TrkA Protein Research Articles

Discovery of novel indazole derivatives as second-generation TRK inhibitors

NTRK gene fusion leads to the activation of downstream signaling pathways, which is a oncogenic driver in various cancers. NTRK fusion-positive cancers can be treated with the first-generation TRK inhibitors, larotrectinib and entrectinib. Unfortunately, the patients eventually face the dilemma of no drugs available as the emergence of certain resistance mutations. The development of efficient and broad-spectrum second-generation TRK inhibitors is still of great significance. Here, we analyzed the binding modes of compounds 6, 10 with TRKA protein, respectively, a series of novel indazole TRK inhibitors were designed and synthesized using molecular hybridization strategy. Among them, the optimal compound B31 showed strong antiproliferative activities against Km-12, Ba/F3-TRKAG595R, and Ba/F3-TRKAG667C cell lines with IC50 values of 0.3, 4.7, and 9.9 nM, respectively. And the inhibitory effect against TRKAG667C (IC50 = 9.9 nM) was better than that of selitrectinib (IC50 = 113.1 nM). Further, compound B31 exhibited moderate kinase selectivity and excellent plasma stability (t1/2 > 480 min). In vivo pharmacokinetic studies in Sprague-Dawley rats showed that B31 had acceptable pharmacokinetic properties.

Genetic etiology study in a large cohort with congenital insensitivity to pain with anhidrosis.

Pathogenic variations in the NTRK1 can cause congenital insensitivity to pain with anhidrosis (CIPA), a rare autosomal recessive inherited neuropathy. The precise diagnosis of CIPA relies on the identification of pathogenic genotypes. Therefore, it is essential to expand the NTRK1 variation spectrum and improve molecular diagnosis methods. In this study, 74 probands with typical manifestations of CIPA but unknown genotypes were recruited. A comprehensive molecular genetic analysis was performed to identify variations in the NTRK1 , using techniques including Sanger and next-generation sequencing, bioinformatic analysis, quantitative polymerase chain reaction (qPCR), gap-PCR, short tandem repeat (STR) genotyping, and reverse-transcription PCR. In addition, functional assays were conducted to determine the pathogenicity of variants of uncertain significance (VUS) and further characterized changes in glycosylation and phosphorylation of 14 overexpressed mutant vectors with variants at different domains in the TrkA protein, which is encoded by NTRK1 . A total of 48 variations in the NTRK1 were identified, including 22 novel ones. When combined with data from another 53 CIPA patients examined in our previous work, this study establishes the largest genotypic and phenotypic spectra of CIPA worldwide, including 127 CIPA families. Moreover, functional studies indicated that the pathogenicity of VUS mainly affected insufficient glycosylation in the extracellular domain and abnormal phosphorylation in the intracellular domain. This study not only provides important evidence for precise diagnosis of CIPA but also further enriches our understanding of the pathogenesis of this disease.

Design, synthesis and anticancer evaluation of novel arylhydrazones of active methylene compounds

Nerve growth factor (NGF) and its receptor, tropomyosin kinase receptor kinase type A (TrkA) is emerging as an important target for Glioblastoma (GBM) treatment. TrkA is the cancer biomarker majorly involved in tumor invasion and migration into nearby normal tissue. However, currently, available Trk inhibitors exhibit many adverse effects in cancer patients, thus demanding a novel class of ligands to regulate Trk signaling. Here, we exploited the role of TrkA (NTRK1) expression from the 651 datasets of brain tumors. RNA sequence analysis identified overexpression of NTRK1 in GBM, recurrent GBM as well in Oligoastrocytoma patients. Also, TrkA expression tends to increase over the higher grades of GBM. TrkA protein targeting hydrazone derivatives, R48, R142, and R234, were designed and their mode of interaction was studied using molecular docking and dynamic simulation studies. Ligands' stability and binding assessment reveals R48, 2 2-(2-(2-hydroxy-4-nitrophenyl) hydrazineylidene)-1-phenylbutane-1,3-dione, as a potent ligand that interacts well with TrkA's hydrophobic residues, Ile, Phe, Leu, Ala, and Val. R48- TrkA exhibits stable binding potentials with an average RMSD value <0.8 nm. R48 obeyed Lipinski's rule of five and possessed the best oral bioavailability, suggesting R48 as a potential compound with drug-likeness properties. In-vitro analysis also revealed that R48 exhibited a higher cytotoxicity effect for U87 GBM cells than TMZ with the IC50 value of 68.99 μM. It showed the lowest percentage of cytotoxicity to the non-cancerous TrkA expressing MEF cells. However, further SiRNA analysis validates the non-specific binding of R48, necessitating structural alteration for the development of R48-based TrkA inhibitor for GBM therapeutics.

