TrkA Gene Research Articles

A TaqMan-Based qRT-PCR Assay for Accurate Evaluation of the Oncogenic TrkAIII Splice Variant in Tumor cDNAs

Background: Alternative NTRK1/TrkA splicing resulting in TrkAIII expression, originally discovered in advanced-stage metastatic neuroblastomas, is also pronounced in prostate, medullary thyroid, glioblastoma multiforme, MCPyV-positive Merkel cell, cutaneous malignant melanoma, and pituitary neuroendocrine tumor subsets. In tumor models, TrkAIII exhibits actionable oncogenic activity equivalent to the TrkT3-fused oncogene, and in tumor cell lines, alternative TrkAIII splicing is promoted by hypoxia, nutrient deprivation, endoplasmic reticulum stress, and SV40 large T antigen, implicating tumor microenvironmental conditions and oncogenic polyoma viruses in tumor-associated TrkAIII expression. Collectively, these observations characterize TrkAIII as a potentially frequent, actionable oncogenic alternative to TrkA gene fusion in different tumor types. Currently, therapeutic approval for efficacious Trk inhibitors is restricted to Trk-fused gene positive tumors and not for tumors potentially driven by TrkAIII. Methods: With the therapeutically relevant aim of improving the identification of tumors potentially driven by TrkAIII, we have developed a TaqMan-based qRT-PCR assay for evaluating TrkAIII expression in tumor cDNAs. Results: This assay, validated using gel-purified fs-TrkA and TrkAIII cDNAs alone and in complex cDNA mixtures, employs primers and probes designed from fs-TrkA and TrkAIII sequences, with specificity provided by a TaqMan probe spanning the TrkAIII exon 5–8 splice junction. It is highly efficient, reproducible, and specific and can detect as few as 10 TrkAIII copies in complex RNAs extracted from either fresh or FFPE tumor tissues. Conclusions: Inclusion of this assay into precision oncology algorithms, when paired with fs-TrkA qRT-PCR and TrkA immune histochemistry, will make it easier to identify patients with therapy-resistant, advanced-stage metastatic Trk-fused gene-negative tumors potentially driven by TrkAIII, for whom approval of third-line effective Trk inhibitors could be extended.

The genetic characterization of germplasm and identification of the litter size trait associated candidate genes in Dexin mutton and fine-wool sheep.

Xinjiang is a major province of sheep breeding in China, which plays an important role in meeting people's needs for meat products, increasing farmers' income and sustainable development of animal husbandry. However, the genetic differentiation relationship between breeds was not clear, and most sheep had low fecundity, which seriously restricted the efficient development of sheep industry. Therefore, this study used the whole genome resequencing to detect the genetic variation of Dexin mutton and fine-wool sheep, explored the selected regions and important genes of the litter size traits, analyzed the genetic mechanism of reproductive traits, and provided new insights for the high fecundity breeding of sheep. A total of 5,236.338 G genome data and 35,884,037 SNPs were obtained. Furthermore, we identified 39 selection signals spanning candidate genes, 99 genes were significantly associated related to growth, reproduction and immunity, among which, BRIP1, BMPR1B, BMP4, NGF, etc. genes, and MAKP signaling pathway, Fanconi anemia pathway and Thyroid hormone signaling pathway and other signaling pathways were significantly correlated with litter size trait. Among them, we identified NGF, TrKA and BRIP1 genes was the important genes for sheep litter size traits and the mutation frequencies of 9 SNPs in BRIP1 gene were significantly different in domestic sheep in the world. The research provided new insights for the breeding of self-cultivated meat fine-wool sheep.

TrkA serves as a virulence modulator in Porphyromonas gingivalis by maintaining heme acquisition and pathogenesis.

