular signals, which function in a temporal and spatial fashion to control mesenchymal progenitor cell (MPC) fate. Here, we show the genetic inhibition of tropomyosin receptor kinase A (TrkA) sensory nerve innervation of the developing cranium results in premature calvarial suture closure, associated with a decrease in suture MPC proliferation and increased mineralization. In vitro, axons from peripheral afferent neurons derived from dorsal root ganglions (DRGs) of wild-type mice induce MPC proliferation in a spatially restricted manner via a soluble factor when cocultured in microfluidic chambers. Comparative spatial transcriptomic analysis of the cranial sutures in vivo confirmed a positive association between sensory axons and proliferative MPCs. SpatialTime analysis across the developing suture revealed regional-specific alterations in bone morphogenetic protein (BMP) and TGF-β signaling pathway transcripts in response to TrkA inhibition. RNA sequencing of DRG cell bodies, following direct, axonal coculture with MPCs, confirmed the alterations in BMP/TGF-β signaling pathway transcripts. Among these, the BMP inhibitor follistatin-like 1 (FSTL1) replicated key features of the neural-to-bone influence, including mitogenic and anti-osteogenic effects via the inhibition of BMP/TGF-β signaling. Taken together, our results demonstrate that sensory nerve-derived signals, including FSTL1, function to coordinate cranial bone patterning by regulating MPC proliferation and differentiation in the suture mesenchyme.

Background: Suicide Attempts are the main complications of Major Depressive Episodes and are difficult to predict. There is still a lack of knowledge about its neurochemical aspects. There is increasing evidence that Brain-derived neurotrophic factor (BDNF) and Nerve growth factor (NGF) play a role in the pathophysiology and treatment of depression by binding and activating cognate receptors Tyrosine Kinase B (TrkB) and Tyrosie Kinase A (TrkA), respectively. This study was conducted to examine whether BDNF and / or TrkB as well as NGF and / or TrkA expression profiles were changed in the hippocampus of postmortem brain of individuals with depression who committed suicide. Subjects and methods: This study was conducted with the brain tissue of 61 victims who died as a result of suicide due to depression and 25 people who died due to traffic accidents. The psychiatric history of the cases was determined by the psychological autopsy method. Samples were taken from the hippocampus region of the brain at the forensic medicine institution. After storage under appropriate conditions, protein and mRNA levels of BDNF, TrkB, NGF and TrkA were determined in the genetics laboratory. Results: Average age of the suicide group was 30 and the average age of the control group was 24.5. The suicide group consisted of 70.5% male and 29.5% female cases. There was no significant difference between the groups in terms of age (p=0.062) and gender (p=0.718). BDNF, NGF, TrkA and TrkB values were found to be lower in the suicide group compared to the control group and there was a significant difference between the groups (p=

In this article, we demonstrate and validate a new bioassay named the NTAB [NGF-TrkA (nerve growth factor-tropomyosin receptor kinase A) antagonist bioassay] for the determination of the inhibitory potency of NGF-TrkA antagonists, based on the inhibition of NGF-dependent proliferation of the human TF1 erythroleukemic cell line.It is well known that NGF holds great therapeutic potential due to its neurotrophic and neuroprotective properties. NGF is also involved in some pathways, however, principally driven by TrkA that, if not correctly regulated, can lead to unwanted pathological outcomes linked to pain, angiogenesis, and cancer.Indeed, there is an increasing interest, from a therapeutic perspective, in designing new effective molecules (antibodies, antibody fragments, or small molecules) able to inhibit the undesired NGF-TrkA pathway. For these reasons, there is an interest to develop functional cell-based assays for determination of the inhibition potency of compounds inhibiting the NGF-TrkA axis. The NTAB presents significant advantages over other published NGF-TrkA functional bioassays, for these reasons: (1) It is quantitative, (2) it measures a pure TrkA response, (3) it is simpler, (4) it is based on a natural biological response, and (5) it is easily scalable from a lab scale to an automated industrial assay.The NTAB assay was validated with a panel of well-characterized NGF-TrkA inhibitors, yielding characteristic dose-response curves, from which the relative strength of the inhibitors was quantitatively determined and used for comparisons. This new bioassay will be very useful to assist in the validation and prioritization of the best inhibitors among a large number of candidates.

