Differential transcriptomic landscapes of multiple organs from SARS-CoV-2 early infected rhesus macaques.

SARS-CoV-2 infection causes complicated clinical manifestations with variable multi-organ injuries, however, the underlying mechanism, in particular immune responses in different organs, remains elusive. In this study, comprehensive transcriptomic alterations of 14 tissues from rhesus macaque infected with SARS-CoV-2 were analyzed. Compared to normal controls, SARS-CoV-2 infection resulted in dysregulation of genes involving diverse functions in various examined tissues/organs, with drastic transcriptomic changes in cerebral cortex and right ventricle. Intriguingly, cerebral cortex exhibited a hyperinflammatory state evidenced by significant upregulation of inflammation response-related genes. Meanwhile, expressions of coagulation, angiogenesis and fibrosis factors were also up-regulated in cerebral cortex. Based on our findings, neuropilin 1 (NRP1), a receptor of SARS-CoV-2, was significantly elevated in cerebral cortex post infection, accompanied by active immune response releasing inflammatory factors and signal transmission among tissues, which enhanced infection of the central nervous system (CNS) in a positive feedback way, leading to viral encephalitis. Overall, our study depicts a multi-tissue/organ transcriptomic landscapes of rhesus macaque with early infection of SARS-CoV-2, and provides important insights into the mechanistic basis for COVID-19-associated clinical complications.

Azvudine is a thymus-homing anti-SARS-CoV-2 drug effective in treating COVID-19 patients.

Azvudine (FNC) is a nucleoside analog that inhibits HIV-1 RNA-dependent RNA polymerase (RdRp). Recently, we discovered FNC an agent against SARS-CoV-2, and have taken it into Phase III trial for COVID-19 patients. FNC monophosphate analog inhibited SARS-CoV-2 and HCoV-OC43 coronavirus with an EC50 between 1.2 and 4.3 µM, depending on viruses or cells, and selective index (SI) in 15-83 range. Oral administration of FNC in rats revealed a substantial thymus-homing feature, with FNC triphosphate (the active form) concentrated in the thymus and peripheral blood mononuclear cells (PBMC). Treating SARS-CoV-2 infected rhesus macaques with FNC (0.07 mg/kg, qd, orally) reduced viral load, recuperated the thymus, improved lymphocyte profiles, alleviated inflammation and organ damage, and lessened ground-glass opacities in chest X-ray. Single-cell sequencing suggested the promotion of thymus function by FNC. A randomized, single-arm clinical trial of FNC on compassionate use (n = 31) showed that oral FNC (5 mg, qd) cured all COVID-19 patients, with 100% viral ribonucleic acid negative conversion in 3.29 ± 2.22 days (range: 1-9 days) and 100% hospital discharge rate in 9.00 ± 4.93 days (range: 2-25 days). The side-effect of FNC is minor and transient dizziness and nausea in 16.12% (5/31) patients. Thus, FNC might cure COVID-19 through its anti-SARS-CoV-2 activity concentrated in the thymus, followed by promoted immunity.

Dalbavancin binds ACE2 to block its interaction with SARS-CoV-2 spike protein and is effective in inhibiting SARS-CoV-2 infection in animal models.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic worldwide. Currently, however, no effective drug or vaccine is available to treat or prevent the resulting coronavirus disease 2019 (COVID-19). Here, we report our discovery of a promising anti-COVID-19 drug candidate, the lipoglycopeptide antibiotic dalbavancin, based on virtual screening of the FDA-approved peptide drug library combined with in vitro and in vivo functional antiviral assays. Our results showed that dalbavancin directly binds to human angiotensin-converting enzyme 2 (ACE2) with high affinity, thereby blocking its interaction with the SARS-CoV-2 spike protein. Furthermore, dalbavancin effectively prevents SARS-CoV-2 replication in Vero E6 cells with an EC50 of ~12 nM. In both mouse and rhesus macaque models, viral replication and histopathological injuries caused by SARS-CoV-2 infection are significantly inhibited by dalbavancin administration. Given its high safety and long plasma half-life (8-10 days) shown in previous clinical trials, our data indicate that dalbavancin is a promising anti-COVID-19 drug candidate.

RNA-binding Protein UNR Promotes Glioma Cell Migration and Regulates the Expression of Ribosomal Protein L9.

Objective To investigate the role of RNA binding protein─upstream-of-N-Ras (UNR) in the development of glioma and its molecular mechanism.Methods First, bioinformatics analysis of CGGA database was performed to detect UNR expression level and prognosis of patients with glioma. Western blot and real-time PCR were used to detect UNR expression level in glioma cell lines and tissues. Next, UNR siRNAs were transfected in glioma cells, and MTS assay and scratch wound-healing assay were used to detect changes in cell proliferation and migration. Then, the candidate UNR target mRNAs were identified by analyzing the sequencing data of UNR iCLIP-seq, RNA sequencing and ribosome profiling databases of human melanoma. RNA immunoprecipitation and biotin pull-down assays were used to identify the UNR target mRNAs in glioma cells. Finally, western blot was used to detect the effect of UNR knockdown on ribosomal protein L9 (RPL9) and RPL9 protein expression level in glioma cell lines. RPL9 siRNA was transfected in A172 and T98G and the expression of vimentin in the cells was detected with western blot.Results Bioinformatics analysis showed that UNR mRNA expression level was significantly higher in high-grade glioma [Grade 2 (n=126), Grade 3 (n=51), Grade 4 (n=128), P<0.001]. UNR high expression levels were associated with poor prognosis (P=0.0177). UNR had high expression level in glioma cell lines and patient samples compared with normal cell lines and normal brain samples (P<0.01). Knockdown of UNR inhibited glioma cells migration (P<0.05), but did not inhibit glioma cells growth in three glioma cell lines. UNR binded the 3' untranslated region (UTR) of PTEN and RPL9 mRNAs. RPL9 protein was significantly highly expressed in most glioma cell lines (n=9) and knockdown of UNR resulted in a downregulation of RPL9 protein expression. Epithelial-mesenchymal transition (EMT)-related marker─vimentin was positively regulated by RPL9.Conclusions UNR could bind to the 3'UTR of PTEN and RPL9 in glioma cell lines, therefore promoting glioma cell migration and regulating the expression of RPL9. Here, we establish a link between UNR and RPL9 protein, which will provide new ideas for the further study of glioma.

