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BACKGROUND: Neurotrophic tyrosine receptor kinase (NTRK) gene fusions are rare oncogenic drivers prevalent in 0.3% of solid tumors. They are most common in salivary gland cancer (2.6%), thyroid cancer (1.6%), and soft-tissue sarcoma (1.5%). Currently, there are 2 US Food and Drug Administration-approved targeted therapies for NTRK gene fusions: larotrectinib, approved in 2018, and entrectinib, approved in 2019. To date, the real-world uptake of tyrosine receptor kinase inhibitor (TRKi) use for NTRK-positive solid tumors in academic cancer centers remains largely unknown. OBJECTIVE: To describe the demographics, clinical and genomic characteristics, and testing and treatment patterns of patients with NTRK-positive solid tumors treated at US academic cancer centers. METHODS: This was a retrospective chart review study conducted in academic cancer centers in the United States. All patients diagnosed with an NTRK fusion-positive (NTRK1, NTRK2, NTRK3) solid tumor (any stage) and who received cancer treatment at participating sites between January 1, 2012, and July 1, 2023, were included in this study. Patient demographics, clinical characteristics, genomic characteristics, NTRK testing data, and treatment patterns were collected from electronic medical records and analyzed using descriptive statistics as appropriate. RESULTS: In total, 6 centers contributed data for 55 patients with NTRK-positive tumors. The mean age was 49.3 (SD = 20.5) years, 51% patients were female, and the majority were White (78%). The median duration of time from cancer diagnosis to NTRK testing was 85 days (IQR = 44-978). At the time of NTRK testing, 64% of patients had stage IV disease, compared with 33% at cancer diagnosis. Prevalent cancer types in the overall cohort included head and neck (15%), thyroid (15%), brain (13%), lung (13%), and colorectal (11%). NTRK1 fusions were most common (45%), followed by NTRK3 (40%) and NTRK2 (15%). Across all lines of therapy, 51% of patients (n = 28) recei

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

Neurotrophin receptors such as the tropomyosin receptor kinase A receptor (TrkA) and the low-affinity binding p75 neurotrophin receptor p75NTR play a critical role in neuronal survival and their functions are altered in Alzheimer’s disease (AD). Changes in the dynamics of receptors on the plasma membrane are essential to receptor function. However, whether receptor dynamics are affected in different pathophysiological conditions is unexplored. Using live-cell single-molecule imaging, we examined the surface trafficking of TrkA and p75NTR molecules on live neurons that were derived from human-induced pluripotent stem cells (hiPSCs) of presenilin 1 (PSEN1) mutant familial AD (fAD) patients and non-demented control subjects. Our results show that the surface movement of TrkA and p75NTR and the activation of TrkA- and p75NTR-related phosphoinositide-3-kinase (PI3K)/serine/threonine-protein kinase (AKT) signaling pathways are altered in neurons that are derived from patients suffering from fAD compared to controls. These results provide evidence for altered surface movement of receptors in AD and highlight the importance of investigating receptor dynamics in disease conditions. Uncovering these mechanisms might enable novel therapies for AD., source:https://www.mdpi.com/1422-0067/22/24/13260

Gene fusions involving the neurotrophic receptor tyrosine kinase genes NTRK1, NTRK2, and NTRK3, are well established oncogenic drivers in a broad range of pediatric and adult tumors. These fusions are also important actionable markers, predicting often dramatic response to FDA approved kinase inhibitors. Accurate interpretation of the clinical significance of NTRK fusions is a high priority for diagnostic laboratories, but remains challenging and time consuming given the rapid pace of new data accumulation, the diversity of fusion partners and tumor types, and heterogeneous and incomplete information in variant databases and knowledgebases. The ClinGen NTRK Fusions Somatic Cancer Variant Curation Expert Panel (SC-VCEP) was formed to systematically address these challenges and create an expert-curated resource to support clinicians, researchers, patients and their families in making accurate interpretations and informed treatment decisions for NTRK fusion-driven tumors. We describe a system for NTRK fusion interpretation (including compilation of key elements and annotations) developed by the NTRK fusions SC-VCEP. We illustrate this stepwise process on examples of LMNA::NTRK1 and KANK1::NTRK2 fusions. Finally, we provide detailed analysis of current representation of NTRK fusions in public fusion databases and the CIViC knowledgebase, performed by the NTRK fusions SC-VCEP to determine existing gaps and prioritize future curation activities.

[ES] TrkA es un receptor tirosina quinasa que pertenece a la familia de receptores Trk. Dicho receptor se encuentra principalmente en el sistema nervioso periférico, donde participa en el desarrollo y la supervivencia de las neuronas simpáticas y sensoriales. Además, también se encarga del desarrollo de las neuronas colinérgicas del sistema nervioso central y desempeña un papel importante en algunos tejidos no neurales, participando tanto en el desarrollo de células B como en el desarrollo cardíaco y vascular, entre otras funciones. Sin embargo, TrkA no solo resulta interesante por todas las funciones que tiene, sino también por su implicación en el cáncer. Originalmente, TrkA se aisló de un carcinoma de colon, aunque también ha aparecido en carcinomas papilares tiroideos y en casos de leucemia mieloide aguda. En estos cánceres, se puede observar una activación constitutiva del receptor, generalmente por fusiones de su dominio quinasa con el dominio extracelular de otras proteínas. No obstante, también se han descrito algunos casos en los que mutaciones puntuales en el dominio extracelular de TrkA inducen la transformación maligna de las células. Recientemente, en pacientes de diferentes tipos de cáncer se han detectado dos mutaciones distintas en el dominio extracelular de TrkA (S326C y R347C). La primera mutación (S326C) se ha detectado en un caso de cáncer de pulmón, mientras que la segunda (R347C), se ha detectado en un caso de cáncer de ciego del intestino grueso, otro de colon y en un caso de rabdomiosarcoma (cáncer del músculo estriado). Ambas mutaciones cambian un residuo por una cisteína y la forma en que estas mutaciones influyen en la activación de TrkA se desconoce. El objetivo de este trabajo es caracterizar dichas mutaciones, utilizando su expresión en células HeLa, y determinar cómo influyen estas en el desarrollo tumoral., [EN] TrkA is a tyrosine kinase receptor that belongs to the Trk family of receptors. This receptor is found mainly in the peripheral nervous system, where it participates in the development and survival of sympathetic and sensory neurons. In addition, it is also responsible for the development of cholinergic neurons of the central nervous system and plays an important role in some non-neural tissues, participating both in the development of B cells and in cardiac and vascular development, among other functions. However, TrkA is not only interesting for all the functions it has, but also for its involvement in cancer. TrkA was originally isolated from colon carcinoma, although it has also appeared in papillary thyroid carcinomas and in cases of acute myeloid leukemia. In these cancers, a constitutive activation of the receptor can be observed, generally by fusions of its kinase domain with the extracellular domain of other proteins. However, some cases have also been described in which point mutations in the TrkA extracellular domain induce malignant transformation of cells. Recently, two different mutations in the TrkA extracellular domain (S326C and R347C) have been detected in patients with different types of cancer. The first mutation (S326C) has been detected in a case of lung cancer, while the second one (R347C) has been detected in a case of caecum cancer, in a case of colon cancer and in a case of rhabdomyosarcoma (striated muscle cancer). Both mutations exchange a residue for a cysteine and the way in which these mutations influence TrkA activation is unknown. The objective of this work is to characterize these mutations, using their expression in Hela cells, and to determine how they influence tumor development.

