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N-acetylcysteine (NAC), a mucolytic agent and an antidote to acetaminophen intoxication, has been studied in experimental conditions and trials exploring its analgesic activity based on its antioxidant and anti-inflammatory properties. The purpose of this study is to investigate additional mechanisms, namely, the inhibition of nerve growth factor (NGF) and the activation of the Tropomyosin receptor kinase A (TrkA) receptor, which is responsible for nociception. In silico studies were conducted to evaluate dithiothreitol and NAC's interaction with TrkA. We also measured the autophosphorylation of TrkA in SH-SY5Y cells via ELISA to assess NAC's in vitro activity against NGF-induced TrkA activation. The in silico and in vitro tests show that NAC interferes with NGF-induced TrkA activation. In particular, NAC breaks the disulfide-bound Cys 300–345 of TrkA, perturbing the NGF-TrkA interaction and producing a rearrangement of the binding site, inducing a consequent loss of their molecular recognition and spatial reorganization, which are necessary for the induction of the autophosphorylation process. The latter was inhibited by 40% using 20 mM NAC. These findings suggest that NAC could have a role as a TrkA antagonist, an action that may contribute to the activity and use of NAC in various pain states (acute, chronic, nociplastic) sustained by NGF hyperactivity and/or accompanied by spinal cord sensitization. [ABSTRACT FROM AUTHOR]

Intracellular endosomal trafficking controls the balance between protein degradation and synthesis, i.e., proteostasis, but also many of the cellular signaling pathways that emanate from activated growth factor receptors after endocytosis. Endosomal trafficking, sorting, and motility are coordinated by the activity of small GTPases, including Rab proteins, whose function as molecular switches direct activity at endosomal membranes through effector proteins. Rab7 is particularly important in the coordination of the degradative functions of the pathway. Rab7 effectors control endosomal maturation and the properties of late endosomal and lysosomal compartments, such as coordination of recycling, motility, and fusion with downstream compartments. The spatiotemporal regulation of endosomal receptor trafficking is particularly challenging in neurons because of their enormous size, their distinct intracellular domains with unique requirements (dendrites vs. axons), and their long lifespans as postmitotic, differentiated cells. In Charcot–Marie–Tooth 2B disease (CMT2B), familial missense mutations in Rab7 cause alterations in GTPase cycling and trafficking, leading to an ulcero-mutilating peripheral neuropathy. The prevailing hypothesis to account for CMT2B pathologies is that CMT2B-associated Rab7 alleles alter endocytic trafficking of the neurotrophin NGF and its receptor TrkA and, thereby, disrupt normal trophic signaling in the peripheral nervous system, but other Rab7-dependent pathways are also impacted. Here, using TrkA as a prototypical endocytic cargo, we review physiologic Rab7 effector interactions and control in neurons. Since neurons are among the largest cells in the body, we place particular emphasis on the temporal and spatial regulation of endosomal sorting and trafficking in neuronal processes. We further discuss the current findings in CMT2B mutant Rab7 models, the impact of mutations on effector interactions or balance, and how this dysregulation may confer disease. [ABSTRACT FROM AUTHOR]

Prion protein peptide (PrP) has demonstrated neurotoxicity in brain cells, resulting in the progression of prion diseases with spongiform degenerative, amyloidogenic, and aggregative properties. Thymosin beta 4 (Tβ4) plays a role in the nervous system and may be related to motility, axonal enlargement, differentiation, neurite outgrowth, and proliferation. However, no studies about the effects of Tβ4 on prion disease have been performed yet. In the present study, we investigated the protective effect of Tβ4 against synthetic PrP (106–126) and considered possible mechanisms. Hippocampal neuronal HT22 cells were treated with Tβ4 and PrP (106–126) for 24 h. Tβ4 significantly reversed cell viability and reactive oxidative species (ROS) affected by PrP (106–126). Apoptotic proteins induced by PrP (106–126) were reduced by Tβ4. Interestingly, a balance of neurotrophic factors (nerve growth factor and brain-derived neurotrophic factor) and receptors (nerve growth factor receptor p75, tropomyosin related kinase A and B) were competitively maintained by Tβ4 through receptors reacting to PrP (106–126). Our results demonstrate that Tβ4 protects neuronal cells against PrP (106–126) neurotoxicity via the interaction of neurotrophic factors/receptors. [ABSTRACT FROM AUTHOR]

