TrkA Expression Research Articles

Effects of Reactive Oxygen and Nitrogen Species on TrkA Expression and Signalling: Implications for proNGF in Aging and Alzheimer's Disease.

Nerve growth factor (NGF) and its precursor form, proNGF, are critical for neuronal survival and cognitive function. In the brain, proNGF is the only detectable form of NGF. Dysregulation of proNGF in the brain is implicated in age-related memory loss and Alzheimer’s disease (AD). AD is characterized by early and progressive degeneration of the basal forebrain, an area critical for learning, memory, and attention. Learning and memory deficits in AD are associated with loss of proNGF survival signalling and impaired retrograde transport of proNGF to the basal forebrain. ProNGF transport and signalling may be impaired by the increased reactive oxygen and nitrogen species (ROS/RNS) observed in the aged and AD brain. The current literature suggests that ROS/RNS nitrate proNGF and reduce the expression of the proNGF receptor tropomyosin-related kinase A (TrkA), disrupting its downstream survival signalling. ROS/RNS-induced reductions in TrkA expression reduce cell viability, as proNGF loses its neurotrophic function in the absence of TrkA and instead generates apoptotic signalling via the pan-neurotrophin receptor p75NTR. ROS/RNS also interfere with kinesin and dynein motor functions, causing transport deficits. ROS/RNS-induced deficits in microtubule motor function and TrkA expression and signalling may contribute to the vulnerability of the basal forebrain in AD. Antioxidant treatments may be beneficial in restoring proNGF signalling and axonal transport and reducing basal forebrain neurodegeneration and related deficits in cognitive function.

PERINEURAL INVASION IN ORAL SQUAMOUS CELL CARCINOMA: INCIDENCE, CLINICAL IMPACT AND MOLECULAR INSIGHT

<h3>Background</h3> Perineural invasion (PNI) has been associated with poor prognosis in oral squamous cell carcinoma (OSCC). Nerve growth factor (NGF) and its receptor tyrosine kinase A (TrkA) may play a role in PNI in OSCC. <h3>Objective</h3> To characterize the incidence and prognostic correlation of PNI in OSCC and to determine whether NGF and TrkA expressions could be used as biological markers for PNI in OSCC. <h3>Methods</h3> A retrospective review of pathology reports of 430 patients with OSCC treated from 1992 to 2014 in Tayside, Scotland, and followed for 5 years or until death was undertaken. The expression of NGF and TrkA was assessed with immunohistochemistry in 132 tissue sections of OSCC. <h3>Results</h3> PNI was identified in 17.4% of OSCC. High expression of NGF and TrkA was seen in 84% and 92% of OSCC, respectively. PNI-positive OSCC expressed NGF and TrkA with greater frequency than PNI-negative OSCC (<i>P</i> < .05). PNI and high expression of NGF were associated significantly with pain in OSCC (<i>P</i> < .05). PNI-positive OSCC and high NGF/TrkA expression exhibited a greater potential for lymphovascular invasion and depth of invasion >4 mm (<i>P</i> < .05). PNI was significantly associated with advanced size, nodal metastasis, poor differentiation, and stage IV tumors (<i>P</i> < .05). PNI correlated significantly with disease-specific survival in OSCC (<i>P</i> < .05). <h3>Conclusions</h3> Higher level of expression of NGF and TrkA many predict PNI and therefore may be considered as biological markers for PNI in OSCC. Pain could be a predictive for PNI and NGF overexpression. PNI and NGF expression can predict the aggressiveness and prognosis of OSCC.

NGF induces pulmonary arterial hyperreactivity through Connexin 43 increased expression