Auricular Vagal Nerve Stimulation Inhibited Central Nerve Growth Factor/Tropomyosin Receptor Kinase A/Phospholipase C-Gamma Signaling Pathway in Functional Dyspepsia Model Rats With Gastric Hypersensitivity

ObjectiveFunctional dyspepsia (FD), which has a complicated pathophysiologic process, is a common functional gastrointestinal disease. Gastric hypersensitivity is the key pathophysiological factor in patients with FD with chronic visceral pain. Auricular vagal nerve stimulation (AVNS) has the therapeutic effect of reducing gastric hypersensitivity by regulating the activity of the vagus nerve. However, the potential molecular mechanism is still unclear. Therefore, we investigated the effects of AVNS on the brain-gut axis through the central nerve growth factor (NGF)/ tropomyosin receptor kinase A (TrkA)/phospholipase C-gamma (PLC-γ) signaling pathway in FD model rats with gastric hypersensitivity. Materials and MethodsWe established the FD model rats with gastric hypersensitivity by means of colon administration of trinitrobenzenesulfonic acid on ten-day-old rat pups, whereas the control rats were given normal saline. AVNS, sham AVNS, K252a (an inhibitor of TrkA, intraperitoneally), and K252a + AVNS were performed on eight-week-old model rats for five consecutive days. The therapeutic effect of AVNS on gastric hypersensitivity was determined by the measurement of abdominal withdrawal reflex response to gastric distention. NGF in gastric fundus and NGF, TrkA, PLC-γ, and transient receptor potential vanilloid 1 (TRPV1) in the nucleus tractus solitaries (NTS) were detected separately by polymerase chain reaction, Western blot, and immunofluorescence tests. ResultsIt was found that a high level of NGF in gastric fundus and an upregulation of the NGF/TrkA/PLC-γ signaling pathway in NTS were manifested in model rats. Meanwhile, both AVNS treatment and the administration of K252a not only decreased NGF messenger ribonucleic acid (mRNA) and protein expressions in gastric fundus but also reduced the mRNA expressions of NGF, TrkA, PLC-γ, and TRPV1 and inhibited the protein levels and hyperactive phosphorylation of TrkA/PLC-γ in NTS. In addition, the expressions of NGF and TrkA proteins in NTS were decreased significantly after the immunofluorescence assay. The K252a + AVNS treatment exerted a more sensitive effect on regulating the molecular expressions of the signal pathway than did the K252a treatment. ConclusionAVNS can regulate the brain-gut axis effectively through the central NGF/TrkA/PLC-γ signaling pathway in the NTS, which suggests a potential molecular mechanism of AVNS in ameliorating visceral hypersensitivity in FD model rats.

Toward in vivo proof of binding of 18F-labeled inhibitor [18F]TRACK to peripheral tropomyosin receptor kinases