[ES] TrkA es un receptor tirosina quinasa que pertenece a la familia de receptores Trk. Dicho receptor se encuentra principalmente en el sistema nervioso periférico, donde participa en el desarrollo y la supervivencia de las neuronas simpáticas y sensoriales. Además, también se encarga del desarrollo de las neuronas colinérgicas del sistema nervioso central y desempeña un papel importante en algunos tejidos no neurales, participando tanto en el desarrollo de células B como en el desarrollo cardíaco y vascular, entre otras funciones. Sin embargo, TrkA no solo resulta interesante por todas las funciones que tiene, sino también por su implicación en el cáncer. Originalmente, TrkA se aisló de un carcinoma de colon, aunque también ha aparecido en carcinomas papilares tiroideos y en casos de leucemia mieloide aguda. En estos cánceres, se puede observar una activación constitutiva del receptor, generalmente por fusiones de su dominio quinasa con el dominio extracelular de otras proteínas. No obstante, también se han descrito algunos casos en los que mutaciones puntuales en el dominio extracelular de TrkA inducen la transformación maligna de las células. Recientemente, en pacientes de diferentes tipos de cáncer se han detectado dos mutaciones distintas en el dominio extracelular de TrkA (S326C y R347C). La primera mutación (S326C) se ha detectado en un caso de cáncer de pulmón, mientras que la segunda (R347C), se ha detectado en un caso de cáncer de ciego del intestino grueso, otro de colon y en un caso de rabdomiosarcoma (cáncer del músculo estriado). Ambas mutaciones cambian un residuo por una cisteína y la forma en que estas mutaciones influyen en la activación de TrkA se desconoce. El objetivo de este trabajo es caracterizar dichas mutaciones, utilizando su expresión en células HeLa, y determinar cómo influyen estas en el desarrollo tumoral., [EN] TrkA is a tyrosine kinase receptor that belongs to the Trk family of receptors. This receptor is found mainly in the peripheral nervous system, where it participates in the development and survival of sympathetic and sensory neurons. In addition, it is also responsible for the development of cholinergic neurons of the central nervous system and plays an important role in some non-neural tissues, participating both in the development of B cells and in cardiac and vascular development, among other functions. However, TrkA is not only interesting for all the functions it has, but also for its involvement in cancer. TrkA was originally isolated from colon carcinoma, although it has also appeared in papillary thyroid carcinomas and in cases of acute myeloid leukemia. In these cancers, a constitutive activation of the receptor can be observed, generally by fusions of its kinase domain with the extracellular domain of other proteins. However, some cases have also been described in which point mutations in the TrkA extracellular domain induce malignant transformation of cells. Recently, two different mutations in the TrkA extracellular domain (S326C and R347C) have been detected in patients with different types of cancer. The first mutation (S326C) has been detected in a case of lung cancer, while the second one (R347C) has been detected in a case of caecum cancer, in a case of colon cancer and in a case of rhabdomyosarcoma (striated muscle cancer). Both mutations exchange a residue for a cysteine and the way in which these mutations influence TrkA activation is unknown. The objective of this work is to characterize these mutations, using their expression in Hela cells, and to determine how they influence tumor development.

[ES] TrkA es un receptor tirosina quinasa que pertenece a la familia de receptores Trk. Dicho receptor se encuentra principalmente en el sistema nervioso periférico, donde participa en el desarrollo y la supervivencia de las neuronas simpáticas y sensoriales. Además, también se encarga del desarrollo de las neuronas colinérgicas del sistema nervioso central y desempeña un papel importante en algunos tejidos no neurales, participando tanto en el desarrollo de células B como en el desarrollo cardíaco y vascular, entre otras funciones. Sin embargo, TrkA no solo resulta interesante por todas las funciones que tiene, sino también por su implicación en el cáncer. Originalmente, TrkA se aisló de un carcinoma de colon, aunque también ha aparecido en carcinomas papilares tiroideos y en casos de leucemia mieloide aguda. En estos cánceres, se puede observar una activación constitutiva del receptor, generalmente por fusiones de su dominio quinasa con el dominio extracelular de otras proteínas. No obstante, también se han descrito algunos casos en los que mutaciones puntuales en el dominio extracelular de TrkA inducen la transformación maligna de las células. Recientemente, en pacientes de diferentes tipos de cáncer se han detectado dos mutaciones distintas en el dominio extracelular de TrkA (S326C y R347C). La primera mutación (S326C) se ha detectado en un caso de cáncer de pulmón, mientras que la segunda (R347C), se ha detectado en un caso de cáncer de ciego del intestino grueso, otro de colon y en un caso de rabdomiosarcoma (cáncer del músculo estriado). Ambas mutaciones cambian un residuo por una cisteína y la forma en que estas mutaciones influyen en la activación de TrkA se desconoce. El objetivo de este trabajo es caracterizar dichas mutaciones, utilizando su expresión en células HeLa, y determinar cómo influyen estas en el desarrollo tumoral., [EN] TrkA is a tyrosine kinase receptor that belongs to the Trk family of receptors. This receptor is found mainly in the peripheral nervous system, where it participates in the development and survival of sympathetic and sensory neurons. In addition, it is also responsible for the development of cholinergic neurons of the central nervous system and plays an important role in some non-neural tissues, participating both in the development of B cells and in cardiac and vascular development, among other functions. However, TrkA is not only interesting for all the functions it has, but also for its involvement in cancer. TrkA was originally isolated from colon carcinoma, although it has also appeared in papillary thyroid carcinomas and in cases of acute myeloid leukemia. In these cancers, a constitutive activation of the receptor can be observed, generally by fusions of its kinase domain with the extracellular domain of other proteins. However, some cases have also been described in which point mutations in the TrkA extracellular domain induce malignant transformation of cells. Recently, two different mutations in the TrkA extracellular domain (S326C and R347C) have been detected in patients with different types of cancer. The first mutation (S326C) has been detected in a case of lung cancer, while the second one (R347C) has been detected in a case of caecum cancer, in a case of colon cancer and in a case of rhabdomyosarcoma (striated muscle cancer). Both mutations exchange a residue for a cysteine and the way in which these mutations influence TrkA activation is unknown. The objective of this work is to characterize these mutations, using their expression in Hela cells, and to determine how they influence tumor development.

Previously, we reported a family in which bipolar disorder (BD) co-segregates with a Mendelian kidney disorder linked to 1q22. The causative renal gene was later identified as MUC1. Genome-wide linkage analysis of BD in the family yielded a peak at 1q22 that encompassed the NTRK1 and MUC1 genes. NTRK1 codes for TrkA (Tropomyosin-related kinase A) which is essential for development of the cholinergic nervous system. Whole genome sequencing of the proband identified a damaging missense mutation, E492K, in NTRK1. Induced pluripotent stem cells were generated from family members, and then differentiated to neural stem cells (NSCs). E492K NSCs had reduced neurite outgrowth. A conditional knock-in mouse line, harboring the point mutation in the brain, showed depression-like behavior in the tail suspension test following challenge by physostigmine, a cholinesterase inhibitor. These results are consistent with the cholinergic hypothesis of depression. They imply that the NTRK1 E492K mutation, impairs cholinergic neurotransmission, and may convey susceptibility to bipolar disorder.

The R100W mutation in nerve growth factor is associated with hereditary sensory autonomic neuropathy V in a Swedish family. These patients develop severe loss of perception to deep pain but with apparently normal cognitive functions. To better understand the disease mechanism, we examined a knockin mouse model of HSAN V. The homozygous mice showed significant structural deficits in intra-epidermal nerve fibers (IENFs) at birth. These mice had a total loss of pain perception at ∼2 months of age and often failed to survive to adulthood. Heterozygous mutant mice developed a progressive degeneration of small sensory fibers both behaviorally and functionally: they showed a progressive loss of IENFs starting at the age of 9 months accompanied with progressive loss of perception to painful stimuli such as noxious temperature. Quantitative analysis of lumbar 4/5 dorsal root ganglia revealed a significant reduction in small size neurons, while analysis of sciatic nerve fibers revealed the heterozygous mutant mice had no reduction in myelinated nerve fibers. Significantly, the amount of NGF secreted from mouse embryonic fibroblasts were reduced from both heterozygous and homozygous mice compared to their wild-type littermates. Interestingly, the heterozygous mice showed no apparent structural alteration in the brain: neither the anterior cingulate cortex nor the medial septum including NGF-dependent basal forebrain cholinergic neurons. Accordingly, these animals did not develop appreciable deficits in tests for brain function. Our study has thus demonstrated that the NGFR100W mutation likely affects the structure and function of peripheral sensory neurons.

Nerve growth factor (NGF) regulates many aspects of neuronal biology by retrogradely propagating signals along axons to the targets of those axons. How this occurs when axons contain a plethora of proteins that can silence those signals has long perplexed the neurotrophin field. In this issue of the JCI, Li et al. suggest an answer to this vexing problem, while exploring why the Elp1 gene that is mutated in familial dysautonomia (FD) causes peripheral neuropathy. They describe a distinctive function of Elp1 as a protein that is required to sustain NGF signaling by blocking the activity of its phosphatase that shuts off those signals. This finding helps explain the innervation deficits prominent in FD and reveals a unique role for Elp1 in the regulation of NGF-dependent TrkA activity.