Periodontitis is an inflammatory disease of the supporting tissues of the teeth, with polymicrobial infection serving as the major pathogenic factor. As a periodontitis-related keystone pathogen, Porphyromonas gingivalis can orchestrate polymicrobial biofilm skewing into dysbiosis. Some metatranscriptomic studies have suggested that modulation of potassium ion uptake might serve as a signal enhancing microbiota nososymbiocity and periodontitis progression. Although the relationship between potassium transport and virulence has been elucidated in some bacteria, less is mentioned about the periodontitis-related pathogen. Herein, we centered on the virulence modulation potential of TrkA, the potassium uptake regulatory protein of P. gingivalis, and uncovered TrkA as the modulator in the heme acquisition process and in maintaining optimal pathogenicity in an experimental murine model of periodontitis. Hemagglutination and hemolytic activities were attenuated in the case of trkA gene loss, and the entire transcriptomic profiling revealed that the trkA gene can control the expression of genes in relation to electron transport chain activity and translation, as well as some transcriptional factors, including cdhR, the regulator of the heme uptake system hmuYR. Collectively, these results link the heme acquisition process to the potassium transporter, providing new insights into the role of potassium ion in P. gingivalis pathogenesis.

Effects of salt stress on the freeze-drying survival rate of Lactiplantibacillus plantarum LIP-1

In this study, we examined the effects of different salt stress application methods on the Lactiplantibacillus plantarum LIP-1 freeze-drying survival rate. The application of salt stress during the stationary phase significantly improved Lactiplantibacillus plantarum LIP-1 freeze-drying survival rates (P<0.05). The indirect application of salt stress via phosphate-buffered saline containing 0.4mol/L NaCl (NB group) led to significantly higher freeze-drying survival rates compared to when salt stress was directly applied (NA group: the concentration of NaCl is 0.4mol/L) (P<0.05). Following exposure to salt stress, Lactiplantibacillus plantarum LIP-1cells exuded excessive Na+ out of the cell and transported extracellular K+ into the cell, resulting in upregulation of the trkA gene, which is related to K+ transport, thereby significantly upregulating the expression of a lysR-type transcription factor, which increased the cell membrane unsaturated fatty acid content, reducing the degree of cell membrane damage and improving the freeze-drying survival rate. When the concentration of NaCl is 0.4mol/L, compared with direct salt stress application, indirect application led to higher intracellular pH and ATP content, which effectively reduced DNA and cell membrane damage, respectively. Together, these results demonstrate that appropriate indirect salt stress application can improve Lactiplantibacillus plantarum LIP-1 freeze-drying resistance.

Effects of buffer salts on the freeze-drying survival rate of Lactobacillus plantarum LIP-1 based on transcriptome and proteome analyses

Buffer salts are often added to culture medium to promote bacterial growth. However, we found that buffer salts can improve the freeze-drying survival rate. In this experiment, the mechanisms for the effects of different buffer salts on the survival rate of freeze-dried strains were examined. The results showed that buffer salts had important effects on the freeze-drying survival rate of L. plantarum LIP-1 that were related to changes in fatty acid composition. Different buffer salts affected the expression of fatty acid metabolic genes. A new gene cluster that regulates fatty acid metabolism and synthesis was discovered. Potassium ions in buffer salts upregulated the trkA gene and lysR-type transcription factor, and then upregulated the expression of fatty acid synthesis-related acc and fab family genes. These genes help to extend the fatty acid carbon chain and promote the unsaturated fatty acids content, which improves cell membrane fluidity and improves resistance to freeze-drying.

454 Occurrence of TRKAIII splice variant in melanoma: a pilot study

TrkA is a member of the transmembrane neurotrophin receptor family with a critical role in cell proliferation, survival and differentiation. TrkA oncogenic activation is an emerging driver in a wide variety of solid tumors, and TrkA-targeted therapies using inhibitors have been reported to exhibit marked and durable efficacy. An alternative TrkA splice variant, the TrkAIII, characterized by exon 6-7 skipping, exhibits spontaneous aberrant activation. We aimed to explore a potential oncogenic role for TrkA in cutaneous melanoma. Cutaneous melanoma samples from patients of stage III-IV have been collected from the Department of Dermatology of University of L’Aquila. DNA and RNA was extracted from FFPE sections. The cDNA was amplified using TrkA-specific primers. The copy number variation of TrkA gene was evaluated by TaqMan Copy Number assays. Somatic mutations in the region spanning TrkA intron/exon 6 to the intron/exon 8 were analyzed by Sanger Sequencing. Overall, we collected 30 primary melanomas tissue and 12 normal skin specimens. We identified predominant alternative TrkAIII splice variant in 16 of 30 (53%) primary melanomas, while the remaining 14 (47%) cases express mainly the full length TrkA. Normal skin samples exhibit low to undetectable full length TrkA expression. The presence of TrkAIII was always confirmed by sequencing. Somatic DNA mutations in intron/exon boundary that could explain the exons skipping were not found. TrkA gene amplification was detected in 4 of 30 (13%) melanomas. Our results could open the way to new personalized therapeutic approaches for melanoma patients by targeting TrkA oncogenic signals.

