The expression of ProBDNF and its high affinity receptor P75NTR in the neurons of emotion-related brain regions of post-stroke depression rats.

OBJECTIVE: To investigate the expression of the precursor of brain-derived neurotrophic factor (proBDNF) and its high-affinity receptor p75NTR in neurons of emotion-related brain areas (prefrontal cortex, hippocampus, and amygdala) in rats with post-stroke depression (PSD), and to explore the expression levels of proBDNF and p75NTR in neurons of emotion-related brain areas by injecting tissue plasminogen activator (t-PA) into the lateral ventricle of PSD rats, this significantly improved the stress-induced depression-like behavior,thus further validating the above results. METHODS: Rats were randomly divided into four groups: a normal control group (n = 8), a depression group (n = 8), a stroke group (n = 8), and a PSD group (n = 8). The rat model of stroke was established by thread embolism, and the PSD animal model was induced by chronic unpredictable mild stress (CUMS) and solitary feeding. Behavioral tests were conducted, including weight measurement, open field tests, and sucrose preference tests. Immunofluorescence double labeling was used to detect the expression of proBDNF and p75NTR in neurons of emotion-related brain regions in the PSD rat model. Four weeks after CUMS treatment, the PSD group was selected. Rats were infused with t-PA (3 µg dissolved in 6 µL saline, Boehringer Ingelheim), proBDNF (3 µg dissolved in 6 µL saline, Abcam), or equal-volume NS once per day for 7 consecutive days using the syringe pump connecting to injection needles. After 7 days of continuous administration, animal behavior was assessed through scoring, and the expression of proBDNF and p75NTR in the emotion-related brain regions of the PSD rat model was detected using immunofluorescence double labeling. RESULTS: Compared with the normal control group and the stroke group, the body weight, sucrose water consumption, and vertical movement distance in the PSD group were significantly lower (P < 0.05). In contrast, when compared with the proBDNF injection group and saline injection group, the weight, sucrose water consumption, field horizontal movement, and vertical movement distance of the t-PA injection group significantly increased after PSD lateral ventricle intubation.Double immunofluorescence revealed a higher neuronal expression of proBDNF as well as p75NTR in the prefrontal cortex and hippocampus of PSD rats compared to control animals (P < 0.05). In the amygdala, the expression levels of proBDNF and P75NTR were significantly reduced in the PSD group compared to the control group (P < 0.05). The results of the expression levels of proBDNF and P75NTR in the emotion-related brain regions of PSD rats injected with t-PA showed that proBDNF and P75NTR was significantly reduced in the prefrontal cortex, hippocampus, and amygdala of PSD rats compared to those of the NS and proBDNF groups (P < 0.05). CONCLUSIONS: The increased expression of the brain-derived neurotrophic factor precursor proBDNF and its receptor p75NTR in neurons of emotion-related brain regions may play an important role in the pathogenesis of PSD.t-PA reduced the expression of proBDNF and its receptor p75NTR in neurons emotion-related brain regions and significantly improved the stress-induced depression-like behavior. Therefore, it is reasonable to assume that exogenous injection of t-PA may alleviate the depressive symptoms of PSD patients.Reducing the expression of proBDNF by injecting t-PA may provide a novel therapeutic approach for the treatment of stress-related mood disorders.

Breaking NGF-TrkA immunosuppression in melanoma sensitizes immunotherapy for durable memory T cell protection.

Melanoma cells, deriving from neuroectodermal melanocytes, may exploit the nervous system's immune privilege for growth. Here we show that nerve growth factor (NGF) has both melanoma cell intrinsic and extrinsic immunosuppressive functions. Autocrine NGF engages tropomyosin receptor kinase A (TrkA) on melanoma cells to desensitize interferon γ signaling, leading to T and natural killer cell exclusion. In effector T cells that upregulate surface TrkA expression upon T cell receptor activation, paracrine NGF dampens T cell receptor signaling and effector function. Inhibiting NGF, either through genetic modification or with the tropomyosin receptor kinase inhibitor larotrectinib, renders melanomas susceptible to immune checkpoint blockade therapy and fosters long-term immunity by activating memory T cells with low affinity. These results identify the NGF-TrkA axis as an important suppressor of anti-tumor immunity and suggest larotrectinib might be repurposed for immune sensitization. Moreover, by enlisting low-affinity T cells, anti-NGF reduces acquired resistance to immune checkpoint blockade and prevents melanoma recurrence.