BackgroundEntrectinib is a potent inhibitor of tropomyosin receptor kinase (TRK) A, B, and C, which has been shown to have anti-tumour activity against NTRK gene fusion-positive solid tumours, including CNS activity due to its ability to penetrate the blood-brain barrier. We present an integrated efficacy and safety analysis of patients with metastatic or locally advanced solid tumours harbouring oncogenic NTRK1, NTRK2, and NTRK3 gene fusions treated in three ongoing, early-phase trials.MethodsAn integrated database comprised the pivotal datasets of three, ongoing phase 1 or 2 clinical trials (ALKA-372-001, STARTRK-1, and STARTRK-2), which enrolled patients aged 18 years or older with metastatic or locally advanced NTRK fusion-positive solid tumours who received entrectinib orally at a dose of at least 600 mg once per day in a capsule. All patients had an Eastern Cooperative Oncology Group performance status of 0-2 and could have received previous anti-cancer therapy (except previous TRK inhibitors). The primary endpoints, the proportion of patients with an objective response and median duration of response, were evaluated by blinded independent central review in the efficacy-evaluable population (ie, patients with NTRK fusion-positive solid tumours who were TRK inhibitor-naive and had received at least one dose of entrectinib). Overall safety evaluable population included patients from STARTRK-1, STARTRK-2, ALKA-372-001, and STARTRK-NG (NCT02650401; treating young adult and paediatric patients [aged ≤21 years]), who received at least one dose of entrectinib, regardless of tumour type or gene rearrangement. NTRK fusion-positive safety evaluable population comprised all patients who have received at least one dose of entrectinib regardless of dose or follow-up. These ongoing studies are registered with ClinicalTrials.gov, NCT02097810 (STARTRK-1) and NCT02568267 (STARTRK-2), and EudraCT, 2012-000148-88 (ALKA-372-001).FindingsPatients were enrolled in ALKA-372-001 from

ObjectiveTo investigate the TrkA inhibitor, ASP7962, for treatment of painful knee osteoarthritis.DesignPhase 2a, double-blind, placebo- and naproxen-controlled, double-dummy, parallel-group study. Adults with knee osteoarthritis were randomized (2:2:1) to ASP7962 (100 mg), placebo, or naproxen (500 mg) twice daily (BID) for 4 weeks. Primary endpoint: change from baseline to Week 4 in Western Ontario and McMaster Universities Arthritis Index (WOMAC) pain subscale score. Secondary endpoints: change from baseline to Weeks 1, 2, and End of Treatment (EoT) in WOMAC pain subscale score; change from baseline to Weeks 1, 2, 4, and EoT in WOMAC physical function and stiffness subscales, walking pain and WOMAC total scores; and change from baseline in daily average pain score.Results215 participants were randomized (ASP7962 100 mg BID, n = 85; placebo, n = 87; naproxen 500 mg BID, n = 43). No significant difference was observed between ASP7962 and placebo in change from baseline to Week 4 in WOMAC pain subscale score (-0.14; 90% 2-sided CI: -0.62, 0.34; P = 0.316); a significant difference was observed between naproxen and placebo (-0.67; 80% 2-sided CI: -1.12, -0.23; P = 0.027). No differences were observed between ASP7962 and placebo in change from baseline in any WOMAC subscale score; statistically significant changes were observed between naproxen and placebo (P ≤ 0.01, all time points for all WOMAC endpoints). ASP7962 was safe and well-tolerated.ConclusionsFour-week treatment with ASP7962 (100 mg BID) did not improve pain or physical function in individuals with painful knee osteoarthritis. ClinicalTrials.gov, NCT02611466; EudraCT Number, 2014-004996-22.

During the development of the sympathetic nervous system, signals from tropomyosin-related kinase receptors (Trks) and p75 neurotrophin receptors (p75) compete to regulate survival and connectivity. During this process, nerve growth factor (NGF)- TrkA signaling in axons communicates NGF-mediated trophic responses in signaling endosomes. Whether axonal p75 signaling contributes to neuronal death and how signaling endosomes contribute to p75 signaling has not been established. Using compartmentalized sympathetic neuronal cultures (CSCGs) as a model, we observed that the addition of BDNF to axons increased the transport of p75 and induced death of sympathetic neurons in a dynein-dependent manner. In cell bodies, internalization of p75 required the activity of JNK, a downstream kinase mediating p75 death signaling in neurons. Additionally, the activity of Rab5, the key GTPase regulating early endosomes, was required for p75 death signaling. In axons, JNK and Rab5 were required for retrograde transport and death signaling mediated by axonal BDNF-p75 in CSCGs. JNK was also required for the proper axonal transport of p75-positive endosomes. Thus, our findings provide evidence that the activation of JNK by p75 in cell bodies and axons is required for internalization to a Rab5-positive signaling endosome and the further propagation of p75-dependent neuronal death signals.

[ES] Las neurotrofinas son unos ligandos solubles implicados en el desarrollo y supervivencia del sistema nervioso, que ejercen su función mediante la unión a receptores de membrana. TrkA es uno de éstos, un receptor tirosina quinasa implicado en el crecimiento y diferenciación de las neuronas nociceptivas, así como en su supervivencia. La activación de TrkA está mediada así, por la unión a la neurotrofina NGF, que produce su dimerización, autofosoforilación y transfosforilación en el dominio tirosina kinasa intracelular. Durante el estudio de la participación de ciertos residuos en la activación de TrkA, se observó que mutaciones en los residuos N404, S405 y T406 del dominio yuxtamembrana extracelular, disminuían la activación de TrkA por NGF. En este trabajo se estudia los mecanismos de tráfico a membrana y de la inactivación de TrkA con las mutaciones N404A, S405A y T406A. Se estudió la capacidad de transporte a membrana de estos mutantes mediante citometría de flujo y ensayos de inmunofluorescencia, completándolo con estudios de la actividad del receptor a través de ensayos de señalización por actividad quinasa mediante Western Blot, en presencia de NGF. Se observó que especialmente el mutante T406A tiene una capacidad reducida para llegar a la membrana. Aunque el sitio N-S-T (404-406) es un sitio de consenso de N-glicosilación, que podría ser responsable de los efectos observados, se vio que la mutación N404A no afecta a la localización de TrkA en la membrana plasmática, sugiriendo que la inactivación de TrkA-T406A no se debe a una falta en la N-glicosilación del residuo N404. Por otra parte, in silico se predijo un posible sitio de O-glicosilación en la posición T406. Para evaluar dicha posibilidad se realizaron ensayos de deglicosilación. Los resultados finalmente llevaron a descartar esta última hipótesis de Oglicosilación. Sin embargo, determinamos que TrkA necesita de la posición T406 para su correcto tránsito desde las membranas intracelulares hasta la me, [EN] Neurotrophins are soluble ligands implicated in development and survival of the nervous system, working through binding membrane receptors. TrkA is one of these, a tyrosine kinase receptor involved in growth and differentiation of nociceptive neurons, as in their survival. TrkA activation is mediated by NGF neurotrophin binding which leads to dimerization, autophosphorylation and transphosphorylation in the intracellular tyrosine kinase domain. In the study of the role of certain residues in the activation of TrkA, it appeared that mutations in the N404, S405 and T406 residues of the extracellular juxtamembrane domain decreased the activity of TrkA via NGF. This work studies mechanisms of TrkA trafficking and inactivation with N404A, S405A y T406A mutations. Protein trafficking of the receptor in these mutants was studied by flow cytometry and immunofluorescence assays. This was supplemented with activity studies through signalling-kinase activity assays by Western Blot, in the presence of NGF. We observed, especially in the T406A mutant, a reduced ability to reach the membrane, despite the mutation has no effect on activation mechanism of the receptor. Although site N-S-T (404-406) is a N-glycosylation consensus site, that could be responsible of these effects, it was determined that the N404A mutation does not affect TrkA normal location. This suggests that the inactivation of TrkA-T406A is not due to a lack of N-glycan of the N404 residue. Alternatively, a potential O-glycosylation site was predicted in silico at position T406. To evaluate this hypothesis deglycation assays were performed. Results finally led to dismiss this last O-glycosylation hypothesis. However, we determined TrkA needs T406 position for its proper traffic from intracellular membranes to the plasma membrane, suggesting an important post-translational modification, until now unknown in this receptor.

