Spatio-temporal expression profile of NGF and the two-receptor system, TrkA and p75NTR, in experimental autoimmune encephalomyelitis.

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

Effect of mating on mRNA and protein expression of beta nerve growth factor and its receptor, TrKA, in the oviduct of llama (Lama glama).

Copulation produces different stimuli in the female reproductive tract in camelids, which lead to ovulation. Expression of ß-nerve growth factor (ß-NGF) and its specific receptor, tropomyosin receptor kinase A (TrKA), was studied comparing the oviductal microenvironment of mated and nonmated llamas. ß-NGF and TrKA were expressed in the llama ampulla, isthmus, and utero-tubal-junction (UTJ), and they were mainly colocalized in the apical region of the oviductal mucosa. A TrKA immunosignal was also found in muscle cells and blood vessels, with the highest mark in UTJ muscle cells of copulated females. Both ß-NGF and TrKA transcripts were expressed in the three oviductal segments. Relative TrKA abundance did not differ between mated and nonmated females, but relative ß-NGF abundance was higher in the UTJ of copulated females (p < .05). ß-NGF might not be secreted into the oviductal fluid (OF) since the protein was not found in the OF of mated or nonmated females. Therefore, it can be concluded that the llama oviduct expresses the ß-NGF/TrKA system and that an increase in ß-NGF gene expression in the UTJ 24 h after copulation along with an increase in TrKA protein expression may indicate an important role in the gamete transport and fertilization process in llamas.

Evaluation of pan-TRK immunohistochemistry in infantile fibrosarcoma, lipofibromatosis-like neural tumour and histological mimics.

AIMS: Infantile fibrosarcoma is characterised by intersecting fascicles of spindle cells and ETV6-NTRK3 gene fusion in most cases. Given histological overlap with other spindle-cell tumours, the diagnosis can be challenging and often requires molecular confirmation. A recently developed pan-TRK antibody shows promise for identifying tumours with NTRK fusions. The purpose of this study was to evaluate the potential diagnostic utility of pan-TRK immunohistochemistry for infantile fibrosarcoma. METHODS AND RESULTS: We evaluated whole-tissue sections from 210 cases, including 15 infantile fibrosarcomas; five each lipofibromatosis-like neural tumour and lipofibromatosis; 10 each primitive myxoid mesenchymal tumour of infancy (PMMTI) and low-grade myofibroblastic sarcoma; 15 each fibrous hamartoma of infancy (FHI), myofibroma/myofibromatosis and desmoid-type fibromatosis; and 20 each low-grade fibromyxoid sarcoma, synovial sarcoma, spindle-cell rhabdomyosarcoma, malignant peripheral nerve sheath tumour, fibrosarcomatous dermatofibrosarcoma protuberans (F-DFSP) and nodular fasciitis. Immunohistochemistry was performed using a rabbit monoclonal pan-TRK antibody. Immunoreactivity for pan-TRK was observed in all 15 (100%) infantile fibrosarcomas, including diffuse immunoreactivity (>50% of cells) in 14 (93%) cases. Pan-TRK was positive in all five (100%) lipofibromatosis-like neural tumours. Of the 190 histological mimics, diffuse pan-TRK immunoreactivity was noted in 16 (8%) cases, including five PMMTI, five FHI (highlighting predominantly the primitive myxoid spindle-cell components), three F-DFSP, one low-grade myofibroblastic sarcoma, one myofibroma and one spindle-cell rhabdomyosarcoma. CONCLUSIONS: Diffuse pan-TRK immunoreactivity is a highly sensitive but not entirely specific diagnostic marker for infantile fibrosarcoma, and may be helpful in selecting patients for TRK-targeted therapy. As expected, lipofibromatosis-like neural tumours, which harbour NTRK1 fusions, also show diffuse pan-TRK immunoreactivity.

Expression of ß-NGF and high-affinity NGF receptor (TrKA) in llama (Lama glama) male reproductive tract and spermatozoa.

