Nerve growth factor receptor limits inflammation to promote remodeling and repair of osteoarthritic joints.

Osteoarthritis (OA) is a painful, incurable disease affecting over 500 million people. Recent clinical trials of the nerve growth factor (NGF) inhibitors in OA patients have suggested adverse effects of NGF inhibition on joint structure. Here we report that nerve growth factor receptor (NGFR) is upregulated in skeletal cells during OA and plays an essential role in the remodeling and repair of osteoarthritic joints. Specifically, NGFR is expressed in osteochondral cells but not in skeletal progenitor cells and induced by TNFα to attenuate NF-κB activation, maintaining proper BMP-SMAD1 signaling and suppressing RANKL expression in mice. NGFR deficiency hyper-activates NF-κB in murine osteoarthritic joints, which impairs bone formation and enhances bone resorption as exemplified by a reduction in subchondral bone and osteophytes. In human OA cartilage, NGFR is also negatively associated with NF-κB activation. Together, this study suggests a role of NGFR in limiting inflammation for repair of diseased skeletal tissues.

ProNGF processing in adult rat tissues and bioactivity of NGF prodomain peptides.

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.

Neurotrophins and Trk Neurotrophin Receptors in the Retina of Adult Killifish (Nothobranchius guentheri).

Specific subpopulations of neurons in nerve and sensory systems must be developed and maintained, and this is accomplished in significant part by neurotrophins (NTs) and the signaling receptors on which they act, called tyrosine protein kinase receptors (Trks). The neurotrophins-tyrosine protein kinase receptors (NTs/Trks) system is involved in sensory organ regulation, including the visual system. An NTs/Trks system alteration is associated with neurodegeneration related to aging and diseases, including retinal pathologies. An emergent model in the field of translational medicine, for instance, in aging study, is the annual killifish belonging to the Nothobranchius genus, thanks to its short lifespan. Members of this genus, such as Nothobranchius guentheri, and humans share a similar retinal stratigraphy. Nevertheless, according to the authors' knowledge, the occurrence and distribution of the NTs/Trks system in the retina of N. guentheri has never been investigated before. Therefore, the present study aimed to localize neurotrophin BDNF, NGF, and NT-3 and TrkA, TrkB, and TrkC receptors in the N. guentheri retina using the immunofluorescence method. The present investigation demonstrates, for the first time, the occurrence of the NTs/Trks system in N. guentheri retina and, consequently, the potential key role of these proteins in the biology and survival of the retinal cells.

NGFR regulates stromal cell activation in germinal centers.

Nerve growth factor receptor (NGFR) is expressed by follicular dendritic cells (FDCs). However, the role of NGFR in the humoral response is not well defined. Here, we study the effect of Ngfr loss on lymph node organization and function, demonstrating that Ngfr depletion leads to spontaneous germinal center (GC) formation and an expansion of the GC B cell compartment. In accordance with this effect, stromal cells are altered in Ngfr-/- mice with a higher frequency of FDCs, characterized by CD21/35, MAdCAM-1, and VCAM-1 overexpression. GCs are located ectopically in Ngfr-/- mice, with lost polarization together with impaired high-affinity antibody production and an increase in circulating autoantibodies. We observe higher levels of autoantibodies in Bcl2 Tg/Ngfr-/- mice, concomitant with a higher incidence of autoimmunity and lower overall survival. Our work shows that NGFR is involved in maintaining GC structure and function, participating in GC activation, antibody production, and immune tolerance.

Heterogeneity-induced NGF-NGFR communication inefficiency promotes mitotic spindle disorganization in exhausted T cells through PREX1 suppression to impair the anti-tumor immunotherapy with PD-1 mAb in hepatocellular carcinoma.