Simple Summary: NGF was the first growth factor discovered by Rita Levi Montalcini in 1950. TrkA, its high affinity receptor, is an oncogene that is overexpressed in many cancers. However, targeted therapies against TrkA, in particular kinase inhibitors, have not yet demonstrated efficacy in the context of overexpression. In this review, after describing the state-of-the-art TrkA-targeted therapies, we will elicit the failures of these therapies by focusing on non-genomic resistance. Larotrectinib and Entrectinib are specific pan-Trk tyrosine kinase inhibitors (TKIs) approved by the Food and Drug Administration (FDA) in 2018 for cancers with an NTRK fusion. Despite initial enthusiasm for these compounds, the French agency (HAS) recently reported their lack of efficacy. In addition, primary and secondary resistance to these TKIs has been observed in the absence of other mutations in cancers with an NTRK fusion. Furthermore, when TrkA is overexpressed, it promotes ligand-independent activation, bypassing the TKI. All of these clinical and experimental observations show that genetics does not explain all therapeutic failures. It is therefore necessary to explore new hypotheses to explain these failures. This review summarizes the current status of therapeutic strategies with TrkA inhibitors, focusing on the mechanisms potentially involved in these failures and more specifically on the role of TrkA. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF) was the first neurotrophin described. This neurotrophin contributes to organogenesis by promoting sensory innervation and angiogenesis in the endocrine and immune systems. Neuronal and non-neuronal cells produce and secrete NGF, and several cell types throughout the body express the high-affinity neurotrophin receptor TrkA and the low-affinity receptor p75NTR. NGF is essential for glucose-stimulated insulin secretion and the complete development of pancreatic islets. Plus, this factor is involved in regulating lipolysis and thermogenesis in adipose tissue. Immune cells produce and respond to NGF, modulating their inflammatory phenotype and the secretion of cytokines, contributing to insulin resistance and metabolic homeostasis. This neurotrophin regulates the synthesis of gonadal steroid hormones, which ultimately participate in the metabolic homeostasis of other tissues. Therefore, we propose that this neurotrophin's imbalance in concentrations and signaling during metabolic syndrome contribute to its pathophysiology. In the present work, we describe the multiple roles of NGF in immunoendocrine organs that are important in metabolic homeostasis and related to the pathophysiology of metabolic syndrome. [ABSTRACT FROM AUTHOR]

Post-therapeutic relapse, poor survival rates and increasing incidence justify the search for novel therapeutic targets and strategies in cutaneous malignant melanoma (CMM). Within this context, a potential oncogenic role for TrkA in CMM is suggested by reports of NTRK1 amplification, enhanced TrkA expression and intracellular TrkA activation associated with poor prognosis. TrkA, however, exhibits tumour-suppressing properties in melanoma cell lines and has recently been reported not to be associated with CMM progression. To better understand these contradictions, we present the first analysis of potential oncogenic alternative TrkA mRNA splicing, associated with TrkA immunoreactivity, in CMMs, and compare the behaviour of fully spliced TrkA and the alternative TrkAIII splice variant in BRAF(V600E)-mutated A375 melanoma cells. Alternative TrkA splicing in CMMs was associated with unfolded protein response (UPR) activation. Of the several alternative TrkA mRNA splice variants detected, TrkAIII was the only variant with an open reading frame and, therefore, oncogenic potential. TrkAIII expression was more frequent in metastatic CMMs, predominated over fully spliced TrkA mRNA expression in ≈50% and was invariably linked to intracellular phosphorylated TrkA immunoreactivity. Phosphorylated TrkA species resembling TrkAIII were also detected in metastatic CMM extracts. In A375 cells, reductive stress induced UPR activation and promoted TrkAIII expression and, in transient transfectants, promoted TrkAIII and Akt phosphorylation, enhancing resistance to reductive stress-induced death, which was prevented by lestaurtinib and entrectinib. In contrast, fully spliced TrkA was dysfunctional in A375 cells. The data identify fully spliced TrkA dysfunction as a novel mechanism for reducing melanoma suppression, support a causal relationship between reductive stress, UPR activation, alternative TrkAIII splicing and TrkAIII activation and characterise a targetable oncogenic pro-survival role for TrkAIII in CMM. [ABSTRACT FROM AUTHOR]