BackgroundTropomyosin receptor kinases (TrkA, TrkB, TrkC) are a family of tyrosine kinases primarily expressed in neuronal cells of the brain. Identification of oncogenic alterations in Trk expression as a driver in multiple tumor types has increased interest in their role in human cancers. Recently, first- and second-generation 11C and 18F-labeled Trk inhibitors, e.g., [18F]TRACK, have been developed. The goal of the present study was to analyze the direct interaction of [18F]TRACK with peripheral Trk receptors in vivo to prove its specificity for use as a functional imaging probe.MethodsIn vitro uptake and competition experiments were carried out using the colorectal cancer cell line KM12. Dynamic PET experiments were performed with [18F]TRACK, either alone or in the presence of amitriptyline, an activator of Trk, entrectinib, a Trk inhibitor, or unlabeled reference compound TRACK in KM12 tumor-bearing athymic nude mice as well as B6129SF2/J and corresponding B6;129S2-Ntrk2tm1Bbd/J mice. Western blot and immunohistochemistry experiments were done with KM12 tumors, brown adipose tissue (BAT), and brain tissue samples.ResultsUptake of [18F]TRACK was increasing over time reaching 208 ± 72% radioactivity per mg protein (n = 6/2) after 60 min incubation time. Entrectinib and TRACK competitively blocked [18F]TRACK uptake in vitro (IC50 30.9 ± 3.6 and 29.4 ± 9.4 nM; both n = 6/2). [18F]TRACK showed uptake into KM12 tumors (SUVmean,60 min 0.43 ± 0.03; n = 6). Tumor-to-muscle ratio reached 0.9 (60 min) and 1.2 (120 min). In TrkB expressing BAT, [18F]TRACK uptake reached SUVmean,60 min 1.32 ± 0.08 (n = 7). Activation of Trk through amitriptyline resulted in a significant radioactivity increase of 21% in KM12 tumor (SUVmean,60 min from 0.53 ± 0.01 to 0.43 ± 0.03; n = 6; p < 0.05) and of 21% in BAT (SUVmean,60 min from 1.32 ± 0.08; n = 5 to 1.59 ± 0.07; n = 6; p < 0.05) respectively. Immunohistochemistry showed TrkB > TrkA expression on BAT fat cells, but TrkA > TrkB in whole brain. WB analysis showed sevenfold higher TrkB expression in BAT versus KM12 tumor tissue.ConclusionThe present data show that radiotracer [18F]TRACK can target peripheral Trk receptors in human KM12 colon cancer as well as brown adipose tissue as confirmed through in vitro and in vivo blocking experiments. Higher TrkB versus TrkA protein expression was detected in brown adipose tissue of mice confirming a peripheral functional role of brain-derived neurotrophic factor in adipose tissue.

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

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

Cbl-b modulated TrkA ubiquitination and function in the dorsal root ganglion of mice

Casitas B-lineage lymphoma b (Cbl-b) is one of the E3 ubiquitin ligases that ubiquitinate Tropomyosin-related kinase A (TrkA), a key nerve growth factor receptor involved in the pathological pain. Here we found that Cbl-b was abundant in dorsal root ganglion (DRG) neurons of mice and co-localized with TrkA. Ubiquitination of TrkA by Cbl-b exerted a tonic negative control over the protein level of TrkA. Knockdown of Cbl-b caused TrkA accumulation in DRGs and evoked mechanical and heat hypersensitivity in intact mice. Our data showed that knee osteoarthritis induced by destabilization of the medial meniscus (DMM) led to the dissociation of Cbl-b with TrkA in DRG neurons, which impaired the ability of Cbl-b to ubiquitinate TrkA and served as an important mechanism to cause TrkA-dependent pain sensitization. Viral expression of constitutively active Cbl-b in DRGs of osteoarthritic mice effectively repressed TrkA protein level and more importantly, alleviated mechanical allodynia and heat hyperalgesia. Viral delivery of Cbl-b through intra-articular route generated a similar analgesic action. These data suggested that ubiquitination of TrkA by Cbl-b might represent an effective way to treat the osteoarthritic pain.

A multicolor immunosensor for point-of-care testing NTRK1 gene fusion

The gene fusion of Neurotrophic tropomyosin receptor kinase (NTRK) promotes the formation of tumors and is closely related to multiple cancers. Therefore, the detection of NTRK gene fusion markers has been a research hotspot in recent years. However, up to now, there is still lack of a fast and effective detection method for point-of-care testing (POCT). Herein, a multicolor immunosensor for point-of-care testing NTRK1 gene fusion by the naked eye was fabricated. Au nanobipyramids (Au NBPs) can be etched by 3, 3′, 5, 5′-tetramethylbenzidine ions (TMB2+) to produce a variety of aspect ratios, and the blue shift of its longitudinal plasmon band showed different color changes. So, the Au NBPs were combined with horseradish peroxidase (HRP)-TMB immunoassay system for achieving qualitative and semi-quantitative detection of NTRK1 gene fusion marker within 20 min by the naked eyes. The results shows that the relevant linear range for detection of TRKA protein was 7.5 pg/mL to 240 pg/mL. The constructed immunosensor will be expected to be used to quickly detect NTRK gene fusion markers in remote areas with underdeveloped medical conditions.