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.

Etiological Roles of p75NTR in a Mouse Model of Wet Age-Related Macular Degeneration.

Choroidal neovascularization (CNV) is a pathological angiogenesis of the choroidal plexus of the retina and is a key feature in the wet form of age-related macular degeneration. Mononuclear phagocytic cells (MPCs) are known to accumulate in the subretinal space, generating a chronic inflammatory state that promotes the growth of the choroidal neovasculature. However, how the MPCs are recruited and activated to promote CNV pathology is not fully understood. Using genetic and pharmacological tools in a mouse model of laser-induced CNV, we demonstrate a role for the p75 neurotrophin receptor (p75NTR) in the recruitment of MPCs, in glial activation, and in vascular alterations. After laser injury, expression of p75NTR is increased in activated Muller glial cells near the CNV area in the retina and the retinal pigmented epithelium (RPE)-choroid. In p75NTR knockout mice (p75NTR KO) with CNV, there is significantly reduced recruitment of MPCs, reduced glial activation, reduced CNV area, and the retinal function is preserved, as compared to wild type mice with CNV. Notably, a single intravitreal injection of a pharmacological p75NTR antagonist in wild type mice with CNV phenocopied the results of the p75NTR KO mice. Our results demonstrate that p75NTR is etiological in the development of CNV.

p75NTR enhances cognitive dysfunction in a mouse Alzheimer's disease model by inhibiting microRNA-210-3p-mediated PCYT2 through activation of NF-κB.

Alzheimer's disease (AD) is a main cause of dementia and exhibits abnormality in cognitive behaviors. Here, we probed into the role of p75 neurotrophin receptor (p75NTR) in cognitive dysfunction in AD. Primarily, C57BL/6 mouse and neuroblastoma cells were treated by amyloid-beta1-42 (Aß1-42), respectively, to establish the in vivo and in vitro models of AD. The downstream genes of p75NTR were predicted by RNA-sequencing and bioinformatics analysis. Then the interaction among p75NTR, nuclear factor kappa B (NF-κB), microRNA-210-3p (miR-210-3p) and phosphoethanolamine cytidylyltransferase 2 (PYCT2) was verified, followed by analysis of their effects on cognitive behaviors and biological characteristics of hippocampal neurons of mouse with AD-like symptoms. p75NTR knockout alleviated cognitive dysfunction in mice with AD-like symptoms and reduced Aß1-42-induced hippocampal neuron damage and apoptosis. p75NTR up-regulated miR-210-3p expression by activating NF-κB, thereby limiting PCYT2 expression. PCYT2 silencing in p75NTR-/- mice promoted neuronal apoptosis and aggravated cognitive dysfunction in AD mouse models. In summary, p75NTR is capable of accelerating cognitive dysfunction in AD by mediating the NF-κB/miR-210-3p/PCYT2 axis.

Human immunomodulatory ligand B7-1 mediates synaptic remodeling via the p75 neurotrophin receptor.