ß-Nerve growth factor (ß-NGF) is a seminal plasma element, responsible for inducing ovulation in camelids. The main organ of ß-NGF production remains nondescript. The aims of this study were to (a) characterize gene expression and protein localization of ß-NGF and its main receptor tyrosine kinase receptor A (TrKA) in the llama male reproductive tract, and (b) determine whether the seminal ß-NGF interacts with ejaculated sperm by localizing ß-NGF and TrKA in epididymal, ejaculated, and acrosome-reacted (AR) sperms and, additionally, by identifying ß-NGF presence in sperm-adsorbed proteins (SAP). Both ß-NGF and TrkA transcripts are widely expressed along the male reproductive tract, with a higher expression level of ß-NGF at prostate (p < 0.05). ß-NGF immunolabeling was only positive for prostate, whereas TrKA label was present in epithelial and muscular cells of testis, prostate, bulbourethral glands, and epididymis. Using an immunofluorescent technique, ß-NGF was colocalized with TrKA in the middle piece of ejaculated and AR sperm. However, only TrKA was observed in epididymal sperm indicating that ß-NGF could have a seminal origin. This was also confirmed by the identification of four ß-NGF isoforms in SAP. This study extends the knowledge about the participation of ß-NGF/TrkA in llama reproduction, providing evidence that may have roles in the regulation of sperm physiology.

Genetic modifiers of EGFR dependence in non-small cell lung cancer.

Lung adenocarcinomas harboring activating mutations in the epidermal growth factor receptor (EGFR) represent a common molecular subset of non-small cell lung cancer (NSCLC) cases. EGFR mutations predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and thus represent a dependency in NSCLCs harboring these alterations, but the genetic basis of EGFR dependence is not fully understood. Here, we applied an unbiased, ORF-based screen to identify genetic modifiers of EGFR dependence in EGFR-mutant NSCLC cells. This approach identified 18 kinase and kinase-related genes whose overexpression can substitute for EGFR in EGFR-dependent PC9 cells, and these genes include seven of nine Src family kinase genes, FGFR1, FGFR2, ITK, NTRK1, NTRK2, MOS, MST1R, and RAF1. A subset of these genes can complement loss of EGFR activity across multiple EGFR-dependent models. Unbiased gene-expression profiling of cells overexpressing EGFR bypass genes, together with targeted validation studies, reveals EGFR-independent activation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT pathways. Combined inhibition of PI3K-mTOR and MEK restores EGFR dependence in cells expressing each of the 18 EGFR bypass genes. Together, these data uncover a broad spectrum of kinases capable of overcoming dependence on EGFR and underscore their convergence on the PI3K-AKT and MEK-ERK signaling axes in sustaining EGFR-independent survival.

Bex3 Dimerization Regulates NGF-Dependent Neuronal Survival and Differentiation by Enhancing trkA Gene Transcription.

The development of the nervous system is a temporally and spatially coordinated process that relies on the proper regulation of the genes involved. Neurotrophins and their receptors are directly responsible for the survival and differentiation of sensory and sympathetic neurons; however, it is not fully understood how genes encoding Trk neurotrophin receptors are regulated. Here, we show that rat Bex3 protein specifically regulates TrkA expression by acting at the trkA gene promoter level. Bex3 dimerization and shuttling to the nucleus regulate the transcription of the trkA promoter under basal conditions and also enhance nerve growth factor (NGF)-mediated trkA promoter activation. Moreover, qChIP assays indicate that Bex3 associates with the trkA promoter within a 150 bp sequence, immediately upstream from the transcription start site, which is sufficient to mediate the effects of Bex3. Consequently, the downregulation of Bex3 using shRNA increases neuronal apoptosis in NGF-dependent sensory neurons deprived of NGF and compromises PC12 cell differentiation in response to NGF. Our results support an important role for Bex3 in the regulation of TrkA expression and in NGF-mediated functions through modulation of the trkA promoter.