BACKGROUND: The mechanism of decreased T cells infiltrating tumor tissues in hepatocellular carcinoma is poorly understood. METHODS: Cells were separated from the single-cell RNA-sequence dataset of hepatocellular carcinoma patients (GSE149614) for cell-cell communication. Flow cytometry, EDU staining, H3-Ser28 staining, confocal immunofluorescence staining, western blotting and naked microsubcutaneous tumors were performed for the mechanism of NGF-NGFR promoting proliferation. RESULTS: The present study has revealed that during the process of T-cell infiltration from adjacent tissues to tumor tissues, an inefficiency in NGF-NGFR communication occurs in the tumor tissues. Importantly, NGF secreted by tumor cells interacts with NGFR present on the membranes of the infiltrated T cells, thereby promoting the proliferation through the activation of mitotic spindle signals. Mechanistically, the mediation of mitotic spindle signal activation promoting proliferation is executed by HDAC1-mediated inhibition of unclear trans-localization of PREX1. Furthermore, PD-1 mAb acts synergistically with the NGF-NGFR communication to suppress tumor progression in both mouse models and HCC patients. Additionally, NGF-NGFR communication was positively correlates with the PD-1/PDL-1 expression. However, expressions of NGF and NGFR are low in tumor tissues, which is responsible for the invasive clinicopathological features and the disappointing prognosis in HCC patients. CONCLUSION: Inefficiency in NGF-NGFR communication impairs PD-1 mAb immunotherapy and could thus be utilized as a novel therapeutic target in the treatment of HCC patients in clinical practice.

A positive feedback between PDIA3P1 and OCT4 promotes the cancer stem cell properties of esophageal squamous cell carcinoma.

BACKGROUND: Increasing evidence has indicated that long non-coding RNAs (lncRNAs) have been proven to regulate esophageal cancer progression. The lncRNA protein disulfide isomerase family A member 3 pseudogene 1 (PDIA3P1) has been shown to promote cancer stem cell properties; however, its mechanism of action remains unclear. In this study, we investigated the regulation of esophageal cancer stem cell properties by the interaction of PDIA3P1 with proteins. METHODS: The GEPIA2 and Gene Expression Omnibus databases were used to analyze gene expression. PDIA3P1 expression in human esophageal squamous cell carcinoma (ESCC) tissues and cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Loss-of-function experiments were performed to determine the effects of PDIA3P1 on ESCC cell proliferation, migration, and invasion. The sphere formation assay, number of side population cells, and CD271 + /CD44 + cells were detected by flow cytometry to identify the cancer stem cell properties. RNA immunoprecipitation (RIP), RNA pull-down, co-immunoprecipitation (co-IP), dual luciferase reporter, and cleavage under targets and tagmentation (CUT&Tag) assays were performed to elucidate the underlying molecular mechanisms. RESULTS: PDIA3P1 expression was upregulated in ESCC cell lines and tissues. Functionally, higher PDIA3P1 expression promoted cell proliferation, invasion, and metastasis and inhibited apoptosis in esophageal cancer. Importantly, PDIA3P1 promoted cancer stem cell properties in ESCC. Mechanistically, PDIA3P1 interacted with and stabilized octamer-binding transcription factor 4 (OCT4) by eliminating its ubiquitination by the ubiquitinating enzyme WW domain-containing protein 2 (WWP2). Moreover, as a transcription factor, OCT4 bound to the PDIA3P1 promoter and promoted its transcription. CONCLUSIONS: Our research revealed a novel mechanism by which a positive feedback loop exists between PDIA3P1 and OCT4. It also demonstrated that the PDIA3P1-WWP2-OCT4 loop is beneficial for promoting the cancer stem cell properties of ESCC. Owing to this regulatory relationship, the PDIA3P1-WWP2-OCT4-positive feedback loop might be used in the diagnosis and prognosis, as well as in the development of novel therapeutics for esophageal cancer.

Upregulation of proBDNF/p75NTR signaling in immune cells and its correlation with inflammatory markers in patients with major depression.