Cell surface receptors, ligands, and adhesion molecules underlie development, circuit formation, and synaptic function of the central nervous system and represent important therapeutic targets for many neuropathologies. The functional contributions of interactions between cell surface proteins of neurons and nonneuronal cells have not been fully addressed. Using an unbiased protein-protein interaction screen, we showed that the human immunomodulatory ligand B7-1 (hB7-1) interacts with the p75 neurotrophin receptor (p75NTR) and that the B7-1:p75NTR interaction is a recent evolutionary adaptation present in humans and other primates, but absent in mice, rats, and other lower mammals. The surface of hB7-1 that engages p75NTR overlaps with the hB7-1 surface involved in CTLA-4/CD28 recognition, and these molecules directly compete for binding to p75NTR. Soluble or membrane-bound hB7-1 altered dendritic morphology of cultured hippocampal neurons, with loss of the postsynaptic protein PSD95 in a p75NTR-dependent manner. Abatacept, an FDA-approved therapeutic (CTLA-4-hFc fusion) inhibited these processes. In vivo injection of hB7-1 into the murine subiculum, a hippocampal region affected in Alzheimer's disease, resulted in p75NTR-dependent pruning of dendritic spines. Here, we report the biochemical interaction between B7-1 and p75NTR, describe biological effects on neuronal morphology, and identify a therapeutic opportunity for treatment of neuroinflammatory diseases.

Epigenetic Regulation of NGF-Mediated Osteogenic Differentiation in Human Dental Mesenchymal Stem Cells.

Nerve growth factor (NGF) is the best-characterized neurotrophin and is primarily recognized for its key role in the embryonic development of the nervous system and neuronal cell survival/differentiation. Recently, unexpected actions of NGF in bone regeneration have emerged as NGF is able to enhance the osteogenic differentiation of mesenchymal stem cells. However, little is known regarding how NGF signaling regulates osteogenic differentiation through epigenetic mechanisms. In this study, using human dental mesenchymal stem cells (DMSCs), we demonstrated that NGF mediates osteogenic differentiation through p75NTR, a low-affinity NGF receptor. P75NTR-mediated NGF signaling activates the JNK cascade and the expression of KDM4B, an activating histone demethylase, by removing repressive H3K9me3 epigenetic marks. Mechanistically, NGF-activated c-Jun binds to the KDM4B promoter region and directly upregulates KDM4B expression. Subsequently, KDM4B directly and epigenetically activates DLX5, a master osteogenic gene, by demethylating H3K9me3 marks. Furthermore, we revealed that KDM4B and c-Jun from the JNK signaling pathway work in concert to regulate NGF-mediated osteogenic differentiation through simultaneous recruitment to the promoter region of DLX5. We identified KDM4B as a key epigenetic regulator during the NGF-mediated osteogenesis both in vitro and in vivo using the calvarial defect regeneration mouse model. In conclusion, our study thoroughly elucidated the molecular and epigenetic mechanisms during NGF-mediated osteogenesis.

Expression of NGF/proNGF and Their Receptors TrkA, p75NTR and Sortilin in Melanoma.

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

P75NTR Exacerbates SCI-induced Mitochondrial Damage and Neuronal Apoptosis Depending on NTRK3.

OBJECTIVE: The aim of the study was to investigate the mechanism by which p75 neurotrophin receptor (p75NTR) affects mitochondrial damage and neuronal apoptosis in spinal cord injury (SCI). METHODS: After the establishment of SCI rat models, short hairpin (sh) RNA of p75NTR and control sh-RNA were injected into SCI rats, respectively. On days 1, 7 and 21 after SCI, the severity of SCI and cell apoptosis in SCI rats were determined as well as the recovery of hind limb performance and p75NTR expression. After spinal cord neurons were transfected with p75NTR overexpression plasmid or empty plasmid vector or cotransfected with overexpression plasmids of p75NTR and neurotrophic tyrosine receptor kinase3 (NTRK3), the expression levels of p75NTR and NTRK3 were quantified. Moreover, we detected the apoptosis and proliferation rates of the neurons in addition to the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in the neurons. The binding between p75NTR and NTRK3 was confirmed via Co-immunoprecipitation (Co-IP). RESULTS: The rat spinal cords in the Model group were notably damaged after SCI accompanied by increased apoptosis and decreased locomotor function. The expression of p75NTR was significantly upregulated after SCI. The aforementioned injuries were remarkably ameliorated in response to injection of sh-p75NTR. p75NTR overexpression induced mitochondrial damage and neuronal apoptosis in spinal cord neurons, while the promotive effects were perturbed by NTRK3 overexpression. Furthermore, p75NTR directly bound to and downregulated NTRK3. CONCLUSION: Both in vivo and in vitro experiments showed that p75NTR aggravates mitochondrial damage and neuronal apoptosis in SCI through downregulating NTRK3.