Pain is a central feature of soft tissue trauma, which under certain contexts, results in aberrant osteochondral differentiation of tissue-specific stem cells. Here, the role of sensory nerve fibers in this abnormal cell fate decision is investigated using a severe extremity injury model in mice. Soft tissue trauma results in NGF (Nerve growth factor) expression, particularly within perivascular cell types. Consequently, NGF-responsive axonal invasion occurs which precedes osteocartilaginous differentiation. Surgical denervation impedes axonal ingrowth, with significant delays in cartilage and bone formation. Likewise, either deletion of Ngf or two complementary methods to inhibit its receptor TrkA (Tropomyosin receptor kinase A) lead to similar delays in axonal invasion and osteochondral differentiation. Mechanistically, single-cell sequencing suggests a shift from TGFβ to FGF signaling activation among pre-chondrogenic cells after denervation. Finally, analysis of human pathologic specimens and databases confirms the relevance of NGF-TrkA signaling in human disease. In sum, NGF-mediated TrkA-expressing axonal ingrowth drives abnormal osteochondral differentiation after soft tissue trauma. NGF-TrkA signaling inhibition may have dual therapeutic use in soft tissue trauma, both as an analgesic and negative regulator of aberrant stem cell differentiation., Soft tissue trauma can result in aberrant osteochondral differentiation of local mesenchymal progenitor cells. Here the authors show that, in mice, soft tissue trauma results in NGF expression by perivascular cells, which leads to axonal invasion and drives abnormal osteochondral differentiation, and show that this process can be prevented by inhibition of NGF signaling.

Basal forebrain cholinergic neurons (BFCNs) are critical for learning and memory. Profound and early BFCN degeneration is a hallmark of aging and Alzheimer’s disease (AD). BFCNs depend for their survival on the retrograde axonal transport of neurotrophins, proteins critical for neuronal function. Neurotrophins like brain derived neurotrophic factor (BDNF) and pro-nerve growth factor (proNGF) are retrogradely transported to BFCNs from their synaptic targets. In AD, neurotrophin levels are increased within BFCN target areas and reduced in the basal forebrain, implicating dysfunctional neurotrophin transport in AD pathogenesis. However, neurotrophin transport within this highly susceptible neuronal population is currently poorly understood. We began by establishing protocols for the accurate quantification of axonal transport in BFCNs using microfluidic culture. We then determined the effect of age on neurotrophin transport. BFCNs were left in culture for up to 3 weeks to model aging in vitro. BFCNs initially displayed robust neurotrophin transport, which diminished with in vitro age. We observed that the levels of proNGF receptor tropomyosin-related kinase-A (TrkA) were reduced in aged neurons. Additionally, neurotrophin transport in BFCNs derived from 3xTg-AD mice, an AD model, was also impaired. Next, we sought to determine a mechanism for these transport deficits. First, we determined that proNGF transport was solely contingent upon the levels of TrkA. We then found that elevation of oxidative stress, an established AD contributor, significantly reduced both TrkA levels and proNGF retrograde transport. TrkA levels are partially regulated by protein tyrosine phosphatase-1B (PTP1B), an enzyme whose activity is reduced by oxidation. PTP1B antagonism significantly reduced TrkA levels and proNGF retrograde transport in BFCNs. Treatment of BFCNs with PTP1B-activating antioxidants rescued TrkA levels, proNGF transport, and proNGF-mediated axonal degeneration. Our results suggest that oxidative stress contributes to BFCN degeneration in aging and AD by impairing retrograde neurotrophin transport via oxidative PTP1B-mediated TrkA loss. Thesis Doctor of Philosophy (PhD) During aging and Alzheimer’s disease (AD), the connections between neurons, a type of brain cell, break down, causing memory loss. This breakdown begins in a brain area called the basal forebrain. Basal forebrain neurons rely upon the transport of nutrients along their connections with other neurons, called axons, for proper function. This transport process becomes impaired in AD. Our goal was to understand why this happens. First, we determined that axonal transport was impaired with age and in basal forebrain neurons of mice genetically predisposed to develop AD. We recreated these impairments by increasing the levels of harmful molecules called reactive oxidative species (ROS). ROS levels increase with age and become abnormally high during AD. We found that increased ROS impair axonal transport and contribute to the breakdown of basal forebrain neurons. Our work suggests that reducing ROS will help prevent the breakdown of basal forebrain neurons in AD.