Breast cancer represents the most common type of cancer and is the leading cause of death due to cancer among women. Thus, the prevention and early diagnosis of breast cancer is of primary urgency, as well as the development of new treatments able to improve its prognosis. Nerve Growth Factor (NGF) is a neurotrophic factor involved in the regulation of neuronal functions through the binding of the Tropomyosin receptor kinase A (TrkA) and the Nerve Growth Factor receptor or Pan-Neurotrophin Receptor 75 (NGFR/p75NTR). In addition, its precursor (pro-NGF) can extert biological activity by forming a trimeric complex with NGFR/p75NTR and sortilin, or by binding to TrkA receptors with low affinity. Several examples of in vitro and in vivo evidence show that NGF is both synthesized and released by breast cancer cells, and has mitogen, antiapoptotic and angiogenic effects on these cells through the activation of different signaling cascades that involve TrkA and NGFR/p75NTR receptors. Conversely, pro-NGF signaling has been related to breast cancer invasion and metastasis. Other studies suggested that NGF and its receptors could represent a good diagnostic and prognostic tool, as well as promising therapeutic targets for breast cancer. In this paper, we comprehensively summarize and systematically review the current experimental evidence on this topic. INPLASY ID: INPLASY2022100017. [ABSTRACT FROM AUTHOR]

Among the factors involved in diabetic retinopathy (DR), nerve growth factor (NGF) and vascular endothelial growth factor A (VEGFA) have been shown to affect both neuronal survival and vascular function, suggesting that their crosstalk might influence DR outcomes. To address this question, the administration of eye drops containing NGF (ed-NGF) to adult Sprague Dawley rats receiving streptozotocin (STZ) intraperitoneal injection was used as an experimental paradigm to investigate NGF modulation of VEGFA and its receptor VEGFR2 expression. We show that ed-NGF treatment prevents the histological and vascular alterations in STZ retina, VEGFR2 expression decreased in GCL and INL, and preserved the co-expression of VEGFR2 and NGF-tropomyosin-related kinase A (TrkA) receptor in retinal ganglion cells (RGCs). The WB analysis confirmed the NGF effect on VEGFR2 expression and activation, and showed a recovery of VEGF isoform dysregulation by suppressing STZ-induced VEGFA121 expression. Reduction in inflammatory and pro-apoptotic intracellular signals were also found in STZ+NGF retina. These findings suggest that ed-NGF administration might favor neuroretina protection, and in turn counteract the vascular impairment by regulating VEGFR2 and/or VEGFA isoform expression during the early stages of the disease. The possibility that an increase in the NGF availability might contribute to the switch from the proangiogenic/apoptotic to the neuroprotective action of VEGF is discussed. [ABSTRACT FROM AUTHOR]