ProBDNF is the precursor protein of brain-derived neurotrophic factor (BDNF) expressed in the central nervous system and peripheral tissues. Previous studies showed that the blood levels of both proBDNF and p75 neurotrophic receptors (p75NTR) in major depressive disorder (MDD) were increased, but which blood cell types express proBDNF and its receptors is not known. Furthermore, the relationship between proBDNF/p75NTR and inflammatory cytokines in peripheral blood of MDD is unclear. Peripheral blood mononuclear cells (PBMCs) and serum were obtained from depressive patients (n = 32) and normal donors (n = 20). We examined the expression of proBDNF and inflammatory markers and their correlative relationship in patients with major depression. Using flow cytometry analysis, we examined which blood cells express proBDNF and its receptors. Finally, the role of proBDNF/p75NTR signal in inflammatory immune activity of PBMCs was verified in vitro experiments. Inflammatory cytokines in PBMC from MDD patients were increased and correlated with the major depression scores. The levels of IL-1ß and IL-10 were also positively correlated with the major depression scores, while the levels of TNF-α and IL-6 were negatively correlated with the major depression scores. Intriguingly, the levels of sortilin were positively correlated with IL-1ß. Q-PCR and Western blots showed proBDNF, p75NTR, and sortilin levels were significantly increased in PBMCs from MDD patients compared with that from the normal donors. Flow cytometry studies showed that proBDNF and p75NTR were present mainly in CD4+ and CD8+ T cells. The number of proBDNF and p75NTR positive CD4+ and CD8+ T cells from MDD patients was increased and subsequently reversed after therapeutic management. Exogenous proBDNF protein or p75ECD-Fc treatment of cultured PBMC affected the release of inflammatory cytokines in vitro. ProBDNF promoted the expression of inflammatory cytokines, while p75ECD-Fc inhibited the expression of inflammatory cytokines. Given there was an inflammatory response of lymphocytes to proBDNF, it is suggested that proBDNF/p75NTR signaling may upstream inflammatory cytokines in MDD. Our data suggest that proBDNF/p75NTR signaling may not only serve as biomarkers but also may be a potential therapeutic target for MDD.

The Impact of ROS and NGF in the Gliomagenesis and their Emerging Implications in the Glioma Treatment.

Reactive oxygen species (ROS) are highly reactive molecules derived from molecular oxygen (O2). ROS sources can be endogenous, such as cellular organelles and inflammatory cells, or exogenous, such as ionizing radiation, alcohol, food, tobacco, chemotherapeutical agents and infectious agents. Oxidative stress results in damage of several cellular structures (lipids, proteins, lipoproteins, and DNA) and is implicated in various disease states such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging. A large body of studies showed that ROS plays an important role in carcinogenesis. Indeed, increased production of ROS causes accumulation in DNA damage leading to tumorigenesis. Various investigations demonstrated the involvement of ROS in gliomagenesis. The most common type of primary intracranial tumor in adults is represented by glioma. Furthermore, there is growing attention on the role of the Nerve Growth Factor (NGF) in brain tumor pathogenesis. NGF is a growth factor belonging to the family of neurotrophins. It is involved in neuronal differentiation, proliferation and survival. Studies were conducted to investigate NGF pathogenesis's role as a pro- or anti-tumoral factor in brain tumors. It has been observed that NGF can induce both differentiation and proliferation in cells. The involvement of NGF in the pathogenesis of brain tumors leads to the hypothesis of a possible implication of NGF in new therapeutic strategies. Recent studies have focused on the role of neurotrophin receptors as potential targets in glioma therapy. This review provides an updated overview of the role of ROS and NGF in gliomagenesis and their emerging role in glioma treatment.

CD271 promotes proliferation and migration in bladder cancer.

Bladder cancer is a urothelial cancer and effective therapeutic strategies for its advanced stages are limited. Here, we report that CD271, a neurotrophin receptor, promotes the proliferation and migration of bladder cancer cells. CD271 knockdown decreased proliferation in both adherent and spheroid cultures, and vice versa when CD271 was overexpressed in bladder cancer cell lines. CD271 depletion impaired tumorigenicity in vivo. Migration activity was reduced by CD271 knockdown and TAT-Pep5, a known CD271-Rho GDI-binding inhibitor. Apoptosis was induced by CD271 knockdown. Comprehensive gene expression analysis revealed alterations in E2F- and Myc-related pathways upon CD271 expression. In clinical cases, patients with high CD271 expression showed significantly shortened overall survival. In surgically resected specimens, pERK, a known player in proliferation signaling, colocalizes with CD271. These data indicate that CD271 is involved in bladder cancer malignancy by promoting cell proliferation and migration, resulting in poor prognosis.

Genetic and Pharmacological Modulation of P75 Neurotrophin Receptor Attenuate Brain Damage After Ischemic Stroke in Mice.