The evolved divergence of γ-secretase-susceptibility of homologous proteins Ngfrb and Nradd in zebrafish.

OBJECTIVE: NGFR/p75NTR and NRADD/NRH proteins are closely related structurally and are encoded by genes that arose from a duplication event early in vertebrate evolution. The transmembrane domain (TMD) of NGFR is cleaved by γ-secretase but there is conflicting data around the susceptibility to γ-secretase cleavage of NRADD proteins. If NGFR and NRADD show differential susceptibility to γ-secretase, then they can be used to dissect the structural constraints determining substrate susceptibility. We sought to test this differential susceptibility. RESULTS: We developed labelled, lumenally-truncated forms of zebrafish Ngfrb and Nradd and a chimeric protein in which the TMD of Nradd was replaced with the TMD of Ngfrb. We expressed these in zebrafish embryos to test their susceptibility to γ-secretase cleavage by monitoring their stability using western immunoblotting. Inhibition of γ-secretase activity using DAPT increased the stability of only the Ngfrb construct. Our results support that only NGFR is cleaved by γ-secretase. Either NGFR evolved γ-secretase-susceptibility since its creation by gene duplication, or NRADD evolved to be refractory to γ-secretase. Protein structure outside of the TMD of NGFR is likely required for susceptibility to γ-secretase.

Small Endogenous Ligands Modulation of Nerve Growth Factor Bioactivity: A Structural Biology Overview.

Experiments with cell cultures and animal models have provided solid support for the assumption that Nerve Growth Factor (NGF) plays a key role in the regulation of neuronal cell survival and death. Recently, endogenous ligands have been proposed as physiological modulators of NGF biological activity as part of this regulatory cascade. However, the structural and mechanistic determinants for NGF bioactivity remain to be elucidated. We recently unveiled, by an integrated structural biology approach, the ATP binding sites of NGF and investigated the effects on TrkA and p75NTR receptors binding. These results pinpoint ATP as a genuine endogenous modulator of NGF signaling, paving the way to the characterization of not-yet-identified chemical diverse endogenous biological active small molecules as novel modulators of NGF. The present review aims at providing an overview of the currently available 3D structures of NGF in complex with different small endogenous ligands, featuring the molecular footprints of the small molecules binding. This knowledge is essential for further understanding the functional role of small endogenous ligands in the modulation of neurotrophins signaling in physiological and pathological conditions and for better exploiting the therapeutic potentialities of NGF.

Evidence of p75 Neurotrophin Receptor Involvement in the Central Nervous System Pathogenesis of Classical Scrapie in Sheep and a Transgenic Mouse Model.

Neurotrophins constitute a group of growth factor that exerts important functions in the nervous system of vertebrates. They act through two classes of transmembrane receptors: tyrosine-kinase receptors and the p75 neurotrophin receptor (p75NTR). The activation of p75NTR can favor cell survival or apoptosis depending on diverse factors. Several studies evidenced a link between p75NTR and the pathogenesis of prion diseases. In this study, we investigated the distribution of several neurotrophins and their receptors, including p75NTR, in the brain of naturally scrapie-affected sheep and experimentally infected ovinized transgenic mice and its correlation with other markers of prion disease. No evident changes in infected mice or sheep were observed regarding neurotrophins and their receptors except for the immunohistochemistry against p75NTR. Infected mice showed higher abundance of p75NTR immunostained cells than their non-infected counterparts. The astrocytic labeling correlated with other neuropathological alterations of prion disease. Confocal microscopy demonstrated the co-localization of p75NTR and the astrocytic marker GFAP, suggesting an involvement of astrocytes in p75NTR-mediated neurodegeneration. In contrast, p75NTR staining in sheep lacked astrocytic labeling. However, digital image analyses revealed increased labeling intensities in preclinical sheep compared with non-infected and terminal sheep in several brain nuclei. This suggests that this receptor is overexpressed in early stages of prion-related neurodegeneration in sheep. Our results confirm a role of p75NTR in the pathogenesis of classical ovine scrapie in both the natural host and in an experimental transgenic mouse model.