Nerve growth factor (NGF) plays a critical role in pathophysiology of pulmonary hypertension (PH) by promoting pulmonary arterial (PA) inflammation, remodelling and hyperreactivity. To determine the mechanisms of NGF-induced PA hyperreactivity, focusing on Connexin 43 (Cx43), a gap junction protein essential for vascular reactivity. Expression of NGF, its TrkA receptor and Cx43 were evaluated by Western blotting in human PA smooth muscle cells (hPASMC) from control donors or from patients suffering from idiopathic pulmonary arterial hypertension (iPAH). Cx43 expression and localisation were evaluated by Western blotting, immunofluorescence and surface biotinylation, after NGF treatment. Gap junction activity was assessed by FRAP microscopy. Contractions of rat PA were induced by phenylephrine (PHE), with or without NGF pre-treatment. The role of Cx43 in NGF-induced rat PA hyperreactivity was evaluated ex vivo by use of 43 Gap26, a Cx43 blocking peptide, or in vivo by use of an anti-Cx43 siRNA. TrkA expression was increased in hPASMC from iPAH patients compared to control hPASMC. NGF increased Cx43 expression, plasma membrane localisation and phosphorylation, with a higher sensitivity in iPAH cells compared to controls. In both cell types, this increase was abolished with K252a, a TrkA kinase inhibitor. Activity of Cx43-dependent GAP junctions was also increased after NGF treatment, with iPAH hPASMC being more sensitive to NGF than control cells. NGF-induced PA hyperreactivity to PHE was inhibited after treatment with 43 Gap26 or with anti-Cx43 siRNA. NGF, through activation of its TrkA receptor, increases Cx43 expression and plasma membrane localisation in hPASMC. This mechanism enhances Cx43-dependent GAP junction activity and contributes to PA hyperreactivity. Moreover, NGF pathway is upregulated in iPAH hPASMC compared to control hPASMC, and iPAH cells are more sensitive to NGF than control cells regarding Cx43 increased expression and Cx43-dependent GAP junction increased activity.

Disrupted Trigeminal Ganglion Development in a Mouse Model of Familial Dysautonomia

Neurons in the trigeminal ganglion (TG) relay somatosensory information from the face and oral cavity to the central nervous system. Arising from the differentiation of both neural crest cell and ectodermal placode cell precursors during embryogenesis, neurons in the TG ultimately encode one sensory modality (i.e., touch, pain, temperature). TG neuron identity is discernable by particular molecular markers, including the mutually exclusive expression of the neurotrophin receptors TrkA, TrkB, or TrkC, which are required for long-term neuronal function and survival. While little is known about the mechanisms that establish and maintain distinct neuronal subpopulations in the TG, disease studies offer some insight. Familial Dysautonomia (FD) is a hereditary neuropathy caused by a mutation in Elongator complex protein 1 (ELP1), which leads to reduced ELP1 protein levels in sensory and autonomic neurons. FD patients have clinical deficits that point to abnormal TG function, including impaired sensation of facial pain and temperature. Moreover, FD patients have smaller trigeminal nerves compared to healthy controls, in the absence of overt degeneration. Given these findings, we hypothesize that ELP1 is necessary for the development and survival of TG sensory neurons. Our initial results uncovered cytoplasmic Elp1 expression in both placode cell- and neural crest cell-derived neurons of the developing mouse TG starting from embryonic day (E)10.5. Using a conditional knockout mouse model in which Elp1 is deleted from Wnt1-positive neural crest cells and derivatives, including sensory neurons, we next examined the effect of Elp1 loss on TG development. Our preliminary data reveal Elp1 depletion from neural crest cells leads to abnormal TG morphology and fewer trigeminal nerve branches as early as embryonic day (E)11, a phenotype that persists throughout development and suggests defects in both neural crest- and placode cell-derived neurons in the TG. TrkA-expressing neurons, which typically function in pain perception, are especially vulnerable to Elp1 loss during this period of TG formation. By E12.5, we observe decreased TrkA expression in distal trigeminal nerve endings and fewer TrkA-positive neuron cell bodies in the TG. Analyses from TUNEL staining reveal increased cell death in the TG at E12.5, suggesting TrkA-expressing neurons, progenitor cells, and/or supporting cells may undergo aberrant apoptosis in Elp1 conditional knockout TG. Together, our results confirm a role for Elp1 in the development and survival of TG sensory neurons. These findings help explain the loss of facial sensation in FD and may guide future therapeutic interventions to increase quality of life for FD patients.

Fndc5 knockdown significantly decreased the expression of neurotrophins and their respective receptors during neural differentiation of mouse embryonic stem cells.