The dynamics of trkA expression in the bovine ovary are associated with a luteotrophic effect of ovulation-inducing factor/nerve growth factor (OIF/NGF).

BACKGROUND: Ovulation-inducing factor in semen (OIF/NGF) influences ovulation and CL form and function in camelids and, remarkably, in cows. To test the hypothesis that the luteotrophic effect of OIF/NGF is mediated by an increase in trkA receptors in the ovulatory follicle and early CL, a study was designed to characterize the spatial and temporal distribution of trkA in ovarian follicles and CL at known stages of the bovine estrous cycle. METHODS: Sexually mature cattle (n = 14) were examined daily by transrectal ultrasonography to determine the day of ovulation (Day 0), and assigned randomly to be unilaterally ovariectomized on Day 2, 4, 6 or in the pre-ovulatory period just before or after exogenous LH treatment. After a complete interovulatory interval, the cows were re-assigned to a different day-group on which the remaining ovary was removed. Sections of ovarian tissue representing the dominant follicle, largest subordinate follicle, and the CL were processed for immunofluorescent detection and quantification of trkA receptor. RESULTS: TrkA immuno-fluorescence in ovarian tissues was restricted to follicles and the CL (no reaction in stroma or vessels), and was restricted to the cytoplasm (no nuclear staining). The trkA staining intensity, area of staining, and proportion of cells stained was greater in both theca and granulosa layers of dominant follicles than in that of subordinate follicles (P ≤ 0.05) in all day-groups except the Pre-LH group. Among dominant follicles, a progressive reduction in the immuno-positive reaction was detected from Day 2 to Day 6. Among subordinate follicles, immuno-reactivity remained low and unchanged except a rise in the Pre-LH group. The number of immuno-positive cells was greater in early developing CL (Days 2 and 4 combined) than in mature or regressing stage CL (Day 6, Pre- and Post-LH combined; P = 0.01). The intracellular distribution of trkA was more diffuse and widespread in dominant than subordinate follicles, particularly on Day 2 and Post-LH (P < 0.05). CONCLUSIONS: Distinct differences in trkA expression between dominant and subordinate follicles, particularly when circulating progesterone is minimal (early luteal development and after luteolysis) is consistent with a local role of OIF/NGF in follicle selection and early luteogenesis.

LY294002 induces in vitro apoptosis and overexpression of p75NTR in human uterine leiomyosarcoma HTB 114 cells.

Uterine leiomyosarcoma is a severe neoplasia resistant to conventional therapies. In previous studies, we have shown that human SK-UT-1 (ATCC HTB114) uterine leiomyosarcoma cell line secretes nerve growth factor (NGF) and expresses its receptors tyrosine kinase A receptor (TrKA) and low affinity nerve growth factor receptor (p75NTR). Furthermore, we have demonstrated that direct chemical inhibition or IgG neutralization of TrKA receptor induce apoptosis through p75NTR. In the present study, HTB114 cells were exposed to the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 with and without ß-NGF: apoptosis, cell cycle, activation of caspase-3 and protein kinase B (AKT) and TrKA/p75NTR phenotypic expression were evaluated. According to the type of exposure, LY294002 not only induced a relevant increase in apoptosis, but also produced a novel and unexpected phenotypic modulation of the NGF receptors with a downregulation of TrKA and an upregulation of p75NTR. This latter increase enhanced HTB114 apoptosis. Our study confirms that the interference on NGF transduction is a promising therapeutical approach in uterine leiomyosarcoma.

Expression of NGF, BDNF and their high-affinity receptors in ovine mammary glands during development and lactation.