Effect of Aloe vera on the expression of nerve factors, p75 and TrkA receptors in the hippocampus of diabetic rats / Efecto del Aloe vera sobre la expresión de factores de crecimiento nervioso, Receptores p75 y TrkA en el hipocampo de ratas diabéticas

SUMMARY: Diabetes mellitus can lead to structural disorders in the brain. One of the most common complications of diabetes, diabetic neuropathy is associated with central nervous system disorders. Aloe vera has anti-diabetic, antioxidant, and neuroprotective effects. This study was designed to evaluate the effects of Aloe vera gel on the hippocampus changes as well as the expression of nerve growth factor and receptors TrkA and P75 in the hippocampus of streptozotocin (STZ)-induced diabetic rats. 25 male Wistar rats were randomly divided into 5 groups including: control (normal saline), diabetic (normal saline), Aloe vera gel (400 mg/kg/day; gavage), diabetic + Aloe vera gel (400 mg/kg/day; gavage) and diabetic + insulin NPH (10 IU/kg/day; subcutaneous). Experimental diabetes was induced by streptozotocin injection (60 mg/kg; intraperitoneal). All groups treated for 8 weeks. At the end of treatment course, the rat brains were removed for measuring the expression of nerve growth factor, p75 and TrkA receptors were evaluated in the hippocampus. Diabetes induction after 8 weeks caused NGF and P75 expression levels in the diabetic group than other groups significantly increased (p<0.05). The TrkA receptor expression in the diabetic group compared with the control had a significant reduction (p<0.05). On the other hand in the diabetic group receiving Aloe vera gel expression of NGF and P75 expression levels compared to the diabetic group was significantly reduced (p<0.05) and the TrkA receptor expression compared with the diabetic group had a significant increase (p<0.05). The results showed that oral administration of Aloe vera gel in diabetic rats ameliorates diabetes-induced hyperglycemia. On the other hand, Aloe vera gel cause modulation of the expression of NGF neurotrophic factor via increased expression of TrkA receptor-specific and non-specific receptor down-regulation of P75 in the hippocampus of STZ-induced diabetic rats.

RESUMEN: La diabetes mellitus puede provocar trastornos estructurales en el cerebro. Es una de las complicaciones más comunes de la diabetes y la neuropatía diabética y está relacionada con trastornos del sistema nervioso central. El Aloe vera tiene efectos antidiabéticos, antioxidantes y neuroprotectores. Este estudio fue diseñado para evaluar los efectos del gel de Aloe vera en los cambios del hipocampo, así como la expresión del factor de crecimiento nervioso y los receptores TrkA y P75 en el hipocampo de ratas diabéticas inducidas por estreptozotocina (STZ). Se dividieron al azar 25 ratas Wistar macho en 5 grupos de: control (solución salina normal), diabéticos (solución salina normal), gel de Aloe vera (400 mg / kg / día; sonda), diabéticos + gel de Aloe vera (400 mg / kg / día; sonda) y diabéticos + insulina NPH (10 UI / kg / día; subcutánea). La diabetes experimental fue inducida por inyección de estreptozotocina (60 mg / kg; intraperitoneal). Todos los grupos fueron tratados durante 8 semanas. Al final del tratamiento, se extrajeron los cerebros de las ratas para medir la expresión del factor de crecimiento nervioso y se evaluaron los receptores p75 y TrkA en el hipocampo. La inducción de diabetes después de 8 semanas provocó que los niveles de expresión de NGF y P75 en el grupo de diabéticos aumentaran significativamente en comparación con otros grupos (p <0,05). La expresión del receptor TrkA en el grupo diabético comparado con el control tuvo una reducción significativa (p <0,05). Por otro lado, el grupo de ratas diabéticas que recibieron la expresión en gel de Aloe vera de NGF y los niveles de expresión de P75 en comparación con el grupo de ratas diabéticas se redujo significativamente (p <0,05) y la expresión del receptor de TrkA en comparación con el grupo de ratas diabéticas tuvo un aumento significativo (p <0,05). Los resultados mostraron que la administración oral de gel de Aloe vera en ratas diabéticas mejora la hiperglucemia inducida por la diabetes. Por otro lado, el gel de Aloe vera causa modulación de la expresión del factor neurotrófico NGF a través del aumento de la expresión de receptor TrkA específico y no específico y regulación negativa del receptor de P75 en el hipocampo de ratas diabéticas inducidas por STZ.