Fibronectin type III domain-containing-5 (Fndc5) is a trans-membrane protein which is involved in a variety of cellular events including neural differentiation of mouse embryonic stem cells (mESCs) as its knockdown and overexpression diminishes and facilitates this process, respectively. However, downstream targets of Fndc5 in neurogenesis are still unclear. Neurotrophins including NGF, BDNF, NT-3, and NT-4 are the primary regulators of neuronal survival, growth, differentiation, and repair. These biomolecules exert their actions through binding to two different receptor families, Trk and p75NTR. In this study, considering the fact that neurotrophins and their receptors play crucial roles in neural differentiation of ESCs, we sought to evaluate whether knockdown of Fndc5 decreased neural differentiation of mESCs by affecting the neurotrophins and their receptors expression. Results showed that at neural progenitor stage, the mRNA and protein levels of BDNF, Trk, and p75NTR receptors decreased following the Fndc5 knockdown. In mature neural cells, still, the expression of Trk and p75NTR receptors at mRNA and protein levels and BDNF and NGF expression only at protein levels showed a significant decrease in Fndc5 knockdown cells compared to control groups. Taken together, our results suggest that decreased efficiency of neural differentiation following the reduction of Fndc5 expression could be attributed to decreased levels of NGF and BDNF proteins in addition to their cognate receptors.

Implication of TrkC-miR2 in neurotrophin signalling pathway regulation through NGFR transcript targeting.

TrkC and NGFR neurotrophin receptors are associated with cell death, cancer and differentiation. TrkC‐miR2, which is located in TrkC gene, is known to regulate Wnt signalling pathway, and its influence on other signalling pathways is under investigation. Here, through RT‐qPCR, dual‐luciferase assay and Western blotting we reveal that TrkC‐miR2 targets NGFR. Overexpression of TrkC‐miR2 also affected TrkA, TrkC, NFKB, BCL2 and Akt2 expressions involved in neurotrophin signalling pathway, and elevated survival rate of HEK293t and U87 cells was distinguished by flow cytometry and MTT assay. Consistently, an opposite expression correlation was obtained between TrkC‐miR2 and NGFR or TrkC for the duration of NT2 differentiation. Meanwhile, TrkC‐miR2 down‐regulation attenuated NT2 differentiation into neural‐like cells. Overall, here we present in silico and experimental evidence showing TrkC‐miR2 as a new controller in regulation of neurotrophin signalling pathway.

Umbilical mesenchymal stem cell-derived exosomes facilitate spinal cord functional recovery through the miR-199a-3p/145-5p-mediated NGF/TrkA signaling pathway in rats

BackgroundAlthough exosomes, as byproducts of human umbilical cord mesenchymal stem cells (hUC-MSCs), have been demonstrated to be an effective therapy for traumatic spinal cord injury (SCI), their mechanism of action remains unclear.MethodsWe designed and performed this study to determine whether exosomes attenuate the lesion size of SCI by ameliorating neuronal injury induced by a secondary inflammatory storm and promoting neurite outgrowth. We determined the absolute levels of all exosomal miRNAs and investigated the potential mechanisms of action of miR-199a-3p/145-5p in inducing neurite outgrowth in vivo and in vitro.ResultsmiR-199a-3p/145-5p, which are relatively highly expressed miRNAs in exosomes, promoted PC12 cell differentiation suppressed by lipopolysaccharide (LPS) in vitro through modulation of the NGF/TrkA pathway. We also demonstrated that Cblb was a direct target of miR-199a-3p and that Cbl was a direct target of miR-145-5p. Cblb and Cbl gene knockdown resulted in significantly decreased TrkA ubiquitination levels, subsequently activating the NGF/TrkA downstream pathways Akt and Erk. Conversely, overexpression of Cblb and Cbl was associated with significantly increased TrkA ubiquitination level, subsequently inactivating the NGF/TrkA downstream pathways Akt and Erk. Western blot and coimmunoprecipitation assays confirmed the direct interaction between TrkA and Cblb and TrkA and Cbl. In an in vivo experiment, exosomal miR-199a-3p/145-5p was found to upregulate TrkA expression at the lesion site and also promote locomotor function in SCI rats.ConclusionsIn summary, our study showed that exosomes transferring miR-199a-3p/145-5p into neurons in SCI rats affected TrkA ubiquitination and promoted the NGF/TrkA signaling pathway, indicating that hUC-MSC-derived exosomes may be a promising treatment strategy for SCI.

TrkA-mediated endocytosis of p75-CTF prevents cholinergic neuron death upon γ-secretase inhibition.