Mapping of the tyrosine kinase receptors trkA (NTRK1), trkB (NTRK2) and trkC(NTRK3) to human chromosomes 1q22, 9q22 and 15q25 by fluorescence in situ hybridization.

trk (NTRK) genes encode tyrosine kinase transmembrane receptors that are stimulated by neurotrophins, and are responsible for the transduction of signals controlling neuropoiesis and neuron survival in the central and peripheral nervous system, trkA gene has earlier been assigned to three different loci on chromosome 1. To resolve these conflicting results, and confirm the localization of trkB and trkC, probes specific to each of these related genes were constructed and used in fluorescent in situ hybridization on human metaphase cells. Our results indicate that trkA, trkB and trkC are located in chromosome bands 1q22, 9q22 and 15q25, respectively.

Neuroimmune and epigenetic involvement in adolescent binge ethanol-induced loss of basal forebrain cholinergic neurons: Restoration with voluntary exercise.

Binge drinking and alcohol abuse are common during adolescence and cause lasting pathology. Preclinical rodent studies using the adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2‐day on/2‐day off from postnatal day [P]25 to P55) model of human adolescent binge drinking report decreased basal forebrain cholinergic (ie, ChAT+) neurons that persist into adulthood (ie, P56‐P220). Recent studies link AIE‐induced neuroimmune activation to cholinergic pathology, but the underlying molecular mechanisms contributing to the persistent loss of basal forebrain ChAT+ neurons are unknown. We report here that the AIE‐induced loss of cholinergic neuron markers (ie, ChAT, TrkA, and p75NTR), cholinergic neuron shrinkage, and increased expression of the neuroimmune marker pNF‐κB p65 are restored by exercise exposure from P56 to P95 after AIE. Our data reveal that persistently reduced expression of cholinergic neuron markers following AIE is because of the loss of the cholinergic neuron phenotype most likely through an epigenetic mechanism involving DNA methylation and histone 3 lysine 9 dimethylation (H3K9me2). Adolescent intermittent ethanol caused a persistent increase in adult H3K9me2 and DNA methylation at promoter regions of Chat and H3K9me2 of Trka, which was restored by wheel running. Exercise also restored the AIE‐induced reversal learning deficits on the Morris water maze. Together, these data suggest that AIE‐induced adult neuroimmune signaling and cognitive deficits are linked to suppression of Chat and Trka gene expression through epigenetic mechanisms that can be restored by exercise. Exercise restoration of the persistent AIE‐induced phenotypic loss of cholinergic neurons via epigenetic modifications is novel mechanism of neuroplasticity.

Characterization of β-Nerve Growth Factor-TrkA system in male reproductive tract of rabbit and the relationship between β-NGF and testosterone levels with seminal quality during sexual maturation

β-Nerve Growth Factor (β-NGF) is a neurotrophin which acts through its receptors TrkA and p75, performing important actions in male reproductive physiology and its presence in seminal plasma (SP) has been related to male fertility. The aim of the present study was to evaluate the gene expression profile and the immunolocalization of β-NGF and its high-affinity receptor TrkA in sex organs in rabbits during sexual maturation period. β-NGF concentration for both SP and blood plasma (BP) and BP testosterone levels were determined as well as the seminal parameters during such period. Ten New Zealand White x California young rabbits were trained to semen collection since 20 weeks of age and routinely done once a week with two ejaculations per session. At 22 and 37 weeks of age, semen collection was carried out three times a week and seminal parameters were evaluated. Four males were randomly assigned and slaughtered in each age (n = 8); sex organs (prostate, bulbourethral glands and epididymis) were dissected and collected to determine β-NGF and TrkA gene expression and immunolocalization. SP and BP were also taken at each semen collection session to evaluate β-NGF concentration, and testosterone levels were also assessed in BP. The highest β-NGF mRNA expression was observed in prostate compared to bulbourethral glands and epididymis. These two last tissues showed residual β-NGF mRNA expression and limited localization of the neurotrophin. The prostate epithelial cells and lumen were strongly stained with regard to the other sex organs indicating that immunolocalization of β-NGF rely mainly in the prostate. TrkA gene expression was lower but constant and differentially immunolocalized in the sex organ tissues. Finally, β-NGF concentration in SP and BP remained unchanged in accordance to age, while some seminal characteristics such as sperm concentration, percentage of live sperm and mass and progressive motility were enhanced as endowed by BP testosterone variation. β-NGF and its cognate TrkA receptor are expressed and immunolocalized in the male reproductive tract in the two ages studied, independently of the circulating levels of testosterone and β-NGF.