Background Glucose fluctuation promotes neuronal apoptosis, which plays a central role in diabetic encephalopathy (DE). Nerve growth factor (NGF), and its interaction with high-affinity (TrkA) and low-affinity (p75NTR) receptors, are involved in neuronal survival. NGF/TrkA contributes to the activation of the PI3K/AKT pathway, which is beneficial for neuronal survival, and α-Lipoic acid (ALA) exerts clinically favorable neuroprotective effects in the periphery. Whether NGF receptors and the PI3K/AKT pathway are involved in glucose fluctuation-induced neuronal damage, as well as the potential molecular mechanism of ALA in protecting glucose fluctuation-induced neuronal damage, remain unclear. Results The results indicated that constant high glucose (CHG) and intermittent high glucose (IHG) significantly increased the expression of Bax and caspase-3, and decreased the expression of TrkA/p75NTR and p-AKT/AKT, while ALA stimulation reversed the above proteins in PC12 cells. IHG stimulates apoptosis more effectively than CHG in PC12 cells, which is related to the PI3K/AKT pathway but not to the TrkA/p75NTR. Furthermore, neuronal apoptosis induced by IHG was aggravated by the TrkA inhibitor K252a or the PI3K/AKT inhibitor LY294002, but this effect was alleviated by the p75NTR inhibitor TAT-pep5. Conclusion Glucose fluctuation induced cell apoptosis by regulating the TrkA/p75NTR and PI3K/AKT pathway, meanwhile ALA exhibited neuroprotective effects in response to IHG and CHG. These observations indicated that the PI3K/AKT pathway and the balance of TrkA/p75NTR are likely to serve as potential therapeutic targets for DE. In addition, ALA could be a possible therapeutic drug for DE.

SUMMARY The flat bones of the skull are densely innervated during development, but little is known regarding their role during repair. We describe a neurotrophic mechanism that directs sensory nerve transit in the mouse calvaria. Patent cranial suture mesenchyme represents an NGF (nerve growth factor)-rich domain, in which sensory nerves transit. Experimental calvarial injury upregulates Ngf in an IL-1β/TNF-α-rich defect niche, with consequent axonal ingrowth. In calvarial osteoblasts, IL-1β and TNF-α stimulate Ngf and downstream NF-κB signaling. Locoregional deletion of Ngf delays defect site re-innervation and blunted repair. Genetic disruption of Ngf among LysM-expressing macrophages phenocopies these observations, whereas conditional knockout of Ngf among Pdgfra-expressing cells does not. Finally, inhibition of TrkA catalytic activity similarly delays re-innervation and repair. These results demonstrate an essential role of NGF-TrkA signaling in bone healing and implicate macrophage-derived NGF-induced ingrowth of skeletal sensory nerves as an important mediator of this repair., In Brief Meyers et al. describe the role of skeletal sensory nerves in cranial bone repair. The authors demonstrate several necessary aspects of membranous bone healing, including influx of nerve growth factor (NGF)-expressing macrophages after injury, followed by skeletal sensory nerve ingrowth to positively regulate bone repair., Graphical Abstract

In the last decade, Nerve Growth Factor (NGF)-based clinical approaches have lacked specific and efficient Tyrosine Kinase A (TrkA) agonists for brain delivery. Nowadays, the characterization of novel small peptidomimetic is taking centre stage in preclinical studies, in order to overcome the main size-related limitation in brain delivery of NGF holoprotein for Central Nervous System (CNS) pathologies. Here we investigated the NGF mimetic properties of the human NGF 1&ndash, 14 sequence (hNGF1&ndash, 14) and its derivatives, by resorting to primary cholinergic and dorsal root ganglia (DRG) neurons. Briefly, we observed that: 1) hNGF1&ndash, 14 peptides engage the NGF pathway through TrkA phosphorylation at tyrosine 490 (Y490), and activation of ShcC/PI3K and Plc-&gamma, /MAPK signalling, promoting AKT-dependent survival and CREB-driven neuronal activity, as seen by levels of the immediate early gene c-Fos, of the cholinergic marker Choline Acetyltransferase (ChAT), and of Brain Derived Neurotrophic Factor (BDNF), 2) their NGF mimetic activity is lost upon selective TrkA inhibition by means of GW441756, 3) hNGF1&ndash, 14 peptides are able to sustain DRG survival and differentiation in absence of NGF. Furthermore, the acetylated derivative Ac-hNGF1&ndash, 14 demonstrated an optimal NGF mimetic activity in both neuronal paradigms and an electrophysiological profile similar to NGF in cholinergic neurons. Cumulatively, the findings here reported pinpoint the hNGF1&ndash, 14 peptide, and in particular its acetylated derivative, as novel, specific and low molecular weight TrkA specific agonists in both CNS and PNS primary neurons.