[ES] Las neurotrofinas son unos ligandos solubles implicados en el desarrollo y supervivencia del sistema nervioso, que ejercen su función mediante la unión a receptores de membrana. TrkA es uno de éstos, un receptor tirosina quinasa implicado en el crecimiento y diferenciación de las neuronas nociceptivas, así como en su supervivencia. La activación de TrkA está mediada así, por la unión a la neurotrofina NGF, que produce su dimerización, autofosoforilación y transfosforilación en el dominio tirosina kinasa intracelular. Durante el estudio de la participación de ciertos residuos en la activación de TrkA, se observó que mutaciones en los residuos N404, S405 y T406 del dominio yuxtamembrana extracelular, disminuían la activación de TrkA por NGF. En este trabajo se estudia los mecanismos de tráfico a membrana y de la inactivación de TrkA con las mutaciones N404A, S405A y T406A. Se estudió la capacidad de transporte a membrana de estos mutantes mediante citometría de flujo y ensayos de inmunofluorescencia, completándolo con estudios de la actividad del receptor a través de ensayos de señalización por actividad quinasa mediante Western Blot, en presencia de NGF. Se observó que especialmente el mutante T406A tiene una capacidad reducida para llegar a la membrana. Aunque el sitio N-S-T (404-406) es un sitio de consenso de N-glicosilación, que podría ser responsable de los efectos observados, se vio que la mutación N404A no afecta a la localización de TrkA en la membrana plasmática, sugiriendo que la inactivación de TrkA-T406A no se debe a una falta en la N-glicosilación del residuo N404. Por otra parte, in silico se predijo un posible sitio de O-glicosilación en la posición T406. Para evaluar dicha posibilidad se realizaron ensayos de deglicosilación. Los resultados finalmente llevaron a descartar esta última hipótesis de Oglicosilación. Sin embargo, determinamos que TrkA necesita de la posición T406 para su correcto tránsito desde las membranas intracelulares hasta la me, [EN] Neurotrophins are soluble ligands implicated in development and survival of the nervous system, working through binding membrane receptors. TrkA is one of these, a tyrosine kinase receptor involved in growth and differentiation of nociceptive neurons, as in their survival. TrkA activation is mediated by NGF neurotrophin binding which leads to dimerization, autophosphorylation and transphosphorylation in the intracellular tyrosine kinase domain. In the study of the role of certain residues in the activation of TrkA, it appeared that mutations in the N404, S405 and T406 residues of the extracellular juxtamembrane domain decreased the activity of TrkA via NGF. This work studies mechanisms of TrkA trafficking and inactivation with N404A, S405A y T406A mutations. Protein trafficking of the receptor in these mutants was studied by flow cytometry and immunofluorescence assays. This was supplemented with activity studies through signalling-kinase activity assays by Western Blot, in the presence of NGF. We observed, especially in the T406A mutant, a reduced ability to reach the membrane, despite the mutation has no effect on activation mechanism of the receptor. Although site N-S-T (404-406) is a N-glycosylation consensus site, that could be responsible of these effects, it was determined that the N404A mutation does not affect TrkA normal location. This suggests that the inactivation of TrkA-T406A is not due to a lack of N-glycan of the N404 residue. Alternatively, a potential O-glycosylation site was predicted in silico at position T406. To evaluate this hypothesis deglycation assays were performed. Results finally led to dismiss this last O-glycosylation hypothesis. However, we determined TrkA needs T406 position for its proper traffic from intracellular membranes to the plasma membrane, suggesting an important post-translational modification, until now unknown in this receptor.

ProNGF expression has been linked to several types of cancers including breast cancer, and we have previously shown that proNGF stimulates breast cancer invasion in an autocrine manner through membrane receptors sortilin and TrkA. However, little is known regarding TrkA-associated protein partners upon proNGF stimulation. By proteomic analysis and proximity ligation assays, we found that proNGF binding to sortilin induced sequential formation of the functional sortilin/TrkA/EphA2 complex, leading to TrkA-phosphorylation dependent Akt activation and EphA2-dependent Src activation. EphA2 inhibition using siRNA approach abolished proNGF-stimulated clonogenic growth of breast cancer cell lines. Combinatorial targeting of TrkA and EphA2 dramatically reduced colony formation in vitro, primary tumor growth and metastatic dissemination towards the brain in vivo. Finally, proximity ligation assay in breast tumor samples revealed that increased TrkA/EphA2 proximity ligation assay signals were correlated with a decrease of overall survival in patients. All together, these data point out the importance of TrkA/EphA2 functional association in proNGF-induced tumor promoting effects, and provide a rationale to target proNGF/TrkA/EphA2 axis by alternative methods other than the simple use of tyrosine kinase inhibitors in breast cancer.

During the development of the sympathetic nervous system, signals from tropomyosin-related kinase receptors (Trks) and p75 neurotrophin receptors (p75) compete to regulate survival and connectivity. During this process, nerve growth factor (NGF)- TrkA signaling in axons communicates NGF-mediated trophic responses in signaling endosomes. Whether axonal p75 signaling contributes to neuronal death and how signaling endosomes contribute to p75 signaling has not been established. Using compartmentalized sympathetic neuronal cultures (CSCGs) as a model, we observed that the addition of BDNF to axons increased the transport of p75 and induced death of sympathetic neurons in a dynein-dependent manner. In cell bodies, internalization of p75 required the activity of JNK, a downstream kinase mediating p75 death signaling in neurons. Additionally, the activity of Rab5, the key GTPase regulating early endosomes, was required for p75 death signaling. In axons, JNK and Rab5 were required for retrograde transport and death signaling mediated by axonal BDNF-p75 in CSCGs. JNK was also required for the proper axonal transport of p75-positive endosomes. Thus, our findings provide evidence that the activation of JNK by p75 in cell bodies and axons is required for internalization to a Rab5-positive signaling endosome and the further propagation of p75-dependent neuronal death signals.

ObjectiveTo investigate the TrkA inhibitor, ASP7962, for treatment of painful knee osteoarthritis.DesignPhase 2a, double-blind, placebo- and naproxen-controlled, double-dummy, parallel-group study. Adults with knee osteoarthritis were randomized (2:2:1) to ASP7962 (100 mg), placebo, or naproxen (500 mg) twice daily (BID) for 4 weeks. Primary endpoint: change from baseline to Week 4 in Western Ontario and McMaster Universities Arthritis Index (WOMAC) pain subscale score. Secondary endpoints: change from baseline to Weeks 1, 2, and End of Treatment (EoT) in WOMAC pain subscale score; change from baseline to Weeks 1, 2, 4, and EoT in WOMAC physical function and stiffness subscales, walking pain and WOMAC total scores; and change from baseline in daily average pain score.Results215 participants were randomized (ASP7962 100 mg BID, n = 85; placebo, n = 87; naproxen 500 mg BID, n = 43). No significant difference was observed between ASP7962 and placebo in change from baseline to Week 4 in WOMAC pain subscale score (-0.14; 90% 2-sided CI: -0.62, 0.34; P = 0.316); a significant difference was observed between naproxen and placebo (-0.67; 80% 2-sided CI: -1.12, -0.23; P = 0.027). No differences were observed between ASP7962 and placebo in change from baseline in any WOMAC subscale score; statistically significant changes were observed between naproxen and placebo (P ≤ 0.01, all time points for all WOMAC endpoints). ASP7962 was safe and well-tolerated.ConclusionsFour-week treatment with ASP7962 (100 mg BID) did not improve pain or physical function in individuals with painful knee osteoarthritis. ClinicalTrials.gov, NCT02611466; EudraCT Number, 2014-004996-22.

Seminal plasma (SP), the liquid fraction of semen, is not mandatory for conception, but clinical studies suggest that SP improves implantation rates. Prior in vitro studies examining the effects of SP on the endometrium, the site of implantation, surprisingly revealed that SP induces transcriptional profiles associated with neurogenesis. We investigated the presence and activity of neurogenesis pathways in the endometrium, focusing on TrkA, one of the canonical receptors associated with neurotrophic signaling. We demonstrate that TrkA is expressed in the endometrium. To determine if SP activates TrkA signaling, we isolated the two most abundant endometrial cell types-endometrial epithelial cells (eEC) and endometrial stromal fibroblasts (eSF)-and examined TrkA activity in these cells after SP exposure. While SP only moderately activated TrkA in eEC, it potently and rapidly activated TrkA in eSF. This activation occurred in both non-decidualized and decidualized eSF. Blocking this pathway resulted in dysregulation of SP-induced cytokine production by eSF. Surprisingly, while the canonical TrkA agonist nerve growth factor was detected in SP, TrkA activation was principally induced by a 30-100-kDa protein whose identity remains to be established. Our results show that TrkA signaling is highly active in eSF and is rapidly induced by SP.

Seminal plasma (SP), the liquid fraction of semen, is not mandatory for conception, but clinical studies suggest that SP improves implantation rates. Prior in vitro studies examining the effects of SP on the endometrium, the site of implantation, surprisingly revealed that SP induces transcriptional profiles associated with neurogenesis. We investigated the presence and activity of neurogenesis pathways in the endometrium, focusing on TrkA, one of the canonical receptors associated with neurotrophic signaling. We demonstrate that TrkA is expressed in the endometrium. To determine if SP activates TrkA signaling, we isolated the two most abundant endometrial cell types-endometrial epithelial cells (eEC) and endometrial stromal fibroblasts (eSF)-and examined TrkA activity in these cells after SP exposure. While SP only moderately activated TrkA in eEC, it potently and rapidly activated TrkA in eSF. This activation occurred in both non-decidualized and decidualized eSF. Blocking this pathway resulted in dysregulation of SP-induced cytokine production by eSF. Surprisingly, while the canonical TrkA agonist nerve growth factor was detected in SP, TrkA activation was principally induced by a 30-100-kDa protein whose identity remains to be established. Our results show that TrkA signaling is highly active in eSF and is rapidly induced by SP.