The distribution of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their high-affinity tyrosine kinase receptors TrkA and TrkB was investigated by immunohistochemical method in the mammary gland of ewes from prepubertal stage to involution. NGF and BDNF protein expressions were strong during development of glands at prepubertal stage and during pregnancy and decreased during lactation and involution. The expressions localized in both stromal and parenchymal cells of developing gland were mainly arranged in the apical side of secretory cells during lactation. These observations were also confirmed at transcriptional level by RT-PCR analyses. The highest expression of all genes significantly occurred at prepubertal stage. NGF was then down-regulated from pregnancy to involution, and no statistical differences were observed among these stages. The receptor TrkA was also under-expressed from pregnancy to involution, and its expression significantly differed between pregnancy and 30 days of lactation and also between 30 and 60 days of lactation. BDNF was significantly down-regulated at 60 days of lactation in comparison with prepubertal stage and again between pregnancy and 30 days of lactation. The relative abundance of its receptor, TrkB, showed also a significant down-regulation at 60 days of lactation in comparison with pregnancy and involution. Among the myriad of other molecular signals involved in the mammary gland cycle, the local production of neuropeptides and their receptors could be of interest in understanding their potential role in mammary biology.

A novel role of suppressor of cytokine signaling-2 in the regulation of TrkA neurotrophin receptor biology.

Suppressor of cytokine signaling-2 (SOCS2) is a regulator of intracellular responses to growth factors and cytokines. Cultured dorsal root ganglia neurons from neonatal mice with increased or decreased SOCS2 expression were examined for altered responsiveness to nerve growth factor (NGF). In the presence of NGF, SOCS2 over-expression increased neurite length and complexity, whereas loss of SOCS2 reduced neurite outgrowth. Neither loss nor gain of SOCS2 expression altered the relative survival of these cells, suggesting that SOCS2 can discriminate between the differentiation and survival responses to NGF. Interaction studies in 293T cells revealed that SOCS2 immunoprecipitates with TrkA and a juxtamembrane motif of TrkA was required for this interaction. SOCS2 also immunoprecipitated with endogenous TrkA in PC12 Tet-On cells. Over-expression of SOCS2 in PC12 Tet-On cells increased total and surface TrkA expression. In contrast, dorsal root ganglion neurons which over-expressed SOCS2 did not exhibit significant changes in total levels but an increase in surface TrkA was noted. SOCS2-induced neurite outgrowth in PC12 Tet-On cells correlated with increased and prolonged activation of pAKT and pErk1/2 and required an intact SOCS2 SH2 domain and SOCS box domain. This study highlights a novel role for SOCS2 in the regulation of TrkA signaling and biology.

Augmented pain behavioural responses to intra-articular injection of nerve growth factor in two animal models of osteoarthritis.

OBJECTIVES: Nerve growth factor (NGF) is a promising analgesic target, particularly in osteoarthritis (OA) where existing therapies are inadequate. We hypothesised that pain responses to NGF are increased in OA joints. Here, NGF-evoked pain behaviour was compared in two rodent models of OA, and possible mechanisms underlying altered pain responses were examined. METHODS: OA was induced in rat knees by meniscal transection (MNX) or intra-articular monosodium iodoacetate injection (MIA). Once OA pathology was fully established (day 20), we assessed pain behaviour (hindlimb weight-bearing asymmetry and hindpaw mechanical withdrawal thresholds) evoked by intra-articular injection of NGF (10 µg). Possible mechanisms underlying alterations in NGF-induced pain behaviour were explored using indomethacin pretreatment, histopathological evaluation of synovitis, and rtPCR for NGF receptor (tropomyosin receptor kinase (Trk)-A) expression in dorsal root ganglia (DRG). RESULTS: Both the MIA and MNX models of OA displayed reduced ipsilateral weight bearing and hindpaw mechanical withdrawal thresholds, mild synovitis and increased TrkA expression in DRG. NGF injection into OA knees produced a prolonged augmentation of weight-bearing asymmetry, compared to NGF injection in non-osteoarthritic knees. However, hindpaw mechanical withdrawal thresholds were not further decreased by NGF. Pretreatment with indomethacin attenuated NGF-facilitated weight-bearing asymmetry and reversed OA-induced ipsilateral TrkA mRNA up-regulation. CONCLUSIONS: OA knees were more sensitive to NGF-induced pain behaviour compared to non-osteoarthritic knees. Cyclo-oxygenase products may contribute to increased TrkA expression during OA development, and the subsequent increased NGF sensitivity. Treatments that reduce sensitivity to NGF have potential to improve OA pain.