γ-secretase inhibitors (GSI) were developed to reduce the generation of Aβ peptide to find new Alzheimer's disease treatments. Clinical trials on Alzheimer's disease patients, however, showed several side effects that worsened the cognitive symptoms of the treated patients. The observed side effects were partially attributed to Notch signaling. However, the effect on other γ-secretase substrates, such as the p75 neurotrophin receptor (p75NTR) has not been studied in detail. p75NTR is highly expressed in the basal forebrain cholinergic neurons (BFCNs) during all life. Here, we show that GSI treatment induces the oligomerization of p75CTF leading to the cell death of BFCNs, and that this event is dependent on TrkA activity. The oligomerization of p75CTF requires an intact cholesterol recognition sequence (CRAC) and the constitutive binding of TRAF6, which activates the JNK and p38 pathways. Remarkably, TrkA rescues from cell death by a mechanism involving the endocytosis of p75CTF. These results suggest that the inhibition of γ-secretase activity in aged patients, where the expression of TrkA in the BFCNs is already reduced, could accelerate cholinergic dysfunction and promote neurodegeneration.

Ultra-purified alginate gel implantation decreases inflammatory cytokine levels, prevents intervertebral disc degeneration, and reduces acute pain after discectomy

Lumbar intervertebral disc (IVD) herniation causes severe low back pain (LBP), which results in substantial financial and emotional strains. Despite the effectiveness of discectomy, there is no existing treatment for post-operative LBP induced by progressive IVD degeneration. Two key factors of LBP are intradiscal inflammation, indicated by tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), and sensory nerve ingrowth into the inner layer of the annulus fibrosus, triggered by nerve growth factor/high-affinity tyrosine kinase A (TrkA) signalling. In an animal models of discectomy, the bioresorbable ultra-purified alginate (UPAL) gel with an extremely low-toxicity has been effective in acellular tissue repair. We aimed to investigate whether UPAL gel can alleviate LBP using a rat nucleus pulposus (NP) punch model and a rabbit NP aspirate model. In both models, we assessed TNF-α and IL-6 production and TrkA expression within the IVD by immunohistochemistry. Further, histological analysis and behavioural nociception assay were conducted in the rat model. UPAL gel implantation suppressed TNF-α and IL-6 production, downregulated TrkA expression, inhibited IVD degeneration, and reduced nociceptive behaviour. Our results suggest the potential of UPAL gel implantation as an innovative treatment for IVD herniation by reducing LBP and preventing IVD degeneration after discectomy.

Nerve growth factor in the equine joint

Nerve growth factor (NGF) is a neurotrophin with many functions. In humans, it is involved in inflammation, nerve growth, apoptosis and pain signalling. Increased concentrations of NGF in synovial fluid has been shown in humans and dogs with osteoarthritis. Despite osteoarthritis being a common problem in horses, no studies have previously been published on NGF in the equine joint. The aim of this study was to quantify NGF in equine synovial fluid from healthy joints, acutely inflamed septic joints and joints with structural changes associated with osteoarthritis. A secondary aim was to identify the localisation of NGF and its two receptors, TrkA and p75NTR, in healthy and osteoarthritic articular cartilage. NGF concentrations in synovial fluid from osteoarthritic joints (n = 27), septic joints (n = 9) and healthy joints (n = 16) were determined by ELISA. In addition, articular cartilage from osteoarthritic and healthy joints was examined for NGF, TrkA and p75NTR using immunohistochemistry staining.NGF was present in equine synovial fluid and articular cartilage. Compared to synovial fluid from healthy joints, NGF concentration was higher in synovial fluid from joints with structural osteoarthritic changes (P = 0.032) or acute septic inflammation (P = 0.006). In articular cartilage with severe osteoarthritic changes, there was more abundant positive immunohistochemistry staining for NGF and its receptors than in normal articular cartilage. Further studies should focus on identifying precursor forms of NGF, and on receptor expression and downstream signalling of TrkA and P75NTR in health and disease.

Comparison of tyrosine kinase domain properties for the neurotrophin receptors TrkA and TrkB.