The precursor nerve growth factor (ProNGF) and its receptor p75 neurotrophin receptor (p75NTR) are upregulated in several brain diseases, including ischemic stroke. The activation of p75NTR is associated with neuronal apoptosis and inflammation. Thus, we hypothesized that p75NTR modulation attenuates brain damage and improves functional outcomes after ischemic stroke. Two sets of experiments were performed. (1) Adult wild-type (WT) C57BL/6 J mice were subjected to intraluminal suture-middle cerebral artery occlusion (MCAO) to induce cerebral ischemia. Pharmacological inhibitor of p75NTR, LM11A-31 (50 mg/kg), or normal saline was administered intraperitoneally (IP) 1 h post-MCAO, and animals survived for 24 h. (2) Adult p75NTR heterozygous knockout (p75NTR+/-) and WT were subjected to photothrombotic (pMCAO) to induce ischemic stroke, and the animals survived for 72 h. The sensory-motor function of animals was measured using Catwalk XT. The brain samples were collected to assess infarction volume, edema, hemorrhagic transformation, neuroinflammation, and signaling pathway at 24 and 72 h after the stroke. The findings described that pharmacological inhibition and genetic knocking down of p75NTR reduce infarction size, edema, and hemorrhagic transformation following ischemic stroke. Additionally, p75NTR modulation significantly decreased several anti-apoptosis markers and improved sensory motor function compared to the WT mice following ischemic stroke. Our observations exhibit that the involvement of p75NTR in ischemic stroke and modulation of p75NTR could improve the outcome of ischemic stroke by increasing cell survival and enhancing motor performance. LM11A-31 has the potential to be a promising therapeutic agent for ischemic stroke. However, more evidence is needed to illuminate the efficacy of LM11A-31 in ischemic stroke.

ISX-9 Promotes KGF Secretion From MSCs to Alleviate ALI Through NGFR-ERK-TAU-ß-Catenin Signaling Axis.

BACKGROUND: Mesenchymal stem cells (MSCs) have been widely studied to alleviate acute lung injury (ALI) due to their paracrine function. However, the microenvironment of inflammatory outbreaks significantly restricted the factors secreted from MSCs like keratinocyte growth factor (KGF). KGF is a growth factor with tissue-repaired ability. Is there a better therapeutic prospect for MSCs in combination with compounds that promote their paracrine function? Through compound screening, we screened out isoxazole-9 (ISX-9) to promote MSCs derived KGF secretion and investigated the underlying mechanisms of action. METHODS: Compounds that promote KGF secretion were screened by a dual-luciferase reporter gene assay. The TMT isotope labeling quantitative technique was used to detect the differential proteins upon ISX-9 administrated to MSCs. The expressions of NGFR, ERK, TAU, and ß-catenin were detected by Western blot. In the ALI model, we measured the inflammatory changes by HE staining, SOD content detection, RT-qPCR, immunofluorescence, etc. The influence of ISX-9 on the residence time of MSCs transplantation was explored by optical in vivo imaging. RESULTS: We found out that ISX-9 can promote the expression of KGF in MSCs. ISX-9 acted on the membrane receptor protein NGFR, upregulated phosphorylation of downstream signaling proteins ERK and TAU, downregulated phosphorylation of ß-catenin, and accelerated ß-catenin into the nucleus to further increase the expression of KGF. In the ALI model, combined ISX-9 with MSCs treatments upgraded the expression of KGF in the lung, and enhanced the effect of MSCs in reducing inflammation and repairing lung damage compared with MSCs alone. CONCLUSIONS: ISX-9 facilitated the secretion of KGF from MSCs both in vivo and in vitro. The combination of ISX-9 with MSCs enhanced the paracrine function and anti-inflammatory effect of MSCs compared with MSCs applied alone in ALI. ISX-9 played a contributive role in the transplantation of MSCs for the treatment of ALI.

A Vicious NGF-p75NTR Positive Feedback Loop Exacerbates the Toxic Effects of Oxidative Damage in the Human Retinal Epithelial Cell Line ARPE-19.