BackgroundThe highly selective oral tropomyosin receptor kinase (TRK) inhibitor larotrectinib has demonstrated significant activity in adult and pediatric TRK fusion cancers. In the current study, the authors describe the clinical course of children with locally advanced TRK fusion sarcoma who were treated preoperatively with larotrectinib and underwent subsequent surgical resection.MethodsA total of 24 children were treated on a pediatric phase 1 trial of larotrectinib (ClinicalTrials.gov identifier NCT02637687). Five children who had a documented TRK fusion sarcoma and underwent surgical resection were included in the current analysis. Tumor response (Response Evaluation Criteria In Solid Tumors [RECIST] version 1.1) and surgical outcomes were collected prospectively.ResultsA total of 5 patients (median age, 2 years; range, 0.4-12 years) had locally advanced infantile fibrosarcoma (3 patients) or soft-tissue sarcoma (2 patients). Four patients had disease that was refractory to standard therapy. All 5 patients achieved a partial response to larotrectinib by version 1.1 of RECIST and underwent surgical resection after a median of 6 cycles (range, 4-9 cycles) of treatment. Surgical resections were R0 (negative resection margins with no tumor at the inked resection margin) in 3 patients, R1 (microscopic residual tumor at the resection margin) in 1 patient, and R2 (macroscopic residual tumor at the resection margin) in 1 patient. Three patients achieved complete (2 patients) or near-complete (>98% treatment effect; 1 patient) pathologic responses. These patients remained in follow-up and were no longer receiving larotrectinib for a minimum of 7 to 15 months postoperatively. Two patients had viable tumor at the time of surgical resection and positive resection margins and continued to receive adjuvant larotrectinib. No patients experienced postoperative complications or wound healing issues.ConclusionsChildren with locally advanced TRK fusion sarcomas may proceed to

BackgroundGene fusions involving NTRK1, NTRK2, or NTRK3 (TRK fusions) are found in a broad range of paediatric and adult malignancies. Larotrectinib, a highly selective small-molecule inhibitor of the TRK kinases, had shown activity in preclinical models and in adults with tumours harbouring TRK fusions. This study aimed to assess the safety of larotrectinib in paediatric patients.MethodsThis multicentre, open-label, phase 1/2 study was done at eight sites in the USA and enrolled infants, children, and adolescents aged 1 month to 21 years with locally advanced or metastatic solid tumours or CNS tumours that had relapsed, progressed, or were non-responsive to available therapies regardless of TRK fusion status; had a Karnofsky (≥16 years of age) or Lansky (<16 years of age) performance status score of 50 or more, adequate organ function, and full recovery from the acute toxic effects of all previous anticancer therapy. Following a protocol amendment on Sept 12, 2016, patients with locally advanced infantile fibrosarcoma who would require disfiguring surgery to achieve a complete surgical resection were also eligible. Patients were enrolled to three dose cohorts according to a rolling six design. Larotrectinib was administered orally (capsule or liquid formulation), twice daily, on a continuous 28-day schedule, in increasing doses adjusted for age and bodyweight. The primary endpoint of the phase 1 dose escalation component was the safety of larotrectinib, including dose-limiting toxicity. All patients who received at least one dose of larotrectinib were included in the safety analyses. Reported here are results of the phase 1 dose escalation cohort. Phase 1 follow-up and phase 2 are ongoing. This trial is registered with ClinicalTrials.gov, number NCT02637687.FindingsBetween Dec 21, 2015, and April 13, 2017, 24 patients (n=17 with tumours harbouring TRK fusions, n=7 without a documented TRK fusion) with a median age of 4·5 years (IQR 1·3-13·3) were enrolled to thr

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology, 2015., Platelets are the smallest cellular component of circulating blood, and they are critically involved in hemostasis, thrombosis, and inflammation. Diverse pathological conditions impacting platelet production and/or clearance processes may lead to aberrant platelet counts, and pose health risks due to severe hemorrhages, thrombus formation, or impaired immune response. Current therapies for managing these abnormalities are not time- or cost-effective, and other conditions, such as infection and alloimmunization, limit their efficacy. Developing more effective therapies requires a better understanding of the molecular mechanisms underlying thrombopoiesis. Platelet production is a multistage process requiring megakaryocyte maturation and fragmentation in the bone marrow, triggered by an array of growth factors and cytokines. Neurotrophins are among the growth factors that exist in the bone marrow and act through binding tropomyosin receptor kinases (Trks) and/or low affinity receptor p75NTR. In this study, we hypothesized that neurotrophin signaling regulates megakaryopoiesis and thrombopoiesis, and so might prove attractive as a therapeutic target. We have shown that TrkA is expressed by human megakaryocyte progenitors and mature megakaryocytes, and signaling induced by engagement of TrkA receptor with its cognate ligand, nerve growth factor (NGF), increases the number of both cell types, whereas this signaling reduces formation of platelet-like particles. In contrast, treatment with Trk receptor inhibitor, K252a, abrogates these effects and elevates the platelet-like particle counts. In order to study the regulatory role of TrkA signaling, we used Dami cells, a TrkA expressing megakaryocytic cell line, as a model. Modulation of TrkA signaling via K252a-mediated inhibition or through knocking out TrkA in Dami cells increases apoptotic cell death and platelet-like particle counts, which reveals that TrkA is important for megakaryocyte survival and interferes with plate

The tropomyosin-related kinase (Trk) family of receptor tyrosine kinases controls synaptic function, plasticity and sustains differentiation, morphology, and neuronal cell survival. Understanding Trk receptors down-regulation and recycling is a crucial step to point out sympathetic and sensory neuron function and survival. PC12 cells derived from pheochromocytoma of the rat adrenal medulla have been widely used as a model system for studies of neuronal differentiation as they respond to nerve growth factor (NGF) with a dramatic change in phenotype and acquire a number of properties characteristic of sympathetic neurons. In this study we demonstrated that in PC12 cells the TrkA receptor interacts with the deubiquitinating enzyme USP8/UBPy in a NGF-dependent manner and that it is deubiquitinated in vivo and in vitro by USP8. USP8 overexpression blocked NGF-induced neurites outgrowth while the overexpression of the catalytically inactive mutant USP8/UBPyC748A caused a marked increase of cell differentiation. Localization and biochemical experiments have point out that USP8 and TrkA partially co-localize in endosomes after NGF stimulation. Finally we have studied the role played by USP8 on TrkA turnover; using specific siRNA for USP8 we found that USP8 knockdown increases TrkA half-life, suggesting that the deubiquitinating activity of USP8 promotes TrkA degradation.

In both physiological and cell culture systems, EGF-stimulated ERK activity occurs in discrete pulses within individual cells. Many feedback loops are present in the EGF receptor (EGFR)-ERK network, but the mechanisms driving pulsatile ERK kinetics are unknown. Here, we find that in cells that respond to EGF with frequency-modulated pulsatile ERK activity, stimulation through a heterologous TrkA receptor system results in non-pulsatile, amplitude-modulated activation of ERK. We further dissect the kinetics of pulse activity using a combination of FRET- and translocation-based reporters and find that EGFR activity is required to maintain ERK activity throughout the 10-20-minute lifetime of pulses. Together, these data indicate that feedbacks operating within the core Ras-Raf-MEK-ERK cascade are insufficient to drive discrete pulses of ERK activity and instead implicate mechanisms acting at the level of EGFR.

Nerve growth factor (NGF) and its precursor (proNGF) are primarily considered as regulators of neuronal function that induce their responses via the tyrosine kinase receptor TrkA and the pan-neurotrophin receptor p75NTR. It has been generally held that NGF exerts its effects primarily through TrkA, inducing a cascade of tyrosine kinase-initiated responses, while proNGF binds more strongly to p75NTR. When this latter entity interacts with a third receptor, sortilin, apoptotic responses are induced in contrast to the survival/differentiation associated with the other two. Recent studies have outlined portions of the downstream phosphoproteome of TrkA in the neuronal PC12 cells and have clarified the contribution of individual docking sites in the TrkA endodomain. The patterns observed showed a similarity with the profile induced by the epidermal growth factor receptor, which is extensively associated with oncogenesis. Indeed, as with other neurotrophic factors, the distribution of TrkA and p75NTR is not limited to neuronal tissue, thus providing an array of targets outside the nervous systems. One such source is breast cancer cells, in which NGF and proNGF stimulate breast cancer cell survival/growth and enhance cell invasion, respectively. This latter activity is exerted via TrkA (as opposed to p75NTR) in conjunction with sortilin. Another tissue overexpressing proNGF is prostate cancer and here the ability of cancer cells to induce neuritogenesis has been implicated in cancer progression. These studies show that the non-neuronal functions of proNGF/NGF are likely integrated with their neuronal activities and point to the clinical utility of these growth factors and their receptors as biomarkers and therapeutic targets for metastasis and cancer pain.