The tropomyosin-related kinase (Trk) family consists of three receptor tyrosine kinases (RTKs) called TrkA, TrkB, and TrkC. These RTKs are regulated by the neurotrophins, a class of secreted growth factors responsible for the development and function of neurons. The Trks share a high degree of homology and utilize overlapping signaling pathways, yet their signaling is associated with starkly different outcomes in certain cancers. For example, in neuroblastoma, TrkA expression and signaling correlates with a favorable prognosis, whereas TrkB is associated with poor prognoses. To begin to understand how activation of the different Trks can lead to such distinct cellular outcomes, we investigated differences in kinase activity and duration of autophosphorylation for the TrkA and TrkB tyrosine kinase domains (TKDs). We find that the TrkA TKD has a catalytic efficiency that is ∼2-fold higher than that of TrkB, and becomes autophosphorylated in vitro more rapidly than the TrkB TKD. Studies with mutated TKD variants suggest that a crystallographic dimer seen in many TrkA (but not TrkB) TKD crystal structures, which involves the kinase-insert domain, may contribute to this enhanced TrkA autophosphorylation. Consistent with previous studies showing that cellular context determines whether TrkB signaling is sustained (promoting differentiation) or transient (promoting proliferation), we also find that TrkB signaling can be made more transient in PC12 cells by suppressing levels of p75NTR. Our findings shed new light on potential differences between TrkA and TrkB signaling, and suggest that subtle differences in signaling dynamics can lead to substantial shifts in the cellular outcome.

Ultrasonic Imaging Combined with Hysteroscopy in Diagnosis of Endometrial Polyps Based on Multioperator Algorithm and Analysis of Nerve Growth Factor Receptor Transmembrane Protein Expression

The paper aims to understand the expression of nerve growth factor receptor (TrkA) in the ectopic and entopic endometrium of patients with adenomyosis and dysmenorrhea and, at the same time, explore and analyze the combination of hysteroscopy and ultrasound imaging based on multioperator algorithms. The clinical treatment value of color Doppler ultrasound in the diagnosis and treatment of endometrial polyps. The immunohistochemical avidin-peroxidase staining method (SP method) was used to detect 20 cases of ectopic endometrium and entopic endometrium in patients with dysmenorrhea of adenomyosis (study group) and 22 cases of uterine fibroids in the control group. The expression of nerve growth factor receptor in the endometrium of patients with dysmenorrhea and comparative analysis. At the same time, all patients were examined by transvaginal color Doppler ultrasound and hysteroscopy. The scores of endometrial polyps, diagnostic efficiency of the methods, and receiver operating characteristic curve analysis of various diagnostic methods were observed and recorded in the 2 groups. There was no significant difference in the expression of TrkA in the ectopic endometrium and the entopic endometrium in the study group (P > 0.05), but the expression intensity of TrkA in the 2 groups was significantly higher than that in the control group (P = 0.05). In the diagnosis of endometrial polyps, the area of receiver operating characteristic curve in the parallel combined diagnosis of ultrasound and hysteroscopy is significantly larger than that under the combined diagnosis of series. TrkA is widely expressed in the ectopic and entopic endometrium of patients with adenomyosis and dysmenorrhea and may play an important role in the onset of adenomyosis and dysmenorrhea. Hysteroscopy combined with color Doppler ultrasound can effectively improve the diagnostic accuracy of patients with endometrial polyps.

TrkC Overexpression Protects Sevoflurane-Induced Neurotoxicity in Human Induced Pluripotent Stem Cell-Derived Neurons

Background: Inhaled anesthetic sevoflurane (SEVO) may induce cortical neurotoxicity and memory dysfunction in both animals and humans. In this study, we investigated the toxic effects of SEVO on human induced pluripotent stem cell (iPS)-derived neurons. Methods: Human iPS-derived neurons were exposed to SEVO in vitro. SEVO-induced toxic effects were examined with the viability, live caspase 3/7, and neurite density assays, respectively. The effects of SEVO on the receptors of the tyrosine kinases TrkA, TrkB, and TrkC were assessed by qRT-PCR. TrkA, TrkB, and TrkC were ectopically overexpressed in human iPS-derived neurons. Their functional effects on SEVO-induced human iPS-derived neuron toxicity were further investigated. Results: SEVO induced dose-dependent cell death, caspase 3/7 elevation, neurite degeneration, and the downregulation of Trk receptors in human iPS-derived neurons. Adenovirus-mediated Trk receptor overexpression selectively upregulated endogenous TrkA, TrkB, or TrkC gene expressions in human iPS-derived neurons. Specifically, TrkC overexpression, but not TrkA or TrkB overexpression was found to overcome the neurotoxic effects of SEVO in human iPS-derived neurons. Conclusions: SEVO may induce neurotoxicity in human iPS-derived neurons, and its neurotoxic damage could be protected by the overexpression of TrkC.