First-time in-human study of VMD-928, an oral allosteric TrkA selective inhibitor targeting TrkA protein overexpression, in patients with solid tumors or lymphoma.

3081 Background: Tropomyosin receptor kinase A (TrkA) is a protein encoded by the NTRK1 gene. Upregulation of TrkA signal transduction pathways, which can be caused by either NTRK1 gene fusions or intact TrkA protein overexpression, are oncogenic for multiple tumor types. This is clinically validated by demonstrated efficacy of ATP-competitive pan-TrkA/B/C inhibitors, larotrectinib and entrectinib for treatment of advanced solid tumors harboring NTRK1/2/3 gene fusions. VMD-928 is the first oral small-molecule TrkA (NTRK1) selective inhibitor with differentiated allosteric (ATP non-competitive) and irreversible mechanisms of action, acting as a molecular glue which sticks two TrkA proteins together and dose-dependently inhibits TrkA functions and downstream effectors, e.g. activated ERK, a hallmark of cancer. We conducted a first time in human phase 1 trial and completed the dose escalation phase. Methods: This is an open label, Phase 1 study investigating oral VMD-928 in adults with advanced solid tumors or lymphoma. The primary objective is to assess the safety and tolerability of VMD-928 and determine the recommended phase 2 dose. Secondary objectives include characterizing the pharmacokinetics (PK) and pharmacodynamics as well as assessing antitumor activity. Results: Non-biomarker-selected patients (n = 20) were accrued to 4 dose escalation cohorts ranging from 300 mg/day to 2400 mg/day. Three patients were accrued to the 2400 mg dose level with one DLT of elevated bilirubin, AST and ALT. The trough concentrations (Ctrough, ng/mL) ranged from 5.3 to 727. The dose was de-escalated to 1200 mg per day in divided doses and fifteen heavily pretreated patients were accrued with the following tumor types: adenoid cystic carcinoma, cholangiocarcinoma, lung cancer, pancreatic cancer, parotid, and squamous cell carcinoma of head and neck. There were no DLT’s at this dose and one patient with adenoid cystic carcinoma had prolonged stable disease. Common adverse events related to therapy were dark stool (35%), elevated liver enzymes (25%, primarily at 2400 mg/day), fatigue, nausea or vomiting, and decreased appetite (20% each). Conclusions: VMD-928 was well tolerated with mainly gastrointestinal side effects. The recommended phase 2 dose (RP2D) is 600 mg twice (1200 mg) per day. The study is currently accruing in expansion cohorts to evaluate efficacy in biomarker-selected patients with tumors of TrkA protein overexpression. Tumor types with reported high TrkA protein expression including thymic carcinoma (98% with TrkA protein expression without NTRK1/2/3 gene fusions), mesothelioma (81%), squamous cell carcinoma of head and neck (80%), ovarian (80%), hepatocellular (72%), and squamous cell carcinoma of the lung (71%) are being accrued. Clinical trial information: NCT03556228.

MicroRNA-454-mediated NEDD4-2/TrkA/cAMP axis in heart failure: Mechanisms and cardioprotective implications.