Patients with advanced neuroblastoma (NB) receive multimodal clinical therapy, including the potent anthracycline chemotherapy drug doxorubicin (Dox). The acquisition of Dox resistance, however, is a major barrier to a sustained response and leads to a poor prognosis in advanced disease states, reinforcing the need to identify and inhibit Dox resistance mechanisms. In this context, we report on the identification and inhibition of a novel Dox resistance mechanism. This mechanism is characterized by the Dox-induced activation of the oncogenic TrkAIII alternative splice variant, resulting in increased Dox resistance, and is blocked by lestaurtinib, entrectinib, and crizotinib tyrosine kinase and LY294002 IP3-K inhibitors. Using time lapse live cell imaging, conventional and co-immunoprecipitation Western blots, RT-PCR, and inhibitor studies, we report that the Dox-induced TrkAIII activation correlates with proliferation inhibition and is CDK1- and Ca2+-uniporter-independent. It is mediated by ryanodine receptors; involves Ca2+-dependent interactions between TrkAIII, calmodulin and Hsp90; requires oxygen and oxidation; occurs within assembled ERGICs; and does not occur with fully spliced TrkA. The inhibitory effects of lestaurtinib, entrectinib, crizotinib, and LY294002 on the Dox-induced TrkAIII and Akt phosphorylation and resistance confirm roles for TrkAIII and IP3-K consistent with Dox-induced, TrkAIII-mediated pro-survival IP3K/Akt signaling. This mechanism has the potential to select resistant dormant TrkAIII-expressing NB cells, supporting the use of Trk inhibitors during Dox therapy in TrkAIII-expressing NBs. [ABSTRACT FROM AUTHOR]

The Nerve Growth Factor (NGF) belongs to the neurothrophins protein family involved in the survival of neurons in the nervous system. The interaction of NGF with its high-affinity receptor TrkA mediates different cellular pathways related to Alzheimer's disease, pain, ocular dysfunction, and cancer. Therefore, targeting NGF-TrkA interaction represents a valuable strategy for the development of new therapeutic agents. In recent years, experimental studies have revealed that peptides belonging to the N-terminal domain of NGF are able to partly mimic the biological activity of the whole protein paving the way towards the development of small peptides that can selectively target specific signaling pathways. Hence, understanding the molecular basis of the interaction between the N-terminal segment of NGF and TrkA is fundamental for the rational design of new peptides mimicking the NGF N-terminal domain. In this study, molecular dynamics simulation, binding free energy calculations and per-residue energy decomposition analysis were combined in order to explore the molecular recognition pattern between the experimentally active NGF(1–14) peptide and TrkA. The results highlighted the importance of His4, Arg9 and Glu11 as crucial residues for the stabilization of NGF(1–14)-TrkA interaction, thus suggesting useful insights for the structure-based design of new therapeutic peptides able to modulate NGF-TrkA interaction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

N-acetylcysteine (NAC), a mucolytic agent and an antidote to acetaminophen intoxication, has been studied in experimental conditions and trials exploring its analgesic activity based on its antioxidant and anti-inflammatory properties. The purpose of this study is to investigate additional mechanisms, namely, the inhibition of nerve growth factor (NGF) and the activation of the Tropomyosin receptor kinase A (TrkA) receptor, which is responsible for nociception. In silico studies were conducted to evaluate dithiothreitol and NAC's interaction with TrkA. We also measured the autophosphorylation of TrkA in SH-SY5Y cells via ELISA to assess NAC's in vitro activity against NGF-induced TrkA activation. The in silico and in vitro tests show that NAC interferes with NGF-induced TrkA activation. In particular, NAC breaks the disulfide-bound Cys 300-345 of TrkA, perturbing the NGF-TrkA interaction and producing a rearrangement of the binding site, inducing a consequent loss of their molecular recognition and spatial reorganization, which are necessary for the induction of the autophosphorylation process. The latter was inhibited by 40% using 20 mM NAC. These findings suggest that NAC could have a role as a TrkA antagonist, an action that may contribute to the activity and use of NAC in various pain states (acute, chronic, nociplastic) sustained by NGF hyperactivity and/or accompanied by spinal cord sensitization.