Pancreatic cancer (PC) is a continuously high lethal disease, and the tumour microenvironment plays a pivotal role during PC progression. Herein, we focus on that the Nerve growth factor (NGF)/Tropomyosin‐related kinase A (TrkA), in pancreatic stellate cells‐pancreatic cancer cells (PSCs‐PC cells) co‐culture system, influences PC proliferation and invasion. The model of PC cells and PSCs was directly co‐cultured in a no‐touch manner, using the Transwell as the co‐culture system. NGF and TrkA expression was measured in cultured system by real‐time PCR, immunofluorescence, Western blotting analysis or ELISA. Small interfering RNA transfection was used to regulate the expression of TrkA in PC cells. The promotion of cancer invasion was investigated using Matrigel Transwell assay. In our study, NGF/TrkA is overexpressed in PSCs‐PC cells co‐culture system and promotes the invasion and proliferation of PC cells. And the epithelial‐mesenchymal transition‐related genes are influenced by si‐TrkA. What's more, NGF/TrkA regulates the PC cell proliferation and invasion via activation of PI3K/AKT/GSK signalling. The present study demonstrated NGF/TrkA promoted the PC cell proliferation and invasion in the co‐culture system by the activation of the PI3K/AKT/GSK signal cascade, providing a potential therapeutic target for PC patients.

BackgroundNerve growth factor (NGF) and its receptors, tropomyosin receptor kinase A (TrkA) and pan-neurotrophin receptor p75 (p75NTR), are known to play bidirectional roles between the immune and nervous system. There are only few studies with inconclusive results concerning the expression pattern and role of NGF, TrkA, and p75NTR (NGF system) under the neuroinflammatory conditions in multiple sclerosis (MS) and its mouse model, the experimental autoimmune encephalomyelitis (EAE). The aim of this study is to investigate the temporal expression in different cell types of NGF system in the central nervous system (CNS) during the EAE course.MethodsEAE was induced in C57BL/6 mice 6–8 weeks old. CNS tissue samples were collected on specific time points: day 10 (D10), days 20–22 (acute phase), and day 50 (chronic phase), compared to controls. Real-time PCR, Western Blot, histochemistry, and immunofluorescence were performed throughout the disease course for the detection of the spatio-temporal expression of the NGF system.ResultsOur findings suggest that both NGF and its receptors, TrkA and p75NTR, are upregulated during acute and chronic phase of the EAE model in the inflammatory lesions in the spinal cord. NGF and its receptors were co-localized with NeuN+cells, GAP-43+axons, GFAP+cells, Arginase1+cells, and Mac3+cells. Furthermore, TrkA and p75NTR were sparsely detected on CNPase+cells within the inflammatory lesion. Of high importance is our observation that despite EAE being a T-mediated disease, only NGF and p75NTR were shown to be expressed by B lymphocytes (B220+cells) and no expression on T lymphocytes was noticed.ConclusionOur results indicate that the components of the NGF system are subjected to differential regulation during the EAE disease course. The expression pattern of NGF, TrkA, and p75NTR is described in detail, suggesting possible functional roles in neuroprotection, neuroregeneration, and remyelination by direct and indirect effects on the components of the immune system.