Ultra-Purified Alginate Gel Implantation Decreases Inflammatory Cytokine Levels, Prevents Intervertebral Disc Degeneration, and Reduces Acute Pain after Discectomy

Background: Lumbar intervertebral disc (IVD) herniation causes severe low back pain (LBP), which results in substantial financial and emotional strains. Despite the effectiveness of discectomy, there is no existing treatment for post-operative LBP induced by progressive IVD degeneration. Two key factors of LBP are intradiscal inflammation, indicated by tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), and sensory nerve ingrowth into the inner layer of the annulus fibrosus, triggered by nerve growth factor/high-affinity tyrosine kinase A (TrkA) signalling. In an animal models of discectomy, the bioresorbable ultra-purified alginate (UPAL) gel with an extremely low-toxicity has been effective in acellular tissue repair. Methods: We aimed to investigate whether UPAL gel can alleviate LBP using a rat nucleus pulposus (NP) punch model and a rabbit NP aspirate model. In both models, we assessed TNF-α and IL-6 production and TrkA expression within the IVD by immunohistochemistry. Further, histological analysis and behavioural nociception assay were conducted in the rat model. Findings: UPAL gel implantation suppressed TNF-α and IL-6 production, downregulated TrkA expression, inhibited IVD degeneration, and reduced nociceptive behaviour. Interpretation: Our results suggest the potential of UPAL gel implantation as an innovative treatment for IVD herniation by reducing LBP and preventing IVD degeneration after discectomy. Funding Statement: Ministry of Education, Culture, Sports, Science, and Technology of Japan, Akiyama Life Science Foundation, Mochida Pharmaceutical Co., Ltd., and Takeda Science Foundation. Declaration of Interests: The authors declare that no competing interests exist. Ethics Approval Statement: All animal procedures were performed in accordance with the ARRIVE guideline and National Institutes of Health guide for the care and use of Laboratory animals, and the study was approved by the Institutional Animal Care and Use Committee of Hokkaido University (17-0122).

Increased hippocampal TrkA expression ameliorates cranial radiation‑induced neurogenesis impairment and cognitive deficit via PI3K/AKT signaling.

Cognitive deficit is one of the most serious complications of cranial radiotherapy of head and neck cancers. However, the underlying mechanism of this cognitive impairment remains unclear. In the present study, the role of tropomyosin receptor kinase A (TrkA) and its ligand neurotrophin nerve growth factor (NGF) were investigated following whole-brain irradiation (WBI). Young male Sprague-Dawley rats underwent WBI at a single dose of 10 Gy. WBI was determined to result in notable memory decline and substantial neurogenesis impairment in the hippocampus 3 months post-irradiation. Compared with the control group, TrkA protein expression was greater in irradiated rats 1 week after WBI, which then decreased significantly by the 3-month time-point. However, no difference in NGF expression was observed from 1 day to 3 months post-WBI. Overexpression of hippocampal TrkA in rats using adeno-associated virus ameliorated memory decline induced by irradiation. Additionally, upregulating TrkA expression rescued irradiation-induced hippocampal precursor cell proliferation and promoted neurogenesis. PI3K, Akt and ERK1/2 phosphorylation were also revealed to be significantly inhibited by WBI, which was ameliorated by TrkA overexpression. Findings of the present study indicated that the TrkA-dependent signaling pathway may serve a critical role in radiotherapy-induced cognitive deficit and impairments in neurogenesis.

NGF induces pulmonary arterial hyperreactivity through Connexin 43 increased expression