Fascaplysin has been reported to exert anti-cancer effects by inhibiting cyclin-dependent kinase 4 (CDK4); however, the precise mode of action by which fascaplysin suppresses tumor growth is not clear. Here, we found that fascaplysin has stronger anti-cancer effects than other CDK4 inhibitors, including PD0332991 and LY2835219, on lung cancer cells that are wild-type or null for retinoblastoma (RB), indicating that unknown target molecules might be involved in the inhibition of tumor growth by fascaplysin. Fascaplysin treatment significantly decreased tumor angiogenesis and increased cleaved-caspase-3 in xenografted tumor tissues. In addition, survivin and HIF-1α were downregulated in vitro and in vivo by suppressing 4EBP1-p70S6K1 axis-mediated de novo protein synthesis. Kinase screening assays and drug-protein docking simulation studies demonstrated that fascaplysin strongly inhibited vascular endothelial growth factor receptor 2 (VEGFR2) and tropomyosin-related kinase A (TRKA) via DFG-out non-competitive inhibition. Overall, these results suggest that fascaplysin inhibits TRKA and VEGFR2 and downregulates survivin and HIF-1α, resulting in suppression of tumor growth. Fascaplysin, therefore, represents a potential therapeutic approach for the treatment of multiple types of solid cancer. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF) promotes the survival and differentiation of neurons. NGF is initially synthesized as a precursor, proNGF, which is the predominant form in the central nervous system. NGF and proNGF bind to TrkA/p75NTR to mediate cell survival and to sortilin/p75NTR to promote apoptosis. The ratio of TrkA to p75NTR affects whether proNGF and mature NGF signal cell survival or apoptosis. The purpose of this study was to determine whether the loss of TrkA influences p75NTR or sortilin expression levels, and to establish whether proNGF and mature NGF have a similar ability to switch between cell survival and cell death. We systematically altered TrkA receptor levels by priming cells with NGF, using small interfering RNA, and using the mutagenized PC12nnr5 cell line. We found that both NGF and proNGF can support cell survival in cells expressing TrkA, even in the presence of p75NTR and sortilin. However, when TrkA is reduced, proNGF signals cell death, while NGF exhibits no activity. In the absence of TrkA, proNGF-induced cell death occurs, even when p75NTR and sortilin levels are reduced. These results show that proNGF can switch between neurotrophic and apoptotic activity in response to changes in TrkA receptor levels, whereas mature NGF cannot. These results also support the model that proNGF is neurotrophic under normal circumstances, but that a loss in TrkA in the presence of p75NTR and sortilin, as occurs in neurodegenerative disease or injury, shifts proNGF, but not NGF, signalling from cell survival to cell death. [ABSTRACT FROM AUTHOR]

Clostridium perfringens alpha-toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-toxin possesses phospholipase C and sphingomyelinase activities. The toxin is composed of an N-terminal domain (1-250 aa, N-domain), which is the catalytic site, and a C-terminal domain (251-370 aa, C-domain), which is the membrane-binding site. Immunization of mice with the C-domain of alpha-toxin prevents the gas gangrene caused by C. perfringens, whereas immunization with the N-domain has no effect. The central loop domain (55-93 aa), especially H… SW84Y85… G, plays an important role in the interaction with ganglioside GM1a. The toxin binds to lipid rafts in the presence of a GM1a/TrkA complex, and metabolites from phosphatidylcholine to diacylglycerol through the enzymatic activity of alpha-toxin itself. These membrane dynamics leads to the activation of endogenous PLCγ-1 via TrkA. In addition, treatment with alpha-toxin leads to the formation of diacylglycerol at membrane rafts in ganglioside-deficient DonQ cells; this in turn triggers endocytosis and cell death. This article summarizes the current the membrane-binding mechanism of alpha-toxin in detail. [ABSTRACT FROM AUTHOR]