International audience; Mechanical allodynia is a cardinal sign of several inflammatory pain disorders where Nerve Growth Factor, a prototypic neurotrophin, plays a crucial role by binding to TrkA receptors. Here, we took the advantage of our generated knock-in mouse model expressing a chimeric TrkA/TrkC receptor that seems to not specifically develop mechanical allodynia following inflammation, to identify the TrkA downstream pathways involved in this phenomenon. We confirmed and extended that disrupting TrkA specific pathways leads to a specific deficit in mechanical hypersensitivity development following somatic (systemic NGF administration and paw incision) and to a lesser extent, visceral injuries. Despite a deficit in thin, mainly peptidergic, fibres innervation in TrkAC mice, thermal hyperalgesia development was not different from WT mice. Inflammatory reaction (oedema, IL-6 content), pain behaviours following intraplantar capsaicin as well as TRPV1 calcium imaging response of DRG neurons were similar between TrkAC and WT mice. This deficiency in mechanical allodynia development in TrkAC mice is likely due to the alteration of the expression of different TrkA transduction pathways (i.e. Akt, p38 MAPK and c-Jun) especially p38 MAPK, in the DRG cell bodies, ultimately leading to an alteration of at least, ASIC3 channel overexpression, known to participate in nociceptor mechanosensory function.

[ES] TrkA es un receptor tirosina quinasa que pertenece a la familia de receptores Trk. Dicho receptor se encuentra principalmente en el sistema nervioso periférico, donde participa en el desarrollo y la supervivencia de las neuronas simpáticas y sensoriales. Además, también se encarga del desarrollo de las neuronas colinérgicas del sistema nervioso central y desempeña un papel importante en algunos tejidos no neurales, participando tanto en el desarrollo de células B como en el desarrollo cardíaco y vascular, entre otras funciones. Sin embargo, TrkA no solo resulta interesante por todas las funciones que tiene, sino también por su implicación en el cáncer. Originalmente, TrkA se aisló de un carcinoma de colon, aunque también ha aparecido en carcinomas papilares tiroideos y en casos de leucemia mieloide aguda. En estos cánceres, se puede observar una activación constitutiva del receptor, generalmente por fusiones de su dominio quinasa con el dominio extracelular de otras proteínas. No obstante, también se han descrito algunos casos en los que mutaciones puntuales en el dominio extracelular de TrkA inducen la transformación maligna de las células. Recientemente, en pacientes de diferentes tipos de cáncer se han detectado dos mutaciones distintas en el dominio extracelular de TrkA (S326C y R347C). La primera mutación (S326C) se ha detectado en un caso de cáncer de pulmón, mientras que la segunda (R347C), se ha detectado en un caso de cáncer de ciego del intestino grueso, otro de colon y en un caso de rabdomiosarcoma (cáncer del músculo estriado). Ambas mutaciones cambian un residuo por una cisteína y la forma en que estas mutaciones influyen en la activación de TrkA se desconoce. El objetivo de este trabajo es caracterizar dichas mutaciones, utilizando su expresión en células HeLa, y determinar cómo influyen estas en el desarrollo tumoral., [EN] TrkA is a tyrosine kinase receptor that belongs to the Trk family of receptors. This receptor is found mainly in the peripheral nervous system, where it participates in the development and survival of sympathetic and sensory neurons. In addition, it is also responsible for the development of cholinergic neurons of the central nervous system and plays an important role in some non-neural tissues, participating both in the development of B cells and in cardiac and vascular development, among other functions. However, TrkA is not only interesting for all the functions it has, but also for its involvement in cancer. TrkA was originally isolated from colon carcinoma, although it has also appeared in papillary thyroid carcinomas and in cases of acute myeloid leukemia. In these cancers, a constitutive activation of the receptor can be observed, generally by fusions of its kinase domain with the extracellular domain of other proteins. However, some cases have also been described in which point mutations in the TrkA extracellular domain induce malignant transformation of cells. Recently, two different mutations in the TrkA extracellular domain (S326C and R347C) have been detected in patients with different types of cancer. The first mutation (S326C) has been detected in a case of lung cancer, while the second one (R347C) has been detected in a case of caecum cancer, in a case of colon cancer and in a case of rhabdomyosarcoma (striated muscle cancer). Both mutations exchange a residue for a cysteine and the way in which these mutations influence TrkA activation is unknown. The objective of this work is to characterize these mutations, using their expression in Hela cells, and to determine how they influence tumor development.