The tropomyosin-related kinase (Trk) family of receptor tyrosine kinases controls synaptic function, plasticity and sustains differentiation, morphology, and neuronal cell survival. Understanding Trk receptors down-regulation and recycling is a crucial step to point out sympathetic and sensory neuron function and survival. PC12 cells derived from pheochromocytoma of the rat adrenal medulla have been widely used as a model system for studies of neuronal differentiation as they respond to nerve growth factor (NGF) with a dramatic change in phenotype and acquire a number of properties characteristic of sympathetic neurons. In this study we demonstrated that in PC12 cells the TrkA receptor interacts with the deubiquitinating enzyme USP8/UBPy in a NGF-dependent manner and that it is deubiquitinated in vivo and in vitro by USP8. USP8 overexpression blocked NGF-induced neurites outgrowth while the overexpression of the catalytically inactive mutant USP8/UBPyC748A caused a marked increase of cell differentiation. Localization and biochemical experiments have point out that USP8 and TrkA partially co-localize in endosomes after NGF stimulation. Finally we have studied the role played by USP8 on TrkA turnover; using specific siRNA for USP8 we found that USP8 knockdown increases TrkA half-life, suggesting that the deubiquitinating activity of USP8 promotes TrkA degradation.

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.

UnlabelledChronic pain is a major symptom in patients with endometriosis, a common gynecologic condition affecting women in their reproductive years. Although many proalgesic substances are produced by endometriosis lesions, experimental evidence supporting their relative roles is still lacking. Furthermore, it is unclear whether these proalgesic agents directly activate nociceptors to induce endometriosis pain. To determine their relative contribution to pain associated with endometriosis, we evaluated the intrathecal administration of oligodeoxynucleotides (ODNs) antisense to messenger RNA for receptors for 3 pronociceptive mediators known to be produced by the ectopic endometrium. Two weeks after the implant of autologous uterine tissue onto the gastrocnemius muscle, local mechanical hyperalgesia was observed in operated rats. Intrathecal antisense ODN targeting messenger RNA for the interleukin 6 receptor-signaling complex subunit glycoprotein 130 and the nerve growth factor tyrosine kinase receptor A, but not their mismatch ODNs, reversibly attenuated mechanical hyperalgesia at the implant site. In contrast, intrathecal antisense ODN targeting the tumor necrosis factor receptor 1, at a dose that markedly inhibited intramuscularly injected tumor necrosis factor alpha, had only a small antihyperalgesic effect in this model. These results indicate the relative contribution of pronociceptive mediators produced by ectopic endometrial tissue to endometriosis pain. The experimental approach presented here provides a novel method to evaluate for the differential contribution of mediators produced by other painful lesions as well as endometriosis lesions as targets for novel treatment of pain syndromes.PerspectiveThis article presents evidence for the relative contribution of proalgesic mediators to primary hyperalgesia displayed by rats submitted to a model of endometriosis pain. This approach can be used to identify potential targets for the treatment of endometriosis

Receptor-tyrosine kinases (RTKs) are membrane bound receptors characterized by their intrinsic kinase activity. RTK activities play an essential role in several human diseases, including cancer, diabetes and neurodegenerative diseases. RTK activities have been regulated by the expression or silencing of several genes as well as by the utilization of small molecules. Ras Interference 1 (Rin1) is a multifunctional protein that becomes associated with activated RTKs upon ligand stimulation. Rin1 plays a key role in receptor internalization and in signal transduction via activation of Rab5 and association with active form of Ras. This study has two main objectives: (1) It determines the role of Rin1 in the regulation of several RTKs focusing on insulin receptor. This was accomplished by studying the Rin1-insulin receptor interaction using a variety of biochemical and morphological assays. This study shows a novel interaction between the insulin receptor and Rin1 through the Vps9 domain. Two more RTKs (epidermal growth factor receptor and nerve growth factor receptor) also interacted with the SH2 domain of Rin1. The effect of the Rin1-RTK interaction on the activation of both Rab5 and Ras was also studied during receptor internalization and intracellular signaling. Finally, the role of Rin1 was examined in two differentiation processes (adipogenesis and neurogenesis). Rin1 showed a strong inhibitory effect on 3T3-L1 preadipocyte differentiation but it seems to show a modest effect in PC12 neurite outgrowth. These data indicate a selective function and specific interaction of Rin1 toward RTKs. (2) It examines the role of the small molecule Dehydroleucodine (DhL) on several key signaling molecules during adipogenesis. This was accomplished by studying the differentiation of 3T3-L1 preadipocytes exposed to different concentrations of DhL in different days of the adipocyte formation process. The results indicate that DhL selectively blocked adipocyte formation, as well as th

Receptor tyrosine kinases generally act by forming phosphotyrosine-docking sites on their own endodomains that propagate signals through cascades of post-translational modifications driven by the binding of adaptor/effector proteins. The pathways that are stimulated in any given receptor tyrosine kinase are a function of the initial docking sites that are activated and the availability of downstream participants. In the case of the Trk receptors, which are activated by nerve growth factor, there are only two established phosphotyrosine-docking sites (Tyr-490 and Tyr-785 on TrkA) that are known to be directly involved in signal transduction. Taking advantage of this limited repertoire of docking sites and the availability of PC12 cell lines stably transfected with chimeric receptors composed of the extracellular domain of the PDGF receptor and the transmembrane and intracellular domains of TrkA, the downstream TrkA-induced phosphoproteome was assessed for the "native" receptor and mutants lacking Tyr-490 or both Tyr-490 and Tyr-785. Basal phosphorylation levels were compared with those formed after 20 min of stimulation with PDGF. Several thousand phosphopeptides were identified after TiO2 enrichment, and many were up- or down-regulated by receptor activation. The modified proteins in the native sample contained many of the well established participants in TrkA signaling. The results from the mutant receptors allowed grouping of these downstream targets by their dependence on the two characterized docking site(s). A clear subset that was not dependent on either Tyr-490 or Tyr-785 emerged, providing direct evidence that there are other sites on TrkA that are involved in downstream signaling.

Receptor tyrosine kinases (RTKs) function through protein kinase entities located in the intracellular domain of each protomer. Following activation by ligand binding, they selectively form phosphotyrosine residues by autocatalytic modification. Some of these sites are involved in maintaining the active conformation of the kinase, while others become docking sites for various adaptor/effector/scaffold proteins, which, after complexing with the receptor, then initiate further responses through cascades of post-translational modifications and the generation of lipid second messengers. Although there is substantial overlap in the pathways and activities stimulated by this superfamily, the molecular features of the endodomains of the sub-families and the moieties that they interact with to perpetrate their signals are surprisingly distinct, which may play a significant role in the regulation and responses of the individual RTK types. Some use large scaffold proteins as the basis for most, if not all, of their signal-generating interactions, while others have numerous receptor endodomain phosphotyrosine sites that are quite overlapping in specificity. The members of the Trk family of receptors each have several tyrosine residues that are phosphorylated following stimulation, including those in the kinase activation loop, but there are only two established sites (Y490 and Y785 on TrkA) that are known to be directly involved in signal propagation. Taking advantage of this limited repertoire of docking sites, we have applied phosphoproteomic methods to dissect the signaling responses of both the native protein and derivatives that have had these two sites modified. Interestingly, a clear subset that was not dependent on either docking site was identified. A comparison with a similar set of data for EGFR indicates a considerable degree of similarity in the downstream signaling profile between these two RTKs.

Charcot-Marie-Tooth (CMT) Disease is a group of peripheral neuropathies affecting 1 in 2500 individuals. It is characterized by progressive sensory loss and distal muscle weakness affecting mostly the lower extremities that lead to foot deformities. More than thirty genetic loci are affected in this disease, products of which are involved in axonal trafficking. Rab7 is one such gene that gets mutated in CMT Type2B disease. To date four missense mutants of Rab7 have been identified. L129F, K157N, N161T and V162M amino acid substitutions of Rab7 occur in highly conserved regions. Rab7 is a ubiquitous protein but only the peripheral nerves get affected in this disease. This calls for an urgent need to study the pathogenesis of CMT2B disease, the awry molecular mechanisms of which remain unknown. This thesis details for the first time the impaired trafficking, altered endosomal and nuclear signaling caused by Rab7CMT2B mutants through differential interaction with effector proteins. The work provides foundation for identifying potential molecular targets for therapeutic interventions in combating this disease.