Nerve growth factor (NGF) and its high-affinity receptor TRKA are overexpressed in epithelial ovarian cancer (EOC) displaying a crucial role in the disease progression. Otherwise, NGF interacts with its low-affinity receptor P75, activating pro-apoptotic pathways. In neurons, P75 could be cleaved by metalloproteinases (α and γ-secretases), leading to a decrease in P75 signaling. Therefore, this study aimed to evaluate whether the shedding of P75 occurs in EOC cells and whether NGF/TRKA could promote the cleavage of the P75 receptor. The immunodetection of the α-secretase, ADAM17, TRKA, P75, and P75 fragments was assessed by immunohisto/cytochemistry and Western blot in biopsies and ovarian cell lines. The TRKA and secretases' inhibition was performed using specific inhibitors. The results show that P75 immunodetection decreased during EOC progression and was negatively correlated with the presence of TRKA in EOC biopsies. NGF/TRKA increases ADAM17 levels and the fragments of P75 in ovarian cells. This effect is abolished when cells are previously treated with ADAM17, γ-secretase, and TRKA inhibitors. These results indicate that NGF/TRKA promotes the shedding of P75, involving the activation of secretases such as ADAM17. Since ADAM17 has been proposed as a screening marker for early detection of EOC, our results contribute to understanding better the role of ADAM17 and NGF/TRKA in EOC pathogenesis, which includes the NGF/TRKA-mediated cleavage of P75. [ABSTRACT FROM AUTHOR]