Activation of skeletal muscle-resident glial cells upon nerve injury.

Here, we report on the identification of Itga7-expressing muscle-resident glial cells activated by loss of neuromuscular junction (NMJ) integrity. Gene expression analysis at the bulk and single-cell level revealed that these cells are distinct from Itga7-expressing muscle satellite cells. We show that a selective activation and expansion of Itga7+ glial cells occur in response to muscle nerve lesion. Upon activation, muscle glial-derived progenies expressed neurotrophic genes, including nerve growth factor receptor, which enables their isolation by FACS. We show that activated muscle glial cells also expressed genes potentially implicated in extracellular matrix remodeling at NMJs. We found that tenascin C, which was highly expressed by muscle glial cells, activated upon nerve injury and preferentially localized to NMJ. Interestingly, we observed that the activation of muscle glial cells by acute nerve injury was reversible upon NMJ repair. By contrast, in a mouse model of ALS, in which NMJ degeneration is progressive, muscle glial cells steadily increased over the course of the disease. However, they exhibited an impaired neurotrophic activity, suggesting that pathogenic activation of glial cells may be implicated in ALS progression.

Inhibiting the MNK1/2-eIF4E axis impairs melanoma phenotype switching and potentiates antitumor immune responses.

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8+ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti-PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.

Small molecule modulation of the p75 neurotrophin receptor suppresses age- and genotype-associated neurodegeneration in HIV gp120 transgenic mice.

The persistence of HIV in the central nervous system leads to cognitive deficits in up to 50% of people living with HIV even with systemic suppression by antiretroviral treatment. The interaction of chronic inflammation with age-associated degeneration places these individuals at increased risk of accelerated aging and other neurodegenerative diseases and no treatments are available that effectively halt these processes. The adverse effects of aging and inflammation may be mediated, in part, by an increase in the expression of the p75 neurotrophin receptor (p75NTR) which shifts the balance of neurotrophin signaling toward less protective pathways. To determine if modulation of p75NTR could modify the disease process, we treated HIV gp120 transgenic mice with a small molecule ligand designed to engage p75NTR and downregulate degenerative signaling. Daily treatment with 50 mg/kg LM11A-31 for 4 months suppressed age- and genotype-dependent activation of microglia, increased microtubule associated protein-2 (MAP-2), reduced dendritic varicosities and slowed the loss of parvalbumin immunoreactive neurons in the hippocampus. An age related accumulation of microtubule associated protein Tau was identified in the hippocampus in extracellular clusters that co-expressed p75NTR suggesting a link between Tau and p75NTR. Although the significance of the relationship between p75NTR and Tau is unclear, a decrease in Tau-1 immunoreactivity as gp120 mice entered old age (>16 months) suggests that the Tau may transition to more pathological modifications; a process blocked by LM11A-31. Overall, the effects of LM11A-31 are consistent with strong neuroprotective and anti-inflammatory actions that have significant therapeutic potential.

p75NTR silencing inhibits proliferation, migration, and extracellular matrix deposition of hypertrophic scar fibroblasts by activating autophagy through inhibiting the PI3K/Akt/mTOR pathway.