In spite of its variety of biological activities, the clinical exploitation of human NGF (hNGF) is currently limited to ocular pathologies. It is therefore interesting to test the effects of hNGF in preclinical models that may predict their efficacy and safety in the clinical setting of ocular disorders and compare the effects of hNGF with those of its analogs. We used a human retinal pigment cell line, ARPE-19 cells, to investigate the effects of hNGF and its analogs, mouse NGF (mNGF) and painless NGF (pNGF), on cell viability under basal conditions and after exposure to oxidative stimuli, i.e., hydrogen peroxide (H2O2) and ultraviolet (UV)-A rays. The effects of hNGF and pNGF were also tested on the gene expression and protein synthesis of the two NGF receptor subtypes, p75 neurotrophic receptors (p75NTR) and tyrosine kinase A (TrkA) receptors. We drew the following conclusions: (i) the exposure of ARPE-19 cells to H2O2 or UV-A causes a dose-dependent decrease in the number of viable cells; (ii) under baseline conditions, hNGF, but not pNGF, causes a concentration-dependent decrease in cell viability in the range of doses 1-100 ng/mL; (iii) hNGF, but not pNGF, significantly potentiates the toxic effects of H2O2 or of UV-A on ARPE-19 cells in the range of doses 1-100 ng/mL, while mNGF at the same doses presents an intermediate behavior; (iv) 100 ng/mL of hNGF triggers an increase in p75NTR expression in H2O2-treated ARPE-19 cells, while pNGF at the same dose does not; (v) pNGF, but not hNGF (both given at 100 ng/mL), increases the total cell fluorescence intensity for TrkA receptors in H2O2-treated ARPE-19 cells. The present findings suggest a vicious positive feedback loop through which NGF-mediated upregulation of p75NTR contributes to worsening the toxic effects of oxidative damage in the human retinal epithelial cell line ARPE-19. Looking at the possible clinical relevance of these findings, one can postulate that pNGF might show a better benefit/risk ratio than hNGF in the treatment of ocular disorders.

BDNF-TrkB/proBDNF-p75NTR pathway regulation by lipid emulsion rescues bupivacaine-induced central neurotoxicity in rats.

Bupivacaine (BPV) can cause severe central nervous system toxicity when absorbed into the blood circulation system. Rapid intravenous administration of lipid emulsion (LE) could be used to treat local anaesthetic toxicity. This study aimed to investigate the mechanism by which the BDNF-TrkB/proBDNF-p75NTR pathway regulation by LE rescues BPV induced neurotoxicity in hippocampal neurons in rats. Seven- to nine-day-old primary cultured hippocampal neurons were randomly divided into 6 groups: the blank control group (Ctrl), the bupivacaine group (BPV), the lipid emulsion group (LE), the bupivacaine + lipid emulsion group (BPV + LE), the bupivacaine + lipid emulsion + tyrosine kinase receptor B (TrkB) inhibitor group (BPV + LE + K252a), the bupivacaine + lipid emulsion + p75 neurotrophic factor receptor (p75NTR) inhibitor group (BPV + LE + TAT-Pep5). All hippocampal neurons were incubated for 24 h, and their growth state was observed by light microscopy. The relative TrkB and p75NTR mRNA levels were detected by real-time PCR. The protein expression levels of brain-derived neurotrophic factor (BDNF), proBDNF, TrkB, p75NTR and cleaved caspase-3 were detected by western blotting. The results showed that primary hippocampal neuron activity was reduced by BPV. As administration of LE elevated hippocampal neuronal activity, morphology was also somewhat improved. The protein expression and mRNA levels of TrkB and p75NTR were decreased when BPV induced hippocampal neuronal toxicity, while the expression of BDNF was increased. At the same time, BPV increased the original generation of cleaved caspase-3 protein content by hippocampal neurons, while the content of cleaved caspase-3 protein in hippocampal neurons cotreated with LE and BPV was decreased. Thus, this study has revealed LE may reduce apoptosis and promote survival of hippocampal neurons by regulating the BDNF-TrkB pathway and the proBDNF-p75NTR pathway to rescue BPV induced central neurotoxicity in rats.

Investigating In Situ Expression of Neurotrophic Factors and Partner Proteins in Irreversible Pulpitis.

INTRODUCTION: In situ assessments of neurotrophic factors and their associated molecular partners have not been explored to date, particularly in humans. The present investigation aimed to explore the expressional dysregulation of neurotrophic factors (nerve growth factor [NGF], brain derived neurotrophic factor [BDNF], and NT4/5), their receptors (TrkA and TrkB), and their modulators (USP36 and Nedd4-2) directly in irreversibly inflamed human pulp tissues. METHODS: Forty samples each of healthy and irreversibly inflamed pulp were extirpated for the study. Immunohistochemical examinations were carried out for the anatomic changes and expression of neurotrophic factors and partner proteins. Expression was digitally quantified using the IHC profiler module of ImageJ and deduced as optical density. Statistical analyses were carried out by GraphPad Prism. RESULTS: Decrease in nuclear and vessel diameters was observed in irreversibly inflamed pulp tissues. NGF and BDNF were found to be significantly upregulated in symptomatic irreversible pulpitis (SIP), whereas no significant difference was observed in the expression of TrkA and TrkB. Expression of Nedd4-2, USP36, and TrkA was found positively correlated with the NGF in healthy pulp tissues. However, in SIP, positive correlation was only observed between the expression of USP36 and NGF. Among the ligands, BDNF expression was found positively correlated with NGF in healthy pulp but not with NT4/5. In the case of SIP, no correlation was observed between any neurotrophic factors. CONCLUSIONS: Upregulation of NGF, BDNF, USP36 and Nedd4-2 in SIP indicates dysregulation in the molecular events underlying the disease biology and could be exploited as potential markers for the disease diagnosis.