One of the major challenges for cancer therapeutics is the resistance of many tumor cells to induction of cell death due to pro-survival signaling in the cancer cells. Here we review the growing literature which shows that neurotrophins contribute to pro-survival signaling in many different types of cancer. In particular, nerve growth factor, the archetypal neurotrophin, has been shown to play a role in tumorigenesis over the past decade. Nerve growth factor mediates its effects through its two cognate receptors, TrkA, a receptor tyrosine kinase and p75NTR, a member of the death receptor superfamily. Depending on the tumor origin, pro-survival signaling can be mediated by TrkA receptors or by p75NTR. For example, in breast cancer the aberrant expression of nerve growth factor stimulates proliferative signaling through TrkA and pro-survival signaling through p75NTR. This latter signaling through p75NTR promotes increased resistance to the induction of cell death by chemotherapeutic treatments. In contrast, in prostate cells the p75NTR mediates cell death and prevents metastasis. In prostate cancer, expression of this receptor is lost, which contributes to tumor progression by allowing cells to survive, proliferate and metastasize. This review focuses on our current knowledge of neurotrophin signaling in cancer, with a particular emphasis on nerve growth factor regulation of cell death and survival in cancer. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Neuroblastoma (NB) accounts for 15% of all cancer-related deaths of children. While the amplification of the Myc-N proto-oncogene (MYCN) is a major driver of aggressive NB, the expression of the neurotrophin receptor, NTRK1/TrkA, has been shown to be associated with an excellent outcome. MYCN downregulates NTRK1 expression, but it is unknown if the molecular effects of NTRK1 signaling also affect MYCN-induced networks. The aim of this study was to decipher NTRK1 signaling using an unbiased proteome and phosphoproteome approach. To this end, we realized inducible ectopic NTRK1 expression in a NB cell line with MYCN amplification and analyzed the proteomic changes upon NTRK1 activation in a time-dependent manner. In line with the phenotypes observed, NTRK1 activation induced markers of neuronal differentiation and cell cycle arrest. Most prominently, NTRK1 upregulated the expression and phosphorylation of the nuclear lamina component Lamin A/C. Moreover, NTRK1 signaling also induced the aggregation of LMNA within nucleic foci, which accompanies differentiation in other cell types. (1) Background: Neuroblastomas (NBs) are the most common extracranial solid tumors of children. The amplification of the Myc-N proto-oncogene (MYCN) is a major driver of NB aggressiveness, while high expression of the neurotrophin receptor NTRK1/TrkA is associated with mild disease courses. The molecular effects of NTRK1 signaling in MYCN-amplified NB, however, are still poorly understood and require elucidation. (2) Methods: Inducible NTRK1 expression was realized in four NB cell lines with (IMR5, NGP) or without MYCN amplification (SKNAS, SH-SY5Y). Proteome and phosphoproteome dynamics upon NTRK1 activation by its ligand, NGF, were analyzed in a time-dependent manner in IMR5 cells. Target validation by immunofluorescence staining and automated image processing was performed using the three other NB cell lines. (3) Results: In total, 230 proteins and 134 single phosphorylated class I phosphosites were found to be significantly regulated upon NTRK1 activation. Among known NTRK1 targets, Stathmin and the neurosecretory protein VGF were recovered. Additionally, we observed the upregulation and phosphorylation of Lamin A/C (LMNA) that accumulated inside nuclear foci. (4) Conclusions: We provide a comprehensive picture of NTRK1-induced proteome and phosphoproteome dynamics. The phosphorylation of LMNA within nucleic aggregates was identified as a prominent feature of NTRK1 signaling independent of the MYCN status of NB cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer is the leading cancer in American women. Due to the inherent aggressiveness of triple-negative and HER2-enriched breast cancers, it is imperative to identify novel molecular targets for therapeutic intervention. Due to their abnormal activities in metastatic breast cancers, JAK2–STAT3 and TrkA pathways have been individually implicated in aggressive breast tumors. However, their co-activation and signaling interactions have not been thoroughly investigated. Therefore, our study aimed to elucidate the extent of crosstalk between JAK2–STAT3 and TrkA signaling pathways and its impact on breast cancer. Our data revealed a novel interaction between TrkA and STAT3, and that this interaction results in STAT3 phosphorylation and activation by TrkA, leading to enhanced stemness gene expression and stem cell renewal. We further found that the co-activation of JAK2–STAT3 and TrkA pathways is correlated with shorter time to develop overall and organ-specific metastasis, suggesting that this signaling crosstalk underlies the aggressiveness of triple-negative and HER2-enriched breast cancers. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Nerve Growth Factor (NGF) and its high-affinity receptor tropomyosin receptor kinase A (TRKA) increase their expression during the progression of epithelial ovarian cancer (EOC), promoting cell proliferation and angiogenesis through several oncogenic proteins, such as c-MYC and vascular endothelial growth factor (VEGF). The expression of these proteins is controlled by microRNAs (miRs), such as miR-145, whose dysregulation has been related to cancer. The aims of this work were to evaluate in EOC cells whether NGF/TRKA decreases miR-145 levels, and the effect of miR-145 upregulation. The levels of miR-145-5p were assessed by qPCR in ovarian biopsies and ovarian cell lines (human ovarian surface epithelial cells (HOSE), A2780 and SKOV3) stimulated with NGF. Overexpression of miR-145 in ovarian cells was used to evaluate cell proliferation, migration, invasion, c-MYC and VEGF protein levels, as well as tumor formation and metastasis in vivo. In EOC samples, miR-145-5p levels were lower than in epithelial ovarian tumors. Overexpression of miR-145 decreased cell proliferation, migration and invasion of EOC cells, changes that were concomitant with the decrease in c-MYC and VEGF protein levels. We observed decreased tumor formation and suppressed metastasis behavior in mice injected with EOC cells that overexpressed miR-145. As expected, ovarian cell lines stimulated with NGF diminished miR-145-5p transcription and abundance. These results suggest that the tumoral effects of NGF/TRKA depend on the regulation of miR-145-5p levels in EOC cells, and that its upregulation could be used as a possible therapeutic strategy for EOC. [ABSTRACT FROM AUTHOR]

The aim of this study was to identify new potentially active compounds for three protein targets, tropomyosin receptor kinase A (TrkA), N-methyl-d-aspartate (NMDA) receptor, and leucine-rich repeat kinase 2 (LRRK2), that are related to various neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and neuropathic pain. We used a combination of machine learning methods including artificial neural networks and advanced multilinear techniques to develop quantitative structure–activity relationship (QSAR) models for all target proteins. The models were applied to screen more than 13,000 natural compounds from a public database to identify active molecules. The best candidate compounds were further confirmed by docking analysis and molecular dynamics simulations using the crystal structures of the proteins. Several compounds with novel scaffolds were predicted that could be used as the basis for development of novel drug inhibitors related to each target. [ABSTRACT FROM AUTHOR]