The current study aimed to investigate the mechanism by which miR‐454 influences the progression of heart failure (HF) in relation to the neural precursor cell expressed, developmentally downregulated 4‐2 (NEDD4‐2)/tropomyosin receptor kinase A (TrkA)/cyclic adenosine 3',5'‐monophosphate (cAMP) axis. Sprague‐Dawley rats were used to establish a HF animal model via ligation of the left anterior descending branch of the coronary artery. The cardiomyocyte H9c2 cells were treated with H2O2 to stimulate oxidative stress injury in vitro. RT‐qPCR and Western blot assay were subsequently performed to determine the expression patterns of miR‐454, NEDD4‐2, TrkA, apoptosis‐related proteins and cAMP pathway markers. Dual‐luciferase reporter gene assay coupled with co‐immunoprecipitation was performed to elucidate the relationship between miR‐454, NEDD4‐2 and TrkA. Gain‐ or loss‐of‐function experiments as well as rescue experiments were conducted via transient transfection (in vitro) and adenovirus infection (in vivo) to examine their respective functions on H9c2 cell apoptosis and myocardial damage. Our results suggested that miR‐454 was aberrantly downregulated in the context of HF, while evidence was obtained suggesting that it targeted NEDD4‐2 to downregulate NEDD4‐2 in cardiomyocytes. miR‐454 exerted anti‐apoptotic and protective effects on cardiomyocytes through inhibition of NEDD4‐2, while NEDD4‐2 stimulated ubiquitination and degradation of TrkA protein. Furthermore, miR‐454 activated the cAMP pathway via the NEDD4‐2/TrkA axis, which ultimately suppressed cardiomyocyte apoptosis and attenuated myocardial damage. Taken together, the key findings of the current study highlight the cardioprotective role of miR‐454, which is achieved through activation of the cAMP pathway by impairing NEDD4‐2‐induced TrkA ubiquitination.

GATA1, GATA2, and TAL1 Regulate the Expression of Neurotrophic Receptor Tyrosine Kinase in Leukemia Cells

Introduction Acute myeloid leukemia (AML) is a heterogeneous disease with poor overall survival and a high relapse rate. Transcription factors GATA1, GATA2, and TAL1 are key regulators of hematopoietic gene expression during normal embryonic and adult blood cell differentiation. Still, their roles in the regulation of cancer-specific genes during AML progression and therapy response remain unclear. Aims: Here, we aimed at the description of transcriptomic signatures of GATA1, GATA2, and TAL1 and identification of novel therapeutic targets in AML. Methods By the lentiviral transduction we have obtained leukemia (HL-60, Kasumi-1, THP-1) and neuroblastoma (SH-SY5Y) cell lines with overexpression of GATA1 and GATA2. Confocal microscopy was used to detect TrkA protein localization in cells. By qRT-PCR, we measured the expression of genes coding GATA1, GATA2, TAL1, and TrkA at mRNA levels. We used 5-azacytidine (5-aza) to induce changes in gene expression in leukemia cells. Cell viability was measured by trypan blue exclusion in the Neubauer chamber. Results First, we performed extensive data mining: analysis of publicly available transcriptome analysis data of 422 AML patients revealed genes differentially expressed (DEGs) with GATA1, GATA2, and TAL1. We found 125 genes common for GATA1, GATA2, and TAL1 including NTRK1 (encoding TrkA protein). Notably, by k-means clustering, we showed that patients with the induced level of GATA/TAL DEGs have poor overall survival. Previously we showed that leukemia cells have specific intracellular localization of TrkA protein. Ectopic expression of GATA1 and GATA2 in AML cells induced NTRK1 expression at mRNA and protein levels. 5-aza treatment of HL-60 cells resulted in the upregulation of GATA1, TAL1, and NTRK1 at mRNA and protein levels. Also, recombinant NGF (TrkA ligand) was able to rescue AML cells from 5-aza induced death. Conclusions Our data provide a novel mechanism of the GATA1, GATA2, and TAL1 involvement in AML progression. We show that NTRK1 expression is regulated by GATA1, GATA2, and TAL1 in leukemia but not in neuroblastoma cells. Our findings suggest the existence of GATA1, GATA2, and TAL1-driven control of non-hematopoietic genes in AML cells.

Multidisciplinary Treatment, Including Locoregional Chemotherapy, for Merkel-Polyomavirus-Positive Merkel Cell Carcinomas: Perspectives for Patients Exhibiting Oncogenic Alternative Δ exon 6-7 TrkAIII Splicing of Neurotrophin Receptor Tropomyosin-Related Kinase A.