Cortical Brain Injury Causes Retrograde Degeneration of Afferent Basal Forebrain Cholinergic Neurons via the p75NTR.

Traumatic brain injury (TBI) elicits neuronal loss at the site of injury and progressive neuronal loss in the penumbra. However, the consequences of TBI on afferent neurons projecting to the injured tissue from distal locations is unknown. Basal forebrain cholinergic neurons (BFCNs) extend long projections to multiple brain regions including the cortex, regulate many cognitive functions, and are compromised in numerous neurodegenerative disorders. To determine the consequence of cortical injury on these afferent neurons, we used the fluid percussion injury model of traumatic brain injury and assessed the effects on BFCN survival and axon integrity in male and female mice. Survival or death of BF neurons can be regulated by neurotrophins or proneurotrophins, respectively. The injury elicited an induction of proNGF and proBDNF in the cortex and a loss of BFCNs ipsilateral to the injury compared with sham uninjured mice. The p75NTR knock-out mice did not show loss of BFCN neurons, indicating a retrograde degenerative effect of the cortical injury on the afferent BFCNs mediated through p75NTR. In contrast, locus ceruleus neurons, which also project throughout the cortex, were unaffected by the injury, suggesting specificity in retrograde degeneration after cortical TBI. Proneurotrophins (proNTs) provided directly to basal forebrain axons in microfluidic cultures triggered retrograde axonal degeneration and cell death, which did not occur in the absence of p75NTR. This study shows that after traumatic brain injury, proNTs induced in the injured cortex promote BFCN axonal degeneration and retrograde neuron loss through p75NTR.

Knockdown of neurotrophin receptor-interacting melanoma-associated antigen homolog inhibits acute myeloid leukemia cell growth via the ERK pathway.

Neurotrophin receptor-interacting melanoma-associated antigen homolog (NRAGE), a type II melanoma-associated antigen, plays a critical role in cell processes that are involved in the tumorigenesis of various cancers. However, the effect of NRAGE on acute myeloid leukemia (AML) is rarely reported. The expression of NRAGE in AML tissues and the survival rates between different AML groups were obtained from the GEPIA tool. Human AML cell lines were cultured and transfected with siRNA targeting NRAGE. The ability of AML cells to proliferate and cell cycle were examined. Western blotting was performed to detect the activity of the extracellular signal-regulated kinase (ERK) signaling pathway in AML cells. NRAGE expression was enhanced in AML tissues relative to control tissues, and the high NRAGE expression in AML patients is associated with a poor prognosis. The capacity of AML cells to survive and proliferate was significantly decreased and its cell cycle was arrested at the G1 phase after NRAGE was silenced. Furthermore, silencing NRAGE induced the inactivation of the ERK signaling pathway. Furthermore, supplement of tert-Butylhydroquinone, an ERK activator, improved the reduced ability of AML cell survival and proliferation as well as cell cycle arrest induced by NRAGE knockdown. In this study, NRAGE was identified as a tumor promoter in AML, which had an effect on cell proliferation, cell survival, and cell cycle through the ERK signaling pathway in AML cells.

Transcription Pattern of Neurotrophic Factors and Their Receptors in Adult Zebrafish Spinal Cord.

In vertebrates, neurotrophins and their receptors play a fundamental role in the central and peripheral nervous systems. Several studies reported that each neurotrophin/receptor signalling pathway can perform various functions during axon development, neuronal growth, and plasticity. Previous investigations in some fish species have identified neurotrophins and their receptors in the spinal cord under physiological conditions and after injuries, highlighting their potential role during regeneration. In our study, for the first time, we used an excellent animal model, the zebrafish (Danio rerio), to compare the mRNA localization patterns of neurotrophins and receptors in the spinal cord. We quantified the levels of mRNA using qPCR, and identified the transcription pattern of each neurotrophin/receptor pathway via in situ hybridization. Our data show that ngf/trka are the most transcribed members in the adult zebrafish spinal cord.