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.

Intracellular interaction of newly synthesized nerve growth factor and its receptors.

In autocrine cells, both a ligand and its receptors are synthesized in the same cell, but their intracellular interaction is not well known. We examined it using PC84 cells, a mutant PC12 cell line expressing nerve growth factor (NGF). We have already reported that the intracellular precursor of TrkA was phosphorylated and that MAP kinase was phosphorylated in PC84 cells. In this paper we found that the NGF receptors, TrkA and p75NTR, existed mainly as precursors, and most p75NTR localized inside PC84 cells. The phosphorylation of MAP kinase was also observed even when PC84 cells were incubated with anti-NGF antibody to block the extracellular interaction. These results suggest the possibility that newly synthesized NGF could interact intracellularly with the receptors in PC84 cells.

Dissecting the role of sortilin receptor signaling in neurodegeneration induced by NGF deprivation.

Sortilin is a member of the family of vacuolar protein sorting 10 protein domain receptors which has emerged as a co-receptor in cell death and neurodegeneration processes mediated by proneurotrophins. Here we tested the possibility that sortilin deficiency interferes with behavioral and neuropathological endpoints in a chronic Nerve Growth factor (NGF)-deprivation model of Alzheimer's disease (AD), the AD10 anti-NGF mouse. AD10 mice show cholinergic deficit, increased APP processing and tau hyper-phosphorylation, resulting in behavioral deficits in learning and memory paradigms assessed by novel object recognition and Morris water maze tests. Sort1(-/-) mice were crossed with AD10 anti-NGF mice and the neurodegenerative phenotype was studied. We found that the loss of sortilin partially protected AD10 anti-NGF mice from neurodegeneration. A protective effect was observed on non-spatial memory as assessed by novel object recognition, and histopathologically at the level of Aß and BFCNs, while the phosphotau increase was unaltered by knocking out sortilin. We suggest that sortilin might be involved in different aspects of neurodegeneration in a complex way, supporting the view that sortilin functions in the CNS are broader than being a co-receptor in proneurotrophin and neurotrophin signaling.

Messenger RNA expression patterns of p75 neurotrophin receptor and tropomyosin-receptor-kinase A following spinal cord injury.

BACKGROUND: Induction of p75 neurotrophin receptor (p75NTR) could be one of the first steps that initiate apoptotic cascade after injury, or it may indicate regeneration responses undertaken by the injured system, possibly in collaboration with resident tropomyosin-receptor-kinase (Trk). OBJECTIVE: To measure quantitative changes in messenger RNA (mRNA) expression levels of p75NTR, Trk A, and caspase-9 in rat's injured spinal cord (SCI). The reciprocal interaction between Trk and p75NTR signaling pathways can dictate cellular responses to neurotrophins. p75NTR can regulate Trk-dependent responses, but the role of Trk in regulating p75NTR-dependent signaling is not well documented. DESIGN: Using real-time polymerase chain reaction, this study analyzed changes in the mRNA abundance of the mentioned genes at 6, 24, and 72 hours and 7 and 10 days after SCI in adult male rats. SCI was induced at T9 level by transsection. RESULTS: Results show a complicated temporal and spatial pattern of alteration with different degrees and direction (up- or down-regulation) in p75NTR, Trk A, and caspase-9 mRNA expression levels after SCI. The greatest variation was seen in center regions following SCI. This study shows that alteration in p75NTR, Trk A, and caspase-9 expression starts as early as 6 hours after SCI. Alterations in p75NTR, Trk A, and caspase-9 expression within the spinal cord may play a key role in the apoptotic cell death. CONCLUSION: Results suggest that the role of p75NTR is to eliminate damaged cells by activating the apoptotic machinery, especially at the center of damage and during first week after injury.