Merkel cell carcinomas (MCCs) are rare, aggressive, cutaneous neuroendocrine tumours, approximately 80% of which are caused by the genomic integration of Merkel cell polyomavirus (MCPyV). MCPyV-positive MCCs carry poor prognosis in approximately 70% of cases, highlighting the need for greater understanding of the oncogenic mechanisms involved in pathogenesis, progression and post-therapeutic relapse, and translation into novel therapeutic strategies. In a previous pilot study, we reported a potential relationship between MCPyV gene expression and oncogenic alternative Δ exon 6–7 TrkAIII splicing in formalin-fixed paraffin-embedded (FFPE) MCC tissues from a 12-patient cohort of >90% MCPyV-positive MCCs, diagnosed at San Salvatore Hospital, L’Aquila, Italy, characterising a new MCC subgroup and unveiling a novel potential MCPyV oncogenic mechanism and therapeutic target. This, however, could not be fully verified due to poor RNA quality and difficulty in protein extraction from FFPE tissues. Here, therefore, we extend our previous observations to confirm the relationship between MCPyV and oncogenic alternative Δ exon 6–7 TrkAIII splicing in fresh, nonfixed, MCPyV-positive MCC metastasis by detecting sequence-verified RT-PCR products, including full-length Δ exon 6–7 TrkAIII, and by Western blot detection of a 100 kDa TrkA protein isoform of identical size to 100 kDa Δ exon 6–7 TrkAIII expressed by stable transfected SH-SY5Y cells. We also report that in three MCC patients submitted for multidisciplinary treatment, including locoregional chemotherapy, MCPyV large T-antigen mRNA expression, Δ exon 6–7 TrkAIII mRNA expression and intracellular indirect immunofluorescence (IF) TrkA and phosphorylation protein isoform(s) immunoreactivity in FFPE tissues were not reduced in postchemotherapeutic-relapsed MCCs compared to pretherapeutic MCCs, extending the possible roles of this novel potential MCPyV oncogenic mechanism from MCC pathogenesis to post-therapeutic relapse and progression. Detection of alternative Δ exon 6–7 TrkAIII splicing in MCC, therefore, not only characterises a new MCPyV-positive MCC subgroup and unveils a novel potential MCPyV oncogenic mechanism but also identifies patients who may benefit from inhibitors of MCPyV T-antigen and/or TrkAIII expression or clinically approved Trk kinase inhibitors such as larotrectinib or entrectinib, which are known to inhibit activated TrkA oncogenes and to elicit durable responses in TrkA-fusion oncogene-driven cancers, supporting the call for a large-scale multicentre clinical study.

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.

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.

A new porous cationic metal–organic framework: selective sorption of organic dyes and treatment on neural stem cells during spinal cord injury treatment

A novel metal–organic framework containing Co(II) of {(Me2NH2)4[Co12(OH)4(abtc)6 (H2O)12]·14H2O}n (1) has been synthesized via Co(NO3)2·6H2O to react with the 3,3′,5,5′-azobenzenetetracarboxylic acid (H4abtc) ligand under solvothermal conditions. Furthermore, methylene blue (MB) can easily replace free cations; after half a day, more than 90% of the MB (10−5 M) is absorbed selectively, and it makes the title compound become a good medium for the separation and adsorption of the dye. In biological section, the enhancement activity of the compound on the nerve growth factor for the spinal cord injury treatment was evaluated. The relative gene expression of trkA receptor was measured with reverse transcription polymerase chain reaction. In addition to this, the activation of PI3K/Akt signaling pathway in neural stem cells after treated by compound was evaluated with western blot. The results of potential binding models were predicted by molecular docking, and the binding poses provided possible binding mechanisms of the functional groups of the complex to the TRKA protein.

The Impact of Ovariectomy on Olfactory Neuron Regeneration in Mice.