Background A molecule identical to nerve growth factor, with ovulation-inducing properties has been discovered in the seminal plasma of South American camelids (ovulation-inducing factor/nerve growth factor; OIF/NGF). We hypothesize that the ovulatory effect of OIF/NGF is initiated at the level of the hypothalamus, presumably by GnRH neurons. The objective of the present study was to determine the structural relationship between GnRH neurons and neurons expressing high- and low-affinity receptors for NGF (i.e., TrkA and p75, respectively) in the hypothalamus. Methods Mature llamas (n = 4) were euthanized and their hypothalamic tissue was fixed, sectioned, and processed for immunohistochemistry on free-floating sections. Ten equidistant sections per brain were double stained for immunofluorescence detection of TrkA and GnRH, or p75 and GnRH. Results Cells immunoreactive to TrkA were detected in most hypothalamic areas, but the majority of cells were detected in the diagonal band of Broca (part of the ventral forebrain) and the supraoptic nuclei and periventricular area. The number of cells immunoreactive to p75 was highest in the diagonal band of Broca and lateral preoptic areas and least in more caudal areas of the hypothalamus (p

Sea cucumber glucocerebrosides (SCGs), an important sphingolipid, have extensive functions in inhibiting the growth of cancer cells, but their potential to promote neuronal outgrowth hasn’t been well studied. Herein, we evaluated the neuritogenic effects of three Cucumaria frondosa SCGs series (SCG-1, SCG-2, and SCG-3) in NGF-induced PC12 cells. These SCGs exhibited significant neuritogenic effects in a dose-dependent and structure-selective manner, which increased the ratio of neurite-bearing cells and expression of axonal (GAP-43) and synaptic (synaptophysin) proteins. Mechanistic studies suggested that SCGs reinforced the NGF-induced phosphorylation of TrkA, coupled with ERK1/2 activation, then triggered the activation of CREB, finally resulted in a marked increase of BDNF expression. Furthermore, the neuritogenic effects of SCGs could be suppressed when accompanied by the additions of TrkA inhibitor GW441765 or MEK inhibitor U0126. In conclusion, SCGs possess a marked neuritogenic effect that promotes the signalling downstream from TrkA, likely via the MEK/ERK1/2/CREB/BDNF pathway.