Gene rearrangements resulting in the aberrant activity of tyrosine kinases have been identified as drivers of oncogenesis in a variety of cancers. The tropomyosin receptor kinase (TRK) family of tyrosine receptor kinases is emerging as an important target for cancer therapeutics. The TRK family contains three members, TRKA, TRKB, and TRKC, and these proteins are encoded by the genes NTRK1, NTRK2, and NTRK3, respectively. To activate TRK receptors, neurotrophins bind to the extracellular region stimulating dimerization, phosphorylation, and activation of downstream signaling pathways. Major known downstream pathways include RAS/MAPK/ERK, PLCγ, and PI3K/Akt. While being rare in most cancers, TRK fusions with other proteins have been well-established as oncogenic events in specific malignancies, including glioblastoma, papillary thyroid carcinoma, and secretory breast carcinomas. TRK protein amplification as well as alternative splicing events have also been described as contributors to cancer pathogenesis. For patients harboring alterations in TRK expression or activity, TRK inhibition emerges as an important therapeutic target. To date, multiple trials testing TRK-inhibiting compounds in various cancers are underway. In this review, we will summarize the current therapeutic trials for neoplasms involving NTKR gene alterations, as well as the promises and setbacks that are associated with targeting gene fusions. [ABSTRACT FROM AUTHOR]

Fascaplysin has been reported to exert anti-cancer effects by inhibiting cyclin-dependent kinase 4 (CDK4); however, the precise mode of action by which fascaplysin suppresses tumor growth is not clear. Here, we found that fascaplysin has stronger anti-cancer effects than other CDK4 inhibitors, including PD0332991 and LY2835219, on lung cancer cells that are wild-type or null for retinoblastoma (RB), indicating that unknown target molecules might be involved in the inhibition of tumor growth by fascaplysin. Fascaplysin treatment significantly decreased tumor angiogenesis and increased cleaved-caspase-3 in xenografted tumor tissues. In addition, survivin and HIF-1α were downregulated in vitro and in vivo by suppressing 4EBP1-p70S6K1 axis-mediated de novo protein synthesis. Kinase screening assays and drug-protein docking simulation studies demonstrated that fascaplysin strongly inhibited vascular endothelial growth factor receptor 2 (VEGFR2) and tropomyosin-related kinase A (TRKA) via DFG-out non-competitive inhibition. Overall, these results suggest that fascaplysin inhibits TRKA and VEGFR2 and downregulates survivin and HIF-1α, resulting in suppression of tumor growth. Fascaplysin, therefore, represents a potential therapeutic approach for the treatment of multiple types of solid cancer., Competing Interests: The authors declare no conflict of interest.

Clostridium perfringens alpha-toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-toxin possesses phospholipase C and sphingomyelinase activities. The toxin is composed of an N-terminal domain (1-250 aa, N-domain), which is the catalytic site, and a C-terminal domain (251-370 aa, C-domain), which is the membrane-binding site. Immunization of mice with the C-domain of alpha-toxin prevents the gas gangrene caused by C. perfringens, whereas immunization with the N-domain has no effect. The central loop domain (55-93 aa), especially H….SW(84)Y(85)….G, plays an important role in the interaction with ganglioside GM1a. The toxin binds to lipid rafts in the presence of a GM1a/TrkA complex, and metabolites from phosphatidylcholine to diacylglycerol through the enzymatic activity of alpha-toxin itself. These membrane dynamics leads to the activation of endogenous PLCγ-1 via TrkA. In addition, treatment with alpha-toxin leads to the formation of diacylglycerol at membrane rafts in ganglioside-deficient DonQ cells; this in turn triggers endocytosis and cell death. This article summarizes the current the membrane-binding mechanism of alpha-toxin in detail.