Charcot-Marie-Tooth (CMT) Disease is a group of peripheral neuropathies affecting 1 in 2500 individuals. It is characterized by progressive sensory loss and distal muscle weakness affecting mostly the lower extremities that lead to foot deformities. More than thirty genetic loci are affected in this disease, products of which are involved in axonal trafficking. Rab7 is one such gene that gets mutated in CMT Type2B disease. To date four missense mutants of Rab7 have been identified. L129F, K157N, N161T and V162M amino acid substitutions of Rab7 occur in highly conserved regions. Rab7 is a ubiquitous protein but only the peripheral nerves get affected in this disease. This calls for an urgent need to study the pathogenesis of CMT2B disease, the awry molecular mechanisms of which remain unknown. This thesis details for the first time the impaired trafficking, altered endosomal and nuclear signaling caused by Rab7CMT2B mutants through differential interaction with effector proteins. The work provides foundation for identifying potential molecular targets for therapeutic interventions in combating this disease.

Growth factors and their receptors are important for cellular migration as well as axonal guidance and myelination in the brain. They also play a key role in programmed cell death, and are implicated in a number of mental illnesses. Recently, we reported that healthy young adults who carry the T allele variant in the growth factor gene, NTRK1 (at location rs6336), had lower white matter integrity than non-carriers on diffusion images of the brain. Diffusion tensor imaging (DTI) revealed how this single nucleotide polymorphism affects white matter microstructure in human populations; DTI is also used to identify characteristic features of brain connectivity in typically developing children and in patients. Newly discovered links between neuroimaging measures and growth factors whose molecular neuroscience is well known offer an important step in understanding mechanisms that contribute to brain connectivity. Altered fiber connectivity may mediate the relationship between some genetic risk factors and a variety of mental illnesses.

Background: Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive genetic disease characterized by the lack of reaction to noxious stimuli and anhidrosis. It is caused by mutations in the NTRK1 gene, which encodes the high affinity tyrosine kinase receptor I for Neurotrophic Growth Factor (NGF). -- Case Presentation: We present the case of a female patient diagnosed with CIPA at the age of 8 months. The patient is currently 6 years old and her psychomotor development conforms to her age (RMN, SPECT and psychological study are in the range of normality). PCR amplification of DNA, followed by direct sequencing, was used to investigate the presence of NTRK1 gene mutations. Reverse transcriptase (RT)-PCR amplification of RNA, followed by cloning and sequencing of isolated RT-PCR products was used to characterize the effect of the mutations on NTRK1 mRNA splicing. The clinical diagnosis of CIPA was confirmed by the detection of two splice-site mutations in NTRK1, revealing that the patient was a compound heterozygote at this gene. One of these alterations, c.574+1G > A, is located at the splice donor site of intron 5. We also found a second mutation, c.2206-2 A > G, not previously reported in the literature, which is located at the splice acceptor site of intron 16. Each parent was confirmed to be a carrier for one of the mutations by DNA sequencing analysis. It has been proposed that the c.574+1G > A mutation would cause exon 5 skipping during NTRK1 mRNA splicing. We could confirm this prediction and, more importantly, we provide evidence that the novel c.2206-2A > G mutation also disrupts normal NTRK1 splicing, leading to the use of an alternative splice acceptor site within exon 17. As a consequence, this mutation would result in the production of a mutant NTRK1 protein with a seven aminoacid in-frame deletion in its tyrosine kinase domain. --Conclusions: We present the first description of a CIPA-associated NTRK1 mutation causing a sho

Background: Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive genetic disease characterized by the lack of reaction to noxious stimuli and anhidrosis. It is caused by mutations in the NTRK1 gene, which encodes the high affinity tyrosine kinase receptor I for Neurotrophic Growth Factor (NGF). -- Case Presentation: We present the case of a female patient diagnosed with CIPA at the age of 8 months. The patient is currently 6 years old and her psychomotor development conforms to her age (RMN, SPECT and psychological study are in the range of normality). PCR amplification of DNA, followed by direct sequencing, was used to investigate the presence of NTRK1 gene mutations. Reverse transcriptase (RT)-PCR amplification of RNA, followed by cloning and sequencing of isolated RT-PCR products was used to characterize the effect of the mutations on NTRK1 mRNA splicing. The clinical diagnosis of CIPA was confirmed by the detection of two splice-site mutations in NTRK1, revealing that the patient was a compound heterozygote at this gene. One of these alterations, c.574+1G > A, is located at the splice donor site of intron 5. We also found a second mutation, c.2206-2 A > G, not previously reported in the literature, which is located at the splice acceptor site of intron 16. Each parent was confirmed to be a carrier for one of the mutations by DNA sequencing analysis. It has been proposed that the c.574+1G > A mutation would cause exon 5 skipping during NTRK1 mRNA splicing. We could confirm this prediction and, more importantly, we provide evidence that the novel c.2206-2A > G mutation also disrupts normal NTRK1 splicing, leading to the use of an alternative splice acceptor site within exon 17. As a consequence, this mutation would result in the production of a mutant NTRK1 protein with a seven aminoacid in-frame deletion in its tyrosine kinase domain. --Conclusions: We present the first description of a CIPA-associated NTRK1 mutation causing a sho

Background: Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive genetic disease characterized by the lack of reaction to noxious stimuli and anhidrosis. It is caused by mutations in the NTRK1 gene, which encodes the high affinity tyrosine kinase receptor I for Neurotrophic Growth Factor (NGF). -- Case Presentation: We present the case of a female patient diagnosed with CIPA at the age of 8 months. The patient is currently 6 years old and her psychomotor development conforms to her age (RMN, SPECT and psychological study are in the range of normality). PCR amplification of DNA, followed by direct sequencing, was used to investigate the presence of NTRK1 gene mutations. Reverse transcriptase (RT)-PCR amplification of RNA, followed by cloning and sequencing of isolated RT-PCR products was used to characterize the effect of the mutations on NTRK1 mRNA splicing. The clinical diagnosis of CIPA was confirmed by the detection of two splice-site mutations in NTRK1, revealing that the patient was a compound heterozygote at this gene. One of these alterations, c.574+1G > A, is located at the splice donor site of intron 5. We also found a second mutation, c.2206-2 A > G, not previously reported in the literature, which is located at the splice acceptor site of intron 16. Each parent was confirmed to be a carrier for one of the mutations by DNA sequencing analysis. It has been proposed that the c.574+1G > A mutation would cause exon 5 skipping during NTRK1 mRNA splicing. We could confirm this prediction and, more importantly, we provide evidence that the novel c.2206-2A > G mutation also disrupts normal NTRK1 splicing, leading to the use of an alternative splice acceptor site within exon 17. As a consequence, this mutation would result in the production of a mutant NTRK1 protein with a seven aminoacid in-frame deletion in its tyrosine kinase domain. --Conclusions: We present the first description of a CIPA-associated NTRK1 mutation causing a sho

Endophilin B1 is a member of the endophilin family that is localized predominantly to intracellular membranes. Also known as Bax-interacting factor-1 (Bif-1), this protein has been observed to regulate the membrane dynamics of various intracellular compartments, such as the control of mitochondrial morphology and autophagosome formation in fibroblast. Endophilin B1 is expressed in the brain, but its functions in neurons had remained unknown. Recently, we have observed a novel role of endophilin B1 in neurons where it controls the trafficking of TrkA, cognate receptor for the prototypic neurotrophin nerve growth factor (NGF). Knock-down of endophilin B1 expression induces precocious targeting of NGF/TrkA to late endosomes and lysosomes, thereby leading to reduced TrkA levels. This is accompanied by marked attenuation of NGF-induced gene transcription and neurite outgrowth. Our observations suggest that endophilin B1 regulates TrkA level and downstream functions by controlling the movement of TrkA to late endosomes/lysosomes, possibly acting at the level of early endosomes. © 2009 Landes Bioscience.