We have previously shown that the nerve growth factor (NGF) plays a critical role in pathophysiology of pulmonary hypertension (PH) by promoting pulmonary arterial (PA) inflammation, remodelling and hyperreactivity. To characterize mechanisms activated by NGF to induce PA hyperreactivity, focusing on Connexin 43 (Cx43), a gap junction protein essential for vascular reactivity. Expression of NGF and its TrkA receptor were evaluated by Western blotting in human PA smooth muscle cells (hPASMC) from controls or from patients suffering from idiopathic pulmonary arterial hypertension (IPAH). In cells treated or not with NGF (0.1-100 ng/ml, 24 h), Cx43 expression and localisation were evaluated by Western blotting, immunofluorescence and cell surface biotinylation. Gap junction activity was assessed by Lucifer yellow (LY) dye transfer assay. Contractions of control rat PA were induced by phenylephrine (PHE, 10-10-10-4 M) in absence or presence of NGF (100 ng/ml, 24 h). The role of Cx43 in NGF-induced rat PA reactivity was evaluated by use of 43Gap26 (Cx43 blocking peptide, 300 μM), or an anti-Cx43 siRNA (10nmol IV). NGF and TrkA expression were increased in hPASMC from IPAH patients compared to controls. NGF increased Cx43 expression in hPASMC from both controls and IPAH patients, but with a higher sensitivity in IPAH cells (maximum at 1 ng/ml vs. 100 ng/ml). This increase was abolished after treatment with K252a (TrkA kinase inhibitor). In control hPASMC, NGF increased Cx43 plasma membrane localisation and LY diffusion speed through Gap junctions. NGF-induced PA hyperreactivity to PHE was inhibited ex vivo by treatment with 43Gap26 and in vivo after administration of Cx43 siRNA. NGF pathway is unregulated in IPAH cells. NGF increases Cx43 PA expression through activation of its TrkA receptor. Cx43 increased expression at hPASMC plasma membrane upregulates gap junction activity, thus promoting NGF-induced PA hyperreactivity.

Xiaoyao Pills Attenuate Inflammation and Nerve Injury Induced by Lipopolysaccharide in Hippocampal Neurons In Vitro.

Lipopolysaccharides (LPS) are proinflammation mediators that can induce the inflammatory model of the hippocampal neuron, and neuroinflammation participates in the pathophysiology of depression. Xiaoyao Pill is a classical Chinese medicine formula that has been used for the treatment of mental disorders such as depression in China since the Song dynasty. We established a hippocampal neuronal cell inflammation model by LPS and investigate the intervention effect and mechanism of Xiaoyao Pills. The expression levels of IL-6, TNF-α, IDO, 5-HT, brain-derived neurotrophic factor, and β-nerve growth factor were detected by enzyme-linked immunosorbent assay. mRNA levels of IL-6, TNF-α, 5-HT1A, IDO-1, brain-derived neurotrophic factor, nerve growth factor, tropomyosin receptor kinase B, tropomyosin receptor kinase A, and cAMP response element-binding protein were detected by reverse transcription-polymerase chain reaction. To further validate, protein expression was determined by western blot and immunofluorescence. Lipopolysaccharide-induced neuroinflammatory state resulted in the release of IL-6, TNF-α, and IDO and a decrease of BDNF, NGF, TrkB, TrkA, CREB, p-CREB, p-CREB/CREB, and SYP and inhibited hippocampal neurogenesis in the hippocampal neuron. Xiaoyao Pills significantly decreased the levels of IL-6, TNF-α, and IDO in cell supernatant and increased the expression of BDNF, NGF, TrkB, TrkA, CREB, p-CREB, p-CREB/CREB, and SYP as well as the average optical density of BrdU/NeuN double-labelled positive cells. Our study shows that lipopolysaccharides induce inflammation and nerve damage in hippocampal neurons, which are closely related to the pathological mechanism of depression. Xiaoyao Pills (XYW) play an important neuroprotective effect, which is related to its inhibition of neuronal inflammation and promoting the recovery of nerve injury. These results provide a pharmacologic basis for the treatment of depression of XYW in clinical application.

The Receptor Tyrosine Kinase TrkA Is Increased and Targetable in HER2-Positive Breast Cancer.