Expression of tyrosine kinase receptors in cultured dorsal root ganglion neurons in the presence of monosialoganglioside and skeletal muscle cells.

The neurotrophic factor-like activity of monosialoganglioside (GM1) has been shown to activate tyrosine kinase receptors (Trk). Targets of neuronal innervation play a vital role in regulating the survival and differentiation of innervating neurotrophin-responsive neurons. Both GM1 and target skeletal muscle (SKM) cells are essential for the maintenance of the function of neurons. However, much less is known about the effects of GM1 or/and target SKM cells on the expression of Trk receptors in dorsal root ganglion (DRG) neurons. Here we have tested what extent to the expression of TrkA, TrkB, and TrkC receptors in primary cultured of DRG neurons in absence or presence of GM1 or/and SKM cells. In this experiment, we found that: (1) GM1 promoted expression of TrkA and TrkB but not TrkC in primary cultured DRG neurons; (2) target SKM cells promoted expression of TrkC but not TrkA and TrkB in neuromuscular cocultures without GM1 treatment; and (3) GM1 and target SKM cells had additional effects on expression of these three Trk receptors. The results of the present study offered new clues for a better understanding of the association of GM1 and target SKM on the expression of Trk receptors.

Neurotrophin and GDNF family ligand receptor expression in vagal sensory nerve subtypes innervating the adult guinea pig respiratory tract.

We combined retrograde tracing techniques with single-neuron RT-PCR to compare the expression of neurotrophic factor receptors in nodose vs. jugular vagal sensory neurons. The neurons were further categorized based on location of their terminals (tracheal or lungs) and based on expression of the ionotropic capsaicin receptor TRPV1. Consistent with functional studies, nearly all jugular neurons innervating the trachea and lungs expressed TRPV1. With respect to the neurotrophin receptors, the TRPV1-expressing jugular C-fiber neurons innervating both the trachea and lung compartments preferentially expressed tropomyosin-receptor kinase A (TrkA), with only a minority of neurons expressing TrkB or TrkC. The nodose neurons that express TRPV1 (presumed nodose C-fibers) innervate mainly intrapulmonary structures. These neurons preferentially expressed TrkB, with only a minority expressing TrkA or TrkC. The expression pattern in tracheal TRPV1-negative neurons, nodose tracheal presumed Aδ-fiber neurons as well as the intrapulmonary TRPV1-negative presumed Aß-fiber neurons, was similar to that observed in the nodose C-fiber neurons. We also evaluated the expression of GFRα receptors and RET (receptors for the GDNF family ligands). Virtually all vagal sensory neurons innervating the respiratory tract expressed RET and GFRα1. The jugular neurons also categorically expressed GFRα3, as well as ∼50% of the nodose neurons. GFRα2 was expressed in ∼50% of the neurons irrespective of subtype. The results reveal that Trk receptor expression in vagal afferent neurons innervating the adult respiratory tract depends more on the location of the cell bodies (jugular vs. nodose ganglion) than either the location of the terminals or the functional phenotype of the nerve. The data also reveal that in addition to neurotrophins, the GDNF family ligands may be important neuromodulators of vagal afferent nerves innervating the adult respiratory tract.

Tyrosine kinase A receptor (trkA): a potential marker in epithelial ovarian cancer.