No Association of Polymorphisms in Nav1.7 or Nerve Growth Factor Receptor Genes with Trigeminal Neuralgia.

Trigeminal neuralgia is defined as a sudden severe shock-like pain within the distribution of the trigeminal nerve. Pain is a subjective experience that is influenced by gender, culture, environment, psychological traits, and genes. Sodium channels and nerve growth factor play important roles in the transmission of nociceptive signals and pain. The aim of this study was to investigate the occurrence of Nav1.7 sodium channel and nerve growth factor receptor TrkA gene polymorphisms (SCN9A/rs6746030 and NTRK1/rs633, respectively) in trigeminal neuralgia patients. Ninety-six subjects from pain specialty centers in the southeastern region of Brazil were divided into 2 groups: 48 with classical trigeminal neuralgia diagnosis and 48 controls. Pain was evaluated using the visual analog scale and multidimensional McGill Pain Questionnaire. Genomic DNA was obtained from oral swabs in all individuals and was analyzed by real-time polymerase chain reaction. No association was observed between evaluated polymorphisms and trigeminal neuralgia. For allele analyses, patients and controls had similar frequencies for both genes. Genotype distribution or allele frequencies of polymorphisms analyzed here did not correlate to pain scores. Although no association of evaluated polymorphisms and trigeminal neuralgia diagnosis or pain severity was observed, our data do not exclude the possibility that other genotypes affecting the expression of Nav1.7 or TrkA are associated with the disease. Further studies should investigate distinct genetic polymorphisms and epigenetic factors that may be important in expression of these molecules.

Compromised Neurotrophic and Angiogenic Regenerative Capability during Tendon Healing in a Rat Model of Type-II Diabetes.

Metabolic diseases such as diabetes mellitus type-II (DM-II) may increase the risk of suffering painful connective tissue disorders and tendon ruptures. The pathomechanisms, however, by which diabetes adversely affects connective tissue matrix metabolism and regeneration, still need better definition. Our aim was to study the effect of DM-II on expressional changes of neuro- and angiotrophic mediators and receptors in intact and healing Achilles tendon. The right Achilles tendon was transected in 5 male DM-II Goto-Kakizaki (GK) and 4 age-matched Wistar control rats. The left Achilles tendons were left intact. At week 2 post-injury, NGF, BDNF, TSP, and receptors TrkA, TrkB and Nk1 gene expression was studied by quantitative RT-PCR (qRT-PCR) and their protein distribution by immunohistochemistry in intact and injured tendons. The expression of tendon-related markers, Scleraxis (SCX) and Tenomodulin (TNMD), was evaluated by qRT-PCR in intact and injured tendons. Injured tendons of diabetic GK rats exhibited significantly down-regulated Ngf and Tsp1 mRNA and corresponding protein levels, and down-regulated Trka gene expression compared to injured Wistar controls. Intact tendons of DM-II GK rats displayed reduced mRNA levels for Ngf, Tsp1 and Trkb compared to corresponding intact non-diabetic tendons. Up-regulated Scx and Tnmd gene expression was observed in injured tendons of normal and diabetic GK rats compared to intact Wistar controls. However, these molecules were not up-regulated in injured DM-II GK rats compared to their corresponding controls. Our results suggest that DM-II has detrimental effects on neuro- and angiotrophic pathways, and such effects may reflect the compromised repair seen in diabetic Achilles tendon. Thus, novel approaches for regeneration of injured, including tendinopathic, and surgically repaired diabetic tendons may include therapeutic molecular modulation of neurotrophic pathways such as NGF and its receptors.

MicroRNA-4656 is a prognostic factor and tumor suppressor in human pancreatic cancer through a downstream target of TrkA.