The tyrosine kinase receptor A (NTRK1/TrkA) is increasingly regarded as a therapeutic target in oncology. In breast cancer, TrkA contributes to metastasis but the clinicopathological significance remains unclear. In this study, TrkA expression was assessed via immunohistochemistry of 158 invasive ductal carcinomas (IDC), 158 invasive lobular carcinomas (ILC) and 50 ductal carcinomas in situ (DCIS). TrkA was expressed in cancer epithelial and myoepithelial cells, with higher levels of TrkA positively associated with IDC (39% of cases) (p < 0.0001). Interestingly, TrkA was significantly increased in tumours expressing the human epidermal growth factor receptor-2 (HER2), with expression in 49% of HER2-positive compared to 25% of HER2-negative tumours (p = 0.0027). A panel of breast cancer cells were used to confirm TrkA protein expression, demonstrating higher levels of TrkA (total and phosphorylated) in HER2-positive cell lines. Functional investigations using four different HER2-positive breast cancer cell lines indicated that the Trk tyrosine kinase inhibitor GNF-5837 reduced cell viability, through decreased phospho-TrkA (Tyr490) and downstream AKT (Ser473) activation, but did not display synergy with Herceptin. Overall, these data highlight a relationship between the tyrosine kinase receptors TrkA and HER2 and suggest the potential of TrkA as a novel or adjunct target for HER2-positive breast tumours.

Related expression of TRKA and P75 receptors and the changing copy number of MYC-oncogenes determine the sensitivity of brain tumor cells to the treatment of the nerve growth factor in combination with cisplatin and temozolomide.

Objectives Oncological diseases are an urgent medical and social problem. The chemotherapy induces not only the death of the tumor cells but also contributes to the development of their multidrug resistance and death of the healthy cells and tissues. In this regard, the search for the new pharmacological substances with anticancer activity against drug-resistant tumors is of utmost importance. In the present study we primarily investigated the correlation between the expression of TrkA and p75 receptors with the nerve growth factor (NGF) and cisplatin or temozolomide sensitivity of anaplastic astrocytoma (AA), glioblastoma (GB) and medulloblastoma (MB) cell cultures. We then evaluated the changing of copy numbers of MYCC and MYCN and its correlation with cytotoxicity index (CI) in MBcells under NGF exposition. Methods The primary cell cultures were obtained from the tumor biopsy samples of the patients with AA (n=5), GB (n=7) or MB (n=25) prior to radiotherapy and chemotherapy. The cytotoxicity effect of NGF and its combinations with cisplatin or temozolomide, the relative expression of TrkA and p75 receptors, its correlations with CI in AA, GB and MB primary cell cultures were studied by trypan blue cytotoxicity assay and immunofluorescence staining respectively. The effect of NGF on MYCC and MYCN copy numbers in MBcell cultures was studied by fluorescence in situ hybridization. Results We found that the expression of TrkA and p75 receptors (p=0.03) and its ratio (p=0.0004) depends on the sensitivity of AA and GBcells to treatment with NGF and its combinations with cisplatin or temozolomide. NGF reduces (p<0.05) the quantity of MBcells with six or eight copies of MYCN and three or eight copies of MYCC. Besides, NGF increases (p<0.05) the quantity of MBcells containing two copies of both oncogenes. The negative correlation (r=-0.65, p<0.0001) is established between MYCC average copy numbers and CI of NGF in MBcells. Conclusions The relative expression of NGF receptors (TrkA/p75) and its correlation with CI of NGF and its combinations in AA and GBcells point to the mechanism involving a cell death signaling pathway. NGF downregulates (p<0.05) some increased copy numbers of MYCC and MYCN in the human MBcell cultures, and upregulates normal two copies of both oncogenes (p<0.05).

Immunohistochemistry as a screening tool for NTRK gene fusions: results of a first Belgian ring trial

A Belgian ring trial for pan-TRK immunohistochemistry (IHC) staining was organised to harmonise pan-TRK IHC staining protocols and interpretation. As a reference method, the VENTANA pan-TRK Assay (clone EPR17341) on the Benchmark Ultra platform was selected. Six samples were selected: 2 negative, 2 fusion positive and 2 samples with wild-type endogenous TRK expression. Each participating laboratory stained the slides using their routine pan-TRK IHC and reported their results. In addition, they were asked to return one TRK-stained slide from each case. The coordinating lab evaluated these slides, compared them with the reference method and scored them. Two clones were used during the ring trial: A7H6R (Cell Signaling) and EPR17341 (Abcam/Ventana). Seven protocols achieved a sufficient performance mark, and three labs were advised to further optimise the protocol. Interpretation of pan-TRK IHC proved to be challenging in cases with physiological TRK expression. In addition, depending on the NTRK fusion partner, the staining can vary strongly in both intensity and staining pattern. Labs using the Ventana ready-to-use system based on the EPR17341 clone and using the recommended protocol settings scored best. However, given some small optimisation, all labs scored well on the technical staining and the succeeding evaluation.