OBJECTIVES: To evaluate the role of trkA receptor as a potential tumor marker in serous epithelial ovarian cancer and its relationship with the angiogenic factors expression as vascular endothelial growth factor (VEGF) and nerve growth factor (NGF). Additionally, to examine whether NGF and VEGF secreted by epithelial ovarian cancer (EOC) explants and from epithelial ovarian cancer cell line (A2780) are involved in the process of angiogenesis, such as cellular proliferation, migration and differentiation of the human endothelial cell line (EA.hy926). METHODS: The mRNA levels of VEGF, NGF and trkA receptors were measured using PCR in 60 ovarian samples. Cellular localization and semi-quantitative estimation of VEGF, NGF, total trkA and p-trkA was performed using IHC in epithelial cells. NGF, total trkA and p-trkA protein were also evaluated in endothelial cells from the same tissues. Human endothelial cell line EA.hy926 was cultured with conditioned media obtained from both EOC explants and from the A2780 cell line, with or without NGF stimulus. RESULTS: Significantly higher levels of NGF, total trkA and p-trkA protein expressions were observed in epithelial and endothelial cells in poorly differentiated EOC versus normal ovary. Interestingly, the p-trkA receptor expression level showed the most significant difference and its presence was only found in borderline tumor and EOC samples indicating the importance of trkA receptor in EOC as a potential tumor marker. A significant increase in proliferation, migration and differentiation of EA.hy926 cells was observed with NGF, and this effect was significantly reverted when NGF was immuno-blocked and when a trkA inhibitor was used, showing that NGF is an important angiogenic factor in EOC by activating its trkA receptor. CONCLUSION: These results indicate that p-trkA may be considered as a new potential tumor marker in EOC, and that NGF may also act as a direct angiogenic factor in EOC.

Expression of nerve growth factor and its receptors, TrkA and p75, in porcine ovaries.

The cellular localization of nerve growth factor (NGF) and its receptors (TrkA, p75) was investigated during the estrous cycle in gilts. Also, the levels of expression of these factors in walls of tertiary follicles and corpora lutea (CLs) were determined using Western blot. The ovaries from days 3, 7, 16 and 20 of the cycle revealed the presence of NGF and its receptors in oocytes of secondary and tertiary follicles, follicular cells of primary and secondary follicles, thecal and granulosa cells of tertiary follicles and steroidogenic cells of CLs. In wall cells of primary follicles, NGF, TrkA and p75 staining was strongest on day 16, while in secondary follicles, only p75 was more intensely stained on day 16 and 20. In walls of small (to 3 mm in diameter) and medium (4-6 mm in diameter) follicles, NGF staining was lower on day 16, and the p75 reaction was strongest on day 20. On day 20, NGF staining in large follicles (7-10 mm in diameter) was higher than in smaller follicles. The levels of NGF and p75 in small and medium follicles were highest on day 20. The contents of NGF and TrkA in large follicles on day 20 were higher than in smaller follicles. NGF and TrkA contents in CLs were highest on day 7. Our study demonstrates that NGF, TrkA and p75 are expressed in the ovary during the estrous cycle in gilts. These results suggest that NGF and its receptors may be important for ovarian function in cycling gilts.

NTRK fusions and Trk proteins: what are they and how to test for them.

The NTRK genes include a family of three genes, NTRK1, NTRK2, and NTRK3, which are associated with fusions with a variety of partner genes, leading to upregulation of three proteins, TrkA, TrkB, and TrkC. NTRK fusions occur in a variety of solid tumors: at high incidence in secretory carcinoma of the breast and salivary glands, congenital mesoblastic nephroma, and infantile fibrosarcoma; at intermediate incidence in thyroid carcinoma, particularly postradiation carcinomas and a subset of aggressive papillary carcinomas, Spitzoid melanocytic neoplasms, pediatric midline gliomas (particularly pontine glioma), and KIT/PDGFRA/RAS negative gastrointestinal stromal sarcomas; and at a low incidence in many other solid tumors. With new FDA-approved treatments available and effective in treating patients whose tumors harbor NTRK fusions, testing for these fusions has become important. A variety of technologies can be used for testing, including FISH, PCR, DNA, and RNA-based next-generation sequencing, and immunohistochemistry. RNA-based next-generation sequencing represents the gold standard for the identification of NTRK fusions, but FISH using break-apart probes and DNA-based next-generation sequencing also represent adequate approaches. Immunohistochemistry to detect increased levels of Trk protein may be very useful as a screening technology to reduce costs, although it alone does not represent a definitive diagnostic methodology.