Nerve growth factor (NGF) exerts multiple functions on target neurons throughout development. The recent discovery of a point mutation leading to a change from arginine to tryptophan at residue 100 in the mature NGFβ sequence (NGFR100W) in patients with hereditary sensory and autonomic neuropathy, type V (HSAN V), made it possible to distinguish the signaling mechanisms that lead to two functionally different outcomes of NGF: trophic versus nociceptive. We performed extensive biochemical, cellular and live imaging experiments to examine the binding and signaling properties of NGFR100W. Our results show that, similar to the wildtype NGF (wtNGF), the naturally occurring NGFR100Wmutant was capable of binding to and activating the TrkA receptor and its downstream signaling pathways to support neuronal survival and differentiation. However, NGFR100Wfailed to bind and stimulate the 75kD neurotrophic factor receptor (p75NTR)-mediated signaling cascades (i.e. the RhoA-Cofilin pathway). Intraplantar injection of NGFR100Winto adult rats induced neither TrkA-mediated thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia based on agonism for TrkA signaling. Taken together, our studies provide evidence that NGFR100Wretains trophic support capability through TrkA and one aspect of its nociceptive signaling, but fails to engage p75NTRsignaling pathways. Our findings suggest that wtNGF acts through TrkA to regulate the delayed priming of nociceptive responses. The integration of both TrkA and p75NTRsignaling thus appears to regulate neuroplastic effects of NGF in peripheral nociception.Significance StatementIn the present study, we characterized the naturally occurring NGFR100Wmutant that is associated with hereditary sensory and autonomic neuropathy, type V. We have demonstrated for the first time that NGFR100Wretains trophic support capability through TrkA but fails to engage p75NTRsignaling pathways. Furthermore, following Intraplantar injection into adult rats, NGFR100Winduced neither thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia. We have also provided evidence that the integration of both TrkA-and p75NTR-mediated signaling thus appears to regulate neuroplastic effects of NGF in peripheral nociception. Our study with NGFR100Wsuggests that it is possible to uncouple trophic effect from nociceptive function, both induced by wildtype NGF.AbbreviationsNGFnerve growth factor;NGFR100WNGF mutation with a change from tryptophan (W) to arginine (R) at the 100 residue.TrkATropomyosin receptor kinase A;p75NTRthe 75kD neurotrophic factor receptor;HSAN Vhereditary sensory and autonomic neuropathy, type V;BFCNbasal forebrain cholinergic neurons;PNSperipheral nervous system;CNScentral nervous system;BDNFbrain-derived neurotrophic factor;NTneurotrophin;TrkBTropomyosin receptor kinase B;TrkCTropomyosin receptor kinase C;RhoARas homolog gene family, member A;NF-κBnuclear factor kappa B;AktProtein kinase B;JNKc-Jun N-terminal kinases;ADAlzheimer’s disease;CSFcerebrospinal fluid;HIVhuman immunodeficiency virusCIPAcongenital insensitivity to pain with anhidrosis;ERKextracellular signal-related kinase;PI3Kphosphatidylinositol 3-kinase;PLCγand phospholipase Cγ;PGE2Prostaglandin E2;PCRpolymerase chain reaction;GFPgreen fluorescent protein;QDquantum dots;HEK293FTHuman embryonic kidney 293FT cell line;KKENGF mutant protein with mutation at:K32A/K34A/E35A in the mature sequence;Δ9/13NGF mutant protein with deletion of N-terminal 9-13 residues in the mature sequence;DMEMDulbecco’s Modified Eagle’s Medium;PMSFphenylmethylsulfonyl fluoride;SDS-PAGEsodium dodecyl sulfate polyacrylamide gel electrophoresis;DMSOdimethyl sulfoxide;ASanti-sense oligos;MMmis-matched oligos;E15.5 DRGembryonic day 15.5 dorsal root ganglion;ECDextracellular domain;MEMMinimum Essential Medium;TIRFTotal Internal Reflection Fluorescence;EMCCDelectron multiplying charge-coupled device;PC12a cell line derived from a pheochromocytoma of the rat adrenal medulla;BSABovine Serum Albumin;PBSphosphate buffered saline;IgGImmunoglobulin G;DICDays In Culture

Summary Objective Aiming to delineate novel neuro-immune mechanisms for NGF/TrkA signalling in osteoarthritis (OA) pain, we evaluated inflammatory changes in the knee joints following injection of monoiodoacetate (MIA) in mice carrying a TrkA receptor mutation (P782S; TrkA KI mice). Method In behavioural studies we monitored mechanical hypersensitivity following intra-articular MIA and oral prostaglandin D 2 (PGD 2 ) synthase inhibitor treatments. In immunohistochemical studies we quantified joint mast cell numbers, calcitonin gene-related peptide expression in synovia and dorsal root ganglia, spinal cord neuron activation and microgliosis. We quantified joint leukocyte infiltration by flow cytometry analysis, and PGD 2 generation and cyclooxygenase-2 (COX-2) expression in mast cell lines by ELISA and Western blot. Results In TrkA KI mice we observed rapid development of mechanical hypersensitivity and amplification of dorsal horn neurons and microglia activation 7 days after MIA. In TrkA KI knee joints we detected significant leukocyte infiltration and mast cells located in the vicinity of synovial nociceptive fibres. We demonstrated that mast cells exposure to NGF results in up-regulation of COX-2 and increase of PGD 2 production. Finally, we observed that a PGD 2 synthase inhibitor prevented MIA-mechanical hypersensitivity in TrkA KI, at doses which were ineffective in wild type (WT) mice. Conclusion Using the TrkA KI mouse model, we delineated a novel neuro-immune pathway and suggest that NGF-induced production of PGD 2 in joint mast cells is critical for referred mechanical hypersensitivity in OA, probably through the activation of PGD 2 receptor 1 in nociceptors: TrkA blockade in mast cells constitutes a potential target for OA pain.