Estrogen has been shown to affect differentiation and proliferation as a mitogen in various neural systems. Olfactory receptor cells are unique within the nervous system, and have the ability to regenerate even after an individual has reached maturity. Olfactory receptor cells also regenerate after experimentally induced degeneration. The purpose of this study is to observe the influence of estrogen depletion induced by ovariectomy on olfactory nerve regeneration. Female mice underwent bilateral ovariectomy at 8 weeks of age and received intraperitoneal administration of methimazole 1 week later. At 2, 4, and 6 weeks after methimazole administration, the olfactory mucosa was analyzed histochemically to determine olfactory epithelium (OE) thickness, olfactory marker protein distribution, and Ki-67 immunoreactivity. Furthermore, 2 weeks after ovariectomy, trkA protein distribution in the OE and nerve growth factor (NGF) levels in the olfactory bulb were determined by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. Our results showed that in ovariectomized mice OMP, Ki-67, and trkA-immunopositive cells expression decreased at 2 weeks after methimazole injection, a time point at which regeneration is underway. At this same time point, although NGF production in the olfactory bulb had increased before methimazole administration, no differences were observed between the ovx and control groups. These results suggest that estrogen depletion induces a suppressive effect on regeneration of olfactory neurons, and that estrogen may have a potential use in the treatment of sensorineural olfactory dysfunction.

Homology modeling identified for purported drug targets to the neuroprotective effects of levodopa and asiaticoside-D in degenerated cerebral ganglions of Lumbricus terrestris.

CONTEXT:Homology modeling plays role in determining the therapeutic targets dreadful for condition such as neurodegenerative diseases (NDD), which pose challenge in achieving the effective managements. The structures of the serotonin transporter (SERT), aquaporin (AQP), and tropomyosin receptor kinase (TrkA) which are implicated in NDD pathology are still unknown for Lumbricus terrestris, but the three-dimensional (3D) structure of the human counterpart for modeling.AIM:This study aims to generate and evaluate the 3D structure of TrkA, SERT, and AQP proteins and their interaction with the ligands, namely Asiaticoside-D (AD) and levodopa (L-DOPA) the anti-NDD agents.SUBJECTS AND METHODS:Homology modeling for SERT, AQP, and TrkA proteins of Lumbricus terrestris using SWISS-MODEL Server and the modeled structure was validated using Rampage Server. Wet-lab analysis of their correspondent m-RNA levels was also done to validate the in silico data.RESULTS:It was found that TrkA had moderately high homology (67%) to human while SERT and AQP could exhibit 58% and 42%, respectively. The reliability of the model was assessed by Ramachandran plot analysis. Interactions of AD with the SERT, AQP-4, and TrkA showed the binding energies as −9.93, 8.88, and −7.58 of Kcal/mol, respectively, while for L-DOPA did show −3.93, −5.13, and −6.0 Kcal/mol, respectively. The levels of SERT, TrkA, and AQP-4 were significantly reduced (P < 0.001) on ROT induced when compared to those of control worms. On ROT + AD supplementation group (III), m-RNA levels were significantly increased (P < 0.05) when compared to those of ROT induced worms (group II).CONCLUSION:Our pioneering docking data propose the possible of target which is proved useful for therapeutic investigations against the unconquered better of NDD.

Identification of a novel mutation of the NTRK1 gene in patients with congenital insensitivity to pain with anhidrosis (CIPA)

IntroductionCongenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive disorder resulting from NTRK1 mutation. Over 105 NTRK1 mutations have been reported in CIPA patients worldwide. The causative NTRK1 mutations lead to loss of function of the TrkA protein, an important ligand for nerve growth factor (NGF), and therefore induce various clinical phenotypes associated with neuron maturation defects. Materials and methodsThree patients from unrelated families with CIPA were subjected to detailed clinical examinations. Blood samples were collected from all the patients and their available family members, as well as 200 healthy volunteers. Sanger sequencing for all the exons and splicing sites of NTRK1 was performed on all samples. The phenotype-genotype relationship and genetic epidemiology of Chinese CIPA patients were also analysed. ResultsA total of four different NTRK1 mutations [c.851-33T>A, c.44G>A (p.Trp15*), c.287+2dupT, c.1549G>C (p.Gly517Arg)] were identified in these families, and c.1549G>C (p.Gly517Arg) was a novel mutation that had not been reported previously. The ‘mild’ manifestations observed in patients with c.851-33T>A indicated this mutation as a ‘mild’ mutation. After reviewing studies reporting mutations in Chinese CIPA patients, we speculate the mutation c.851-33T>A is one of the founder mutations in the Chinese population. ConclusionsOur research expanded the spectrum of the NTRK1 mutations associated with CIPA patients, provided additional clues relating to the phenotype-genotype relationship in CIPA, and summarized the features of the genetic epidemiology of CIPA in the Chinese ethnic group.