In the present study, we investigated the expression profile and functional mechanism of microRNA-4656 in human pancreatic cancer (PC). MiR-4656 expression in PC tumors was examined using a quantitative reverse transcriptase-polymerase chain reaction in 134 patients. Associations between tumorous miR-4656 expression and clinicopathological parameters of patients, as well as overall survival, were analyzed. MiR-4656 expression was also examined in PC in vitro cell lines. In Capan-1 and AsPC-1 PC cells, lentivirus-induced miR-4656 overexpression or downregulation was applied to investigate its functional regulations on PC in vitro proliferation and invasion, as well as in vivo transplant growth. The association of miR-4656 and its downstream target, the tropomyosin receptor kinase A (TrkA) gene, was investigated in both cell line and clinical pancreatic tumors. In miR-4656-overerxpressed PC cells, TrkA was overexpressed with the aim of investigating its role in miR-4656-induced functional regulation in PC. MiR-4656 was downregulated in PC. Low tumorous miR-4656 expression was associated with a poor prognosis and overall survival of patients. MiR-4656 was also found to be downregulated in PC cell lines. MiR-4656 overexpression in Capan-1 and AsPC-1 cells significantly inhibited cancer proliferation and invasion in vitro, as well as explant growth in vivo, whereas miR-4656 downregulation had no effect on cancer development. The TrkA gene was directly bound by miR-4656, and reversely expressed in PC tumors as miR-4656. TrkA overexpression reversed the inhibitory effect of miR-4656-overexpression on PC proliferation and invasion. MiR-4656 is expressed to a low extent and is a potential biomarker in PC. Overexpressing miR-4656 has tumor suppressive effects on PC development both in vitro and in vivo, likely through its downstream target of the TrkA gene.

Abstract 2643: Discovery and characterization of a selective TrkA inhibitor for the treatment of TrkA-driven cancers

Abstract Kinase gene fusions have been implicated as oncogenic drivers promoting cell survival and proliferation. TrkA gene fusions have been reported in a number of cancer types including lung, colorectal, papillary thyroid, sarcoma, cholangiocarcinoma and other tumors. We have identified novel, potent, selective and orally bioavailable TrkA inhibitors for the treatment of TrkA-driven cancers. One of the inhibitors, GVK-TrkI showed potent activity with an IC50 of 0.8 nM and 1.5 nM in the cell based assays for WT TrkA (AD293 cells expressing TrkA) and TPM3-NTRK1 fusion (KM12 cells) respectively. Potent activity of 12.5 nM IC50 was observed in TrkA Z-lyte kinase assay. Significant change in TrkA Z-lyte kinase IC50 was not observed at multiple ATP concentrations ranging from 100 uM to 1 mM suggesting non-ATP competitive mode of inhibition. Excellent selectivity (&amp;gt;1000x) was observed towards TrkB and TrkC in the cell based assays. GVK-TrkI inhibited TrkA autophosphorylation in a concentration dependent manner up to 24 h in KM12 cells in vitro. GVK-TrkI inhibited cell proliferation in KM12 cells with a GI50 of 34 nM while exhibiting no activity in cell lines without TrkA fusion. Comparable activity was observed in Ba/F3 cell line expressing MPRIP-NTRK1 fusion, demonstrating inhibition of TrkA driven cell proliferation irrespective of the N-terminal fusion with a different gene, MPRIP. In the KM12 xenograft model, treatment with GVK-TrkI at various doses and dosing regimens of 20 mpk bid, 50 mpk bid and 100 mpk qd resulted in dose dependent inhibition with almost 100% tumor growth inhibition (TGI) at 100 mpk qd with 3 out of 8 mice showing complete tumor regression. Besides, at 50 mpk in various dosing regimens of qd, bid and bid alternate day showed good correlation of TGI with compound exposure. GVK-TrkI was well-tolerated, causing no weight loss or death compared with vehicle control. Based on the evidence of ATP binding site mutations contributing to clinical resistance with kinase inhibitor drugs, GVK-TrkI is predicted to be effective as a second generation TrkA inhibitor with a non-ATP binding site mechanism compared to pan-Trk inhibitors currently being pursued in clinic. These results suggest that GVK-TrkI is an effective therapeutic option in TrkA driven cancers with TrkA gene fusions, activating mutations and cancers with NGF/TrkA overexpression resulting in constitutive activation of TrkA pathway. Citation Format: Vijaya G. Tirunagaru, Kumaragurubaran Nagaswamy, Sayan Mitra, Srinivas Maddi, Ram Sudheer Adluri, Hemant Joshi, Jang B. Gupta. Discovery and characterization of a selective TrkA inhibitor for the treatment of TrkA-driven cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2643.