Neurotrophin receptors such as the tropomyosin receptor kinase A receptor (TrkA) and the low-affinity binding p75 neurotrophin receptor p75NTR play a critical role in neuronal survival and their functions are altered in Alzheimer’s disease (AD). Changes in the dynamics of receptors on the plasma membrane are essential to receptor function. However, whether receptor dynamics are affected in different pathophysiological conditions is unexplored. Using live-cell single-molecule imaging, we examined the surface trafficking of TrkA and p75NTR molecules on live neurons that were derived from human-induced pluripotent stem cells (hiPSCs) of presenilin 1 (PSEN1) mutant familial AD (fAD) patients and non-demented control subjects. Our results show that the surface movement of TrkA and p75NTR and the activation of TrkA- and p75NTR-related phosphoinositide-3-kinase (PI3K)/serine/threonine-protein kinase (AKT) signaling pathways are altered in neurons that are derived from patients suffering from fAD compared to controls. These results provide evidence for altered surface movement of receptors in AD and highlight the importance of investigating receptor dynamics in disease conditions. Uncovering these mechanisms might enable novel therapies for AD.

Background Aging is associated with some cognitive decline and enhanced risk of development of neurodegenerative diseases. It is assumed that altered metabolism and functions of neurotrophin systems may underlie these age-related functional and structural modifications. CerebrolysinTM (CBL) is a neuropeptide mixture with neurotrophic effects, which is widely used for the treatment of stroke and traumatic brain injury patients. It is also evident that CBL has an overall beneficial effect and a favorable benefit-risk ratio in patients with dementia. However, the effects of CBL on cognition and brain neurotrophin system in normal aging remain obscure. Objective The aim of the present study was to examine the age-related modifications of endogenous neurotrophin systems in the brain of male Wistar rats and the effects of CBL on learning and memory as well as the levels neurotrophins and their receptors. Methods Old (23-24 months) and young (2-3 months) male Wistar rats were used for the study. A half of animals were subjected to CBL course (2.5 ml/kg, 20 i.p. injections). Behavior of rats was studied using the open field test and simple water maze training. The contents of NGF and BDNF were studied using enzyme-linked immunosorbent assay; the expression of neurotrophin receptors was estimated by Western-blot analysis. Results CBL treatment did not affect general status, age-related weight changes, general locomotor activity as well as general brain histology. In a water maze task, a minor effect of CBL was observed in old rats at the start of training and no effect on memory retention was found. Aging induced a decrease in neurotrophin receptors TrkA, TrkB, and p75NTR in the neocortex. CBL counteracted effects of aging on neocortical TrkA and p75NTR receptors and decreased expression of proNGF without influencing overall NGF levels. BDNF system was not significantly affected by CBL. Conclusion The pro-neuroplastic "antiaging" effects of CBL in the neocortex of old animals were generally related to the NGF rather than the BDNF system.

Neurotrophins constitute a family of growth factors initially characterized as predominant mediators of nervous system development, neuronal survival, regeneration and plasticity. Their biological activity is promoted by the binding of two different types of receptors, leading to the generation of multiple and variegated signaling cascades in the target cells. Increasing evidence indicates that neurotrophins are also emerging as crucial regulators of metabolic processes in both neuronal and non-neuronal cells. In this context, it has been reported that neurotrophins affect redox balance, autophagy, glucose homeostasis and energy expenditure. Additionally, the trophic support provided by these secreted factors may involve the regulation of cholesterol metabolism. In this review, we examine the neurotrophins’ signaling pathways and their effects on metabolism by critically discussing the most up-to-date information. In particular, we gather experimental evidence demonstrating the impact of these growth factors on cholesterol metabolism.

Osteoarthritis (OA) is the most common form of articular joint arthritis and a cause of significant morbidity. In this review, we present the role of nerve growth factor (NGF) in pain generation, relationship between NGF and OA pain, and pathogenic factors (interleukin-1β, transforming growth factor-β1, mechanical loading, and adipokines) involved in OA development. Since NGF blocking is an efficient way to inhibit OA-associated pain, we summarize four categories of drugs that target NGF/tropomyosin receptor kinase A (TrkA) signaling. In addition, we discuss the future of NGF/TrkA antagonists and underline their potential for use in OA pain relief. A better understanding of the causes and treatment of OA will facilitate the development of more effective methods of OA pain management.