Effect of Electroacupuncture at Different Acupoints on Expression of NGF and TrkA in Cerebral Cortex in Rats with Myocardial Ischemia

To compare the therapeutic effect of electroacupuncture (EA) of "Shenmen" (HT 7, Yuan point of the Heart Meridian), "Zhizheng" (SI 7, Luo point of the Small Intestine Meridian) and "Xinshu" (BL15, Back-shu point) on the expression of nerve growth factor (NGF) and its receptor, tyrosine kinase A (TrkA) in the cerebral cortex of myocardial ischemia (MI) rats. A total of 70 male SD rats were randomized into sham operation (sham, n=10), model, HT 7, SI 7, and BL15 groups (n=15 in each of the latter 4 groups). The MI model was established by ligation of the anterior descending branch of the left coronary artery. EA (1 mA, 2 Hz) was respectively applied to HT 7, SI 7 and BL15 for 15 min, once per day for 1 week. Immunohistochemistry and real-time fluorescent quantitative PCR were used to detect the expression of NGF and TrkA proteins and genes in the cerebral cortex. Compared with the sham group, no significant changes were found in the number of NGF immune-reaction (IR)-positive and TrkA IR-positive cells, and the expression levels of NGF mRNA and TrkA mRNA in the model group (P>0.05). After EA intervention, the number of NGF and TrkA IR-positive cells and the expression of NGF mRNA and TrkA mRNA in each of the 3 EA groups were significantly increased relevant to the model group (P<0.01, P<0.05). The effect of EA at BL 15 was significantly superior to that of EA at HT 7 and SI 7 in increasing the number of NGF and TrkA positive cells and up-regulating the expression of their mRNAs (P<0.01, P<0.05). EA at HT 7, SI 7 and BL 15 can increase the levels of expression of NGF and TrkA proteins and mRNAs in the cerebral cortex of subacute MI rats and the effects of EA-BL 15 are obviously superior to those of EA-HT 7 and EA-SI 7, suggesting a relative specificity of the effect of EA at different acupoints.

Lin28a functionally modulates bupivacaine-induced dorsal root ganglion neuron apoptosis through TrkA activation

PurposeLin-28 Homolog A gene (Lin28a) is an important regulator in nerve system. In this study, we investigated the functional mechanism of Lin28a during the process of bupivacaine (BUP)-induced neuronal apoptosis of spinal cord dorsal root ganglion neurons (DRGNs). MethodsYoung mouse DRGNs were cultured in vitro and treated with series concentrations of BUP. Apoptosis was evaluated by TUNEL assay. Corresponding Lin28a mRNA and protein expressions were evaluated by qRT-PCR and western blot (WB) assays. Lin28a was downregulated by siRNA and its effect on BUP (5 mM)-induced DRGN apoptosis was measured by qRT-PCR, WB and TUNEL assays, respectively. Alternatively, Lin28a was upregulated in DRGNs. It’s effect on BUP (0.1 mM)-induced DRGN apoptosis was also measured. Finally, WB was used to examine Caspase-3/9 and TrkA protein expressions in Lin28a-downregualted and BUP-injured DRGN to explore Lin28a-associated signaling pathways. ResultsIn DRGN in vitro culture, 0.1 mM BUP induced moderate neuronal apoptosis while 5 mM BUP induced significant apoptosis. Lin28a mRNA and protein were both upregulated by BUP, in concentration-dependent manner. Functional assays showed that Lin28a downregulation rescued 5 mM BUP-induced neuronal apoptosis, whereas Lin28a upregulation aggravated 0.1 mM BUP-induced neuronal apoptosis in DRGNs. WB showed that Lin28a downregulation reduced Caspase-3/9 proteins and activated TrkA through phosphorylation in BUP-injured DRGNs. ConclusionLin28a is a potent regulator in BUP-induced neuronal apoptosis in DRGNs. The apoptotic protection by Lin28a inhibition is likely through the activation of TrkA signaling pathway.