AraC interacts with p75NTR transmembrane domain to induce cell death of mature neurons.

Cytosine arabinoside (AraC) is one of the main therapeutic treatments for several types of cancer, including acute myeloid leukaemia. However, after a high-dose AraC chemotherapy regime, patients develop severe neurotoxicity and cell death in the central nervous system leading to cerebellar ataxia, dysarthria, nystagmus, somnolence and drowsiness. AraC induces apoptosis in dividing cells. However, the mechanism by which it leads to neurite degeneration and cell death in mature neurons remains unclear. We hypothesise that the upregulation of the death receptor p75NTR is responsible for AraC-mediated neurodegeneration and cell death in leukaemia patients undergoing AraC treatment. To determine the role of AraC-p75NTR signalling in the cell death of mature neurons, we used mature cerebellar granule neurons' primary cultures from p75NTR knockout and p75NTRCys259 mice. Evaluation of neurite degeneration, cell death and p75NTR signalling was done by immunohistochemistry and immunoblotting. To assess the interaction between AraC and p75NTR, we performed cellular thermal shift and AraTM assays as well as Homo-FRET anisotropy imaging. We show that AraC induces neurite degeneration and programmed cell death of mature cerebellar granule neurons in a p75NTR-dependent manner. Mechanistically, Proline 252 and Cysteine 256 residues facilitate AraC interaction with the transmembrane domain of p75NTR resulting in uncoupling of p75NTR from the NFκB survival pathway. This, in turn, exacerbates the activation of the cell death/JNK pathway by recruitment of TRAF6 to p75NTR. Our findings identify p75NTR as a novel molecular target to develop treatments for counteract AraC-mediated cell death of mature neurons.

Neurotrophin Analog ENT-A044 Activates the p75 Neurotrophin Receptor, Regulating Neuronal Survival in a Cell Context-Dependent Manner.

Neuronal cell fate is predominantly controlled based on the effects of growth factors, such as neurotrophins, and the activation of a variety of signaling pathways acting through neurotrophin receptors, namely Trk and p75 (p75NTR). Despite their beneficial effects on brain function, their therapeutic use is compromised due to their polypeptidic nature and blood-brain-barrier impermeability. To overcome these limitations, our previous studies have proven that DHEA-derived synthetic analogs can act like neurotrophins, as they lack endocrine side effects. The present study focuses on the biological characterization of a newly synthesized analog, ENT-A044, and its role in inducing cell-specific functions of p75NTR. We show that ENT-A044 can induce cell death and phosphorylation of JNK protein by activating p75NTR. Additionally, ENT-A044 can induce the phosphorylation of TrkB receptor, indicating that our molecule can activate both neurotrophin receptors, enabling the protection of neuronal populations that express both receptors. Furthermore, the present study demonstrates, for the first time, the expression of p75NTR in human-induced Pluripotent Stem Cells-derived Neural Progenitor Cells (hiPSC-derived NPCs) and receptor-dependent cell death induced via ENT-A044 treatment. In conclusion, ENT-A044 is proposed as a lead molecule for the development of novel pharmacological agents, providing new therapeutic approaches and research tools, by controlling p75NTR actions.

Generation of Human 293-F Suspension NGFR Knockout Cells Using CRISPR/Cas9 Coupled to Fluorescent Protein Expression.

Suspension cells derived from human embryonic kidney cells (HEK 293) are attractive cell lines for retroviral vector production in gene therapeutic development studies and applications. The low-affinity nerve growth factor receptor (NGFR) is a genetic marker frequently used as a reporter gene in transfer vectors to detect and enrich genetically modified cells. However, the HEK 293 cell line and its derivatives endogenously express the NGFR protein. To eradicate the high background NGFR expression in future retroviral vector packaging cells, we here employed the CRISPR/Cas9 system to generate human suspension 293-F NGFR knockout cells. The expression of a fluorescent protein coupled via a 2A peptide motif to the NGFR targeting Cas9 endonuclease enabled the simultaneous depletion of cells expressing Cas9 and remaining NGFR-positive cells. Thus, a pure population of NGFR-negative 293-F cells lacking persistent Cas9 expression was obtained in a simple and easily applicable procedure.