Hypertrophic scar (HS) results from abnormal wound healing, accompanied by excessive hypercellularity, migration, and extracellular matrix (ECM) deposition. Autophagy dysregulation plays crucial roles during HS formation. The overexpressed p75 neurotrophin receptor (p75NTR) in injured skin tissue after wound healing becomes a factor aggravating scar. This study was designed to investigate the role of p75NTR and p75NTR-mediated autophagy in the process of HS. The results revealed that p75NTR expression was significantly upregulated while that of autophagy proteins was downregulated in cicatrix at 3 and 6 months after a burn, which was recovered at 12 months. p75NTR silencing inhibited proliferation, migration, and ECM deposition of hypertrophic scar fibroblasts (HSF), whereas p75NTR overexpression presented the opposite results. Silencing of p75NTR reduced the expression of PI3K/Akt/mTOR signaling molecules while enhancing that of autophagy proteins. Importantly, PI3K agonist (IGF-1) intervention notably decreased the levels of LC3B II/I and Beclin-1 and restored the inhibitory effects of p75NTR silencing on proliferation, migration, and ECM deposition of HSF. Concurrently, autophagy inhibitor 3-methyladenine (3-MA) treatment exhibited the same variation trends with IGF-1. Taken together, these findings demonstrated that p75NTR silencing inhibits proliferation, migration, and ECM deposition of HSF by activating autophagy by inhibiting the PI3K/Akt/mTOR pathway.

Neuroanatomical basis of the nerve growth factor ovulation-induction pathway in llamas†.

The objective of the study was to characterize the anatomical framework and sites of action of the nerve growth factor (NGF)-mediated ovulation-inducing system of llamas. The expression patterns of NGF and its receptors in the hypothalamus of llamas (n = 5) were examined using single and double immunohistochemistry/immunofluorescence. We also compare the expression pattern of the P75 receptor in the hypothalamus of llama and a spontaneous ovulator species (sheep, n = 5). Both NGF receptors (TrkA and P75) were highly expressed in the medial septum and diagonal band of Broca, and populations of TrkA cells were observed in the periventricular and dorsal hypothalamus. Unexpectedly, we found NGF immunoreactive cell bodies with widespread distribution in the hypothalamus but not in areas endowed with NGF receptors. The organum vasculosum of the lamina terminalis (OVLT) and the median eminence displayed immunoreactivity for P75. Double immunofluorescence using vimentin, a marker of tanycytes, confirmed that tanycytes were immunoreactive to P75 in the median eminence and in the OVLT. Additionally, tanycytes were in close association with GnRH and kisspeptin in the arcuate nucleus and median eminence of llamas. The choroid plexus of llamas contained TrkA and NGF immunoreactivity but no P75 immunoreactivity. Results of the present study demonstrate sites of action of NGF in the llama hypothalamus, providing support for the hypothesis of a central effect of NGF in the ovulation-inducing mechanism in llamas.

Nardilysin controls cardiac sympathetic innervation patterning through regulation of p75 neurotrophin receptor.

Cardiac sympathetic innervation is critically involved in the regulation of circulatory dynamics. However, the molecular mechanism for the innervation patterning has remained elusive. Here, we demonstrate that nardilysin (NRDC, Nrdc), an enhancer of ectodomain shedding, regulates cardiac sympathetic innervation. Nardilysin-deficient (Nrdc-/- ) mice show hypoplastic hearts, hypotension, bradycardia, and abnormal sympathetic innervation patterning. While the innervation of left ventricle (LV) of wild-type mice is denser in the subepicardium than in the subendocardium, Nrdc-/- LV lacks such a polarity and is uniformly and more abundantly innervated. At the molecular level, the full-length form of p75 neurotrophin receptor (p75NTR , Ngfr) is increased in Nrdc-/- LV due to the reduced ectodomain shedding of p75NTR . Importantly, the reduction of p75NTR rescued the abnormal innervation phenotype of Nrdc-/- mice. Moreover, sympathetic neuron-specific, but not cardiomyocyte-specific deletion of Nrdc recapitulated the abnormal innervation patterning of Nrdc-/- mice. In conclusion, neuronal nardilysin critically regulates cardiac sympathetic innervation and circulatory dynamics via modulation of p75NTR .

Nerve growth factor receptor role on rabbit sperm storage.

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).