Expression of Nerve Growth Factor (NGF) and Its Receptors TrkA and p75 in the Reproductive Organs of Laying Hens

In order to investigate the expression levels of nerve growth factor (NGF) and its receptors (TrkA and p75) in prehierarchical follicles and oviducts of hens, five 130-day-old laying hens were examined by immunohistochemistry and RT-PCR analysis. NGF and its receptors were expressed in theca cells and granulosa cells of prehierarchical follicles, and they were also expressed in the epithelial cells of oviducts. The expression of the genes NGF, TrkA and p75 were significantly different in prehierarchical follicles (p<0.05 or p<0.01), and NGF and TrkA gene expression was significantly different in different parts of oviduct (p<0.05 or p<0.01). The expression of NGF and p75 mRNA levels was highest in large white follicle (LWF), as well as the expression of TrkA in small yellow follicle (SYF). In the oviduct, the expression of NGF was the highest in infundibulum, and lowest in isthmus. These results suggest that NGF may play an important role in the regulation of hen reproduction.

Novel transcripts reveal a complex structure of the human TRKA gene and imply the presence of multiple protein isoforms.

BackgroundTropomyosin-related kinase A (TRKA) is a nerve growth factor (NGF) receptor that belongs to the tyrosine kinase receptor family. It is critical for the correct development of many types of neurons including pain-mediating sensory neurons and also controls proliferation, differentiation and survival of many neuronal and non-neuronal cells. TRKA (also known as NTRK1) gene is a target of alternative splicing which can result in several different protein isoforms. Presently, three human isoforms (TRKAI, TRKAII and TRKAIII) and two rat isoforms (TRKA L0 and TRKA L1) have been described.ResultsWe show here that human TRKA gene is overlapped by two genes and spans 67 kb—almost three times the size that has been previously described. Numerous transcription initiation sites from eight different 5′ exons and a sophisticated splicing pattern among exons encoding the extracellular part of TRKA receptor indicate that there might be a large variety of alternative protein isoforms. TrkA genes in rat and mouse appear to be considerably shorter, are not overlapped by other genes and display more straightforward splicing patterns. We describe the expression profile of alternatively spliced TRKA transcripts in different tissues of human, rat and mouse, as well as analyze putative endogenous TRKA protein isoforms in human SH-SY5Y and rat PC12 cells. We also characterize a selection of novel putative protein isoforms by portraying their phosphorylation, glycosylation and intracellular localization patterns. Our findings show that an isoform comprising mainly of TRKA kinase domain is capable of entering the nucleus.ConclusionsResults obtained in this study refer to the existence of a multitude of TRKA mRNA and protein isoforms, with some putative proteins possessing very distinct properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-015-0215-x) contains supplementary material, which is available to authorized users.

TrkA is amplified in malignant melanoma patients and induces an anti-proliferative response in cell lines.

BackgroundThe nerve growth factor (NGF) receptor tyrosine-kinase TrkA is a well-known determinant of the melanocytic lineage, through modulation of the MAPK and AKT cascades. While TrkA gene is frequently rearranged in cancers, its involvement in malignant melanoma (MM) development is still unclear.MethodsWe analyzed a dataset of primary cutaneous MM (n = 31) by array comparative genomic hybridization (aCGH), to identify genomic amplifications associated with tumor progression. The analysis was validated by genomic quantitative PCR (qPCR) on an extended set of cases (n = 64) and the results were correlated with the clinical outcome. To investigate TrkA molecular pathways and cellular function, we generated inducible activation of the NGF-TrkA signaling in human MM cell lines.ResultsWe identified amplification of 1q23.1, where the TrkA locus resides, as a candidate hotspot implicated in the progression of MM. Across 40 amplicons detected, segmental amplification of 1q23.1 showed the strongest association with tumor thickness. By validation of the analysis, TrkA gene amplification emerged as a frequent event in primary melanomas (50 % of patients), and correlated with worse clinical outcome. However, experiments in cell lines revealed that induction of the NGF-TrkA signaling produced a phenotype of dramatic suppression of cell proliferation through inhibition of cell division and pronounced intracellular vacuolization, in a way straightly dependent on NGF activation of TrkA. These events were triggered via MAPK activity but not via AKT, and involved p21cip1 protein increase, compatibly with a mechanism of oncogene-induced growth arrest.ConclusionsTaken together, our findings point to TrkA as a candidate oncogene in MM and support a model in which the NGF-TrkA-MAPK pathway may mediate a trade-off between neoplastic transformation and adaptive anti-proliferative response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1791-y) contains supplementary material, which is available to authorized users.