先天性無痛無汗症(CIPA)は温覚・痛覚と発汗機能を欠如し、精神遅滞を伴う常染色体劣性遺伝の疾患である。私たちは、1996年にこの疾患の原因をはじめて明らかにし、報告した。CIPAの原因は、神経成長因子(NGF)に対する受容体タンパク質をコードする遺伝子TRKA(NTRK1)の機能喪失性変異である。本研究では、CIPAの分子病態を解析することにより、NGFが内感覚神経と交感神経の両者の生存・維持に必須であることを示唆する結果を得た。このことは、疼痛のメカニズムを考える上で、新たな視点を開く可能性がある。

Endophilin B1 is a member of the endophilin family that is localized predominantly to intracellular membranes. Also known as Bax-interacting factor-1 (Bif-1), this protein has been observed to regulate the membrane dynamics of various intracellular compartments, such as the control of mitochondrial morphology and autophagosome formation in fibroblast. Endophilin B1 is expressed in the brain, but its functions in neurons had remained unknown. Recently, we have observed a novel role of endophilin B1 in neurons where it controls the trafficking of TrkA, cognate receptor for the prototypic neurotrophin nerve growth factor (NGF). Knock-down of endophilin B1 expression induces precocious targeting of NGF/TrkA to late endosomes and lysosomes, thereby leading to reduced TrkA levels. This is accompanied by marked attenuation of NGF-induced gene transcription and neurite outgrowth. Our observations suggest that endophilin B1 regulates TrkA level and downstream functions by controlling the movement of TrkA to late endosomes/lysosomes, possibly acting at the level of early endosomes. © 2009 Landes Bioscience.

GIPC is a PDZ protein located on peripheral endosomes that binds to the juxtamembrane region of the TrkA nerve growth factor (NGF) receptor and has been implicated in NGF signaling. We establish here that endogenous GIPC binds to the C terminus of APPL, a Rab5 binding protein, which is a marker for signaling endosomes. When PC12(615) cells are treated with either NGF or antibody agonists to activate TrkA, GIPC and APPL translocate from the cytoplasm and bind to incoming, endocytic vesicles carrying TrkA concentrated at the tips of the cell processes. GIPC, but not APPL, dissociates from these peripheral endosomes prior to or during their trafficking from the cell periphery to the juxtanuclear region, where they acquire EEA1. GIPC's interaction with APPL is essential for recruitment of GIPC to peripheral endosomes and for TrkA signaling, because a GIPC PDZ domain mutant that cannot bind APPL or APPL knockdown with small interfering RNA inhibits NGF-induced GIPC recruitment, mitogen-activated protein kinase activation, and neurite outgrowth. GIPC is also required for efficient endocytosis and trafficking of TrkA because depletion of GIPC slows down endocytosis and trafficking of TrkA and APPL to the early EEA1 endosomes in the juxtanuclear region. We conclude that GIPC, following its recruitment to TrkA by APPL, plays a key role in TrkA trafficking and signaling from endosomes.

GIPC is a PDZ protein located on peripheral endosomes that binds to the juxtamembrane region of the TrkA nerve growth factor (NGF) receptor and has been implicated in NGF signaling. We establish here that endogenous GIPC binds to the C terminus of APPL, a Rab5 binding protein, which is a marker for signaling endosomes. When PC12(615) cells are treated with either NGF or antibody agonists to activate TrkA, GIPC and APPL translocate from the cytoplasm and bind to incoming, endocytic vesicles carrying TrkA concentrated at the tips of the cell processes. GIPC, but not APPL, dissociates from these peripheral endosomes prior to or during their trafficking from the cell periphery to the juxtanuclear region, where they acquire EEA1. GIPC's interaction with APPL is essential for recruitment of GIPC to peripheral endosomes and for TrkA signaling, because a GIPC PDZ domain mutant that cannot bind APPL or APPL knockdown with small interfering RNA inhibits NGF-induced GIPC recruitment, mitogen-activated protein kinase activation, and neurite outgrowth. GIPC is also required for efficient endocytosis and trafficking of TrkA because depletion of GIPC slows down endocytosis and trafficking of TrkA and APPL to the early EEA1 endosomes in the juxtanuclear region. We conclude that GIPC, following its recruitment to TrkA by APPL, plays a key role in TrkA trafficking and signaling from endosomes.

Mast cells have a long life in tissues, where they are involved in inflammatory and hypersensitivity reactions. Activation through cross-linking of the high-affinity IgE-receptor (FcεRI) is a primary stimulus capable of degranulating mast cells, contributing to symptoms of allergy. The aim of this study was to investigate the mechanisms by which mast cells survive the allergic activation. The study confirmed on a continual single-cell basis that mast cells could indeed recover from the IgE-mediated degranulation and be activated again. For the second activation, kinetics of synthesis and release of certain mediators paralleled those of the initial activation. Mast cell survival was promoted due to a decreased level of apoptosis as a result of activation by FcεRI, suggesting that the activation initiated an anti-apoptotic program in mast cells. Given the potential survival- promoting role of nerve growth factor (NGF) in many cell types, we determined whether mast cells released and responded to NGF. It was found that mast cells expressed functional TrkA, the high-affinity receptor for NGF. Furthermore, the release of NGF was increased from degranulating mast cells in response to FcεRI cross-linking. Next, the possible effects of anti-apoptotic Bcl-2 family genes on mast cell survival were examined. Following FcεRI aggregation, transcripts of the pro-survival Bcl-2 homologue Al were remarkably up-regulated in mast cells. In contrast, degranulating mast cells from Al knock-out mice failed to exhibit survival advantage. These data strongly suggest that Al is required for mast call survival following allergic activation. In conclusion, stimuli causing an allergic reaction trigger mast cells to release NGF and up- regulate Al, which may contribute to the survival of mast cells. Our finding may be of significance in understanding the mechanisms underlying the long life-span of mast cells during allergic reactions and hence indicate possible therapeutic interventions.

Congenital insensitivity to pain with anhidrosis is an autosomal recessive hereditary disorder characterized by recurrent episodic fever, anhidrosis (inability to sweat), absence of reaction to noxious stimuli, self-mutilating behavior, and mental retardation. The human TRKA gene (NTRK1), located on chromosome 1q21-q22 encodes the receptor tyrosine kinase for nerve growth factor. We reported that TRKA is the gene responsible for CIPA and we developed a comprehensive strategy to screen for TRKA mutations and polymorphisms, as based on the gene's structure and organization. Here we report eight novel mutations detected as either a homozygous or heterozygous state in nine CIPA families from five countries. Mendelian inheritance of the mutations was confirmed in seven families for which samples from either parent were available. However, non-mendelian inheritance seems likely for the family when only samples from the mother and siblings, (but not from the father) were available. A paternal uniparental disomy for chromosome 1 is likely to be the cause of reduction to homozygosity of the TRKA gene mutation in this family. Interestingly, a Hispanic patient from the USA has two autosomal genetic disorders, CIPA and pyruvate kinase deficiency, whose genetic loci are both mapped to a closely linked chromosomal region. A splice mutation and a missense mutation were detected in the TRKA and PKLR genes from the homozygous proband, respectively. Thus, concomitant occurrence of two disorders is ascribed to a combination of two separate mutant genes, not a contiguous gene syndrome. This finding suggests a mechanism responsible for two autosomal genetic disorders in one patient. All these data further support findings that TRKA defects can cause CIPA in various ethnic groups. This will aid in diagnosis and genetic counseling of this painless but severe genetic disorder. Hum Mutat.

We have recently reported that retinoic acid (RA) induced the expression of trkA, the high affinity receptor for nerve growth factor (NGF), in human myeloid leukemia KG-1 cells. In the present study, we report that the expression of trkA was also induced by several other differentiation inducers, including 1α, 25-dihydroxyvitamin D3 (Vit D3), 1-β-D-arabinofuranosyl cytosine (Ara-C), sodium butyrate (NaBut), and phorbol 12-myristate 13-acetate (PMA). Interestingly, RA in combination with NaBut or PMA synergistically induced cellular differentiation as well as the expression of trkA in KG-1 cells. Furthermore, activation of the induced trkA receptor by exogenous NGF potentiated the differentiating effects of RA and NaBut. Our results demonstrated that the induction of trkA is an event associated with the differentiation of KG-1 cells. Our findings suggest that NGF, in addition to its pivotal roles in the nervous system, may also play important roles in hematopoietic differentiation.

We have recently reported that retinoic acid (RA) induced the expression of trkA, the high affinity receptor for nerve growth factor (NGF), in human chronic myelogenous leukemia K562 cells. In this paper, we examined the ability of several other differentiation inducers to regulate the expression of trkA and NGF in K562 cells. We found that the expression of trkA was dramatically induced by the two megakaryocyte lineage inducers sodium butyrate (NaBut) and phorbol 12-myristate 13-acetate (PMA), but not by the two erythrocyte lineage inducers hemin or 1-beta-D-arabinofuranosyl cytosine (Ara-C). Furthermore, activation of the up-regulated trkA receptor by exogenous NGF potentiated the megakaryocytic differentiation of K562 cells induced by NaBut and PMA. Our results demonstrated that trkA is one of the essential genes that are up-regulated and involved in the megakaryocytic differentiation of K562 leukemia cells triggered by these differentiation inducers. Our findings suggest that NGF, in addition to its pivotal roles in the nervous system, may also play important roles in hematopoietic differentiation. (C) 2000 Elsevier Science Ltd. All rights reserved.