Differential expression of genes encoding neurotrophic factors and their receptors along the septal-temporal axis of the rat hippocampus.

The hippocampus plays a key role in learning and emotional regulation. The hippocampus' function varies along its septotemporal axis, with the septal pole being more frequently involved in spatial learning and memory, and the temporal pole playing a greater role in emotional behaviors. In this study, we present findings aimed at checking the expression level of the genes encoding neurotrophins and their receptors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and their receptors (TrkA, TrkB and TrkC) in the hippocampus along the septotemporal axis. Using real-time PCR, several different expression patterns were observed. Remarkably, the expression of both NT-3 and TrkA genes in the septal hippocampus was higher than in the middle and temporal hippocampus. Higher expression of NT-3 and TrkA may implicate active neurogenesis in the dentate gyrus (DG) of the septal hippocampus because more neurogenesis occurs in the septal than the temporal DG of rats. Finally, the results obtained in this study emphasize the importance of choosing the hippocampal portion along its septotemporal axis for any hippocampal molecular and biochemical experimental studies.

A case of congenital insensitivity to pain with anhidrosis.

A case of congenital insensitivity to pain with anhidrosis.

From genes to pain: nerve growth factor and hereditary sensory and autonomic neuropathy type V

Hereditary sensory and autonomic neuropathy type V (HSAN V) is an autosomal recessive disorder characterized by the loss of deep pain perception. The anomalous pain and temperature sensations are due to the absence of nociceptive sensory innervation. The neurotrophin nerve growth factor (NGF), by binding to tropomyosin receptor A (TrkA) and p75NTR receptors, is essential for the development and survival of sensory neurons, and for pain perception during adulthood. Recently a homozygous missense mutation (R100W) in the NGF gene has been identified in HSAN V patients. Interestingly, alterations in NGF signalling, due to mutations in the NGF TRKA gene, have also been involved in another congenital insensitivity to pain, HSAN IV, characterized not only by absence of reaction to painful stimuli, but also anhidrosis and mental retardation. These symptoms are absent in HSAN V patients. Unravelling the mechanisms that underlie the differences between HSAN IV and V could assist in better understanding NGF biology. This review highlights the recent key findings in the understanding of HSAN V, including insights into the molecular mechanisms of the disease, derived from genetic studies of patients with this disorder.

Ginsenoside Re and Rd Enhance the Expression of Cholinergic Markers and Neuronal Differentiation in Neuro-2a Cells

In Alzheimer's disease (AD), extensive neuronal loss and a deficiency of the neurotransmitter acetylcholine (ACh) are the major characteristics during pathogenesis in the brain. In the present study, we aimed to investigate whether representative ginsenosides from ginseng can regulate choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), which are required for cholinergic neurotransmission. Our results revealed that Re and Rd induced effectively the expression of ChAT/VAChT genes in Neuro-2a cells as well as ACh elevation. Microtubule-associated protein-2 (MAP-2), nerve growth factor receptor (p75), p21, and TrkA genes and proteins were also significantly expressed. Moreover, both activated extracelullar signal-regulated protein kinase (ERK) and Akt were inhibited by K252a, a selective Trk receptor inhibitor. These findings strongly indicate that Re and Rd play an important role in neuronal differentiation and the nerve growth factor (NGF)-TrkA signaling pathway. High performance liquid chromatography analysis showed that Re and Rd administered orally were transported successfully into brain tissue and increased the level of ChAT and VAChT mRNA. The present study demonstrates that Re and Rd are selective candidates for upregulation of the expression of cholinergic markers, which may counter the symptoms and progress of AD.