Mutation of the cellular adhesion molecule NECL2 is associated with neuromyelitis optica spectrum disorder.

AIMS: To investigate the association of the Nectin/Necl family genes with the risk of developing NMOSD. METHODS: Whole-exome sequencing was performed on two familial NMOSD cases and two unaffected family members. Additionally, 106 patients with sporadic NMOSD and 212 healthy controls (HCs) underwent screening for mutant Necl2. Finally, the molecular weight and cellular localization of mutant NECL2 was examined in transfected HeLa cells. RESULTS: We identified a novel deletion mutation in Necl2 (c.1052_1060delCCACCACCA; p. Thr351_Thr353del), which was associated with disease manifestation in the NMOSD familial cases. The frequency at which the mutation occurred in patients with sporadic NMOSD was significantly higher than for HCs (5.7% and 0, respectively; p<0.01). The mutation was located in the extracellular domain close to the transmembrane region, at a point in the protein sequence characterized by threonine enrichment. The mutant NECL2 had a lower molecular weight and exhibited defective trafficking to the cell surface. CONCLUSIONS: Our results suggest that the Necl2 mutation identified herein may be associated with the risk of developing NMOSD. Furthermore, mutated NECL2 may play a role in the pathogenesis of the disease, potentially through its roles in axonal regeneration and/or via neuron-glia interactions that are relevant to myelination.

Recombinant Expression and Purification of Mouse Nectin-like 4 Glycoprotein in 293ET Cell Line.

Objective To screen the transient and stable cell lines with high production of Nectin-like 4 (Necl-4) protein. Methods First, cDNA sequences encoding the extracellular domain of Necls were cloned into the modified vector pAPtag at the N terminus of alkaline phosphatase (AP) for fusion expression. Next, 293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis. Then, by adding N-glycosylation processing inhibitor kifunensine into the medium, complex glycan was inhibited to generate. The residual glycan of purified protein was removed by endoglycosidase H. Finally, AP protein was removed by using human rhinovirus protease and size exclusion chromatography. The concentration of purified Necl-4 protein was monitored by measuring the absorbance at 280 nm and analyzed by SDS-PAGE. Result The transient and stable cell lines with high production of Necl-4 protein were screened by the color reaction with the AP-tag in the recombinant vector. The soluble and active form of purified Necl-4 protein was obtained after deglycosylation of native N-glycan protein with an expression level of 4 mg/L culture and purity of 95%. Conclusions By using modified AP mammalian protein expression system, we can easily screen the high productive stable cell lines by using AP activity assay. By adding mannosidase inhibitor kifunensine into the medium and cutting purified protein by using endoglycosidase H, we can obtain deglycosylated Necl-4 protein in milligram quantities. Our method might throw a light on the expression and purification of glycoprotein for structural and functional studies.

Astragaloside â £ Protects Against Aß1-42-induced Oxidative Stress, Neuroinflammation and Cognitive Impairment in Rats.

Objective To investigate the neuroprotective action of astragaloside Ⅳ (AS-Ⅳ) on spatial learning and memory impairment induced by amyloid-beta 1-42 (Aß1-42) in rats and elucidate its underlying molecular mechanisms. Methods Adult-male Sprague-Dawley rats (230-250 g) were divided into six groups randomly: control, Aß1-42, AS-Ⅳ, Aß1-42 plus 5 mg/kg·d AS-Ⅳ, Aß1-42 plus 25 mg/kg·d AS-Ⅳ, and Aß1-42 plus 50 mg/kg·d AS-Ⅳ groups. Aß1-42 were delivered by intracerebroventricular injection under the guidance of a brain stereotaxic apparatus. The Morris water maze test (hidden platform test, probe trials, visible platform test) was performed one week after Aß1-42 injection to obtain the ability of rat spatial learning and memory. AS-Ⅳ (5, 25 and 50 mg/kg·d) was administrated intraperitoneally once per day from the 8th day after Aß1-42 injection for 5 consecutive days. Average escape latencies, distances for searching for the platform under water and the percentage of total time elapsed and distance swam in the right quadrant after removing platform were determined by behavior software system. The vision and swim speeds of rats were also determined to exclude the effect of these factors on the parameters of learning and memory. After behavioral tests, the rats were sacrificed immediately by decapitation. Hippocampus were collected. The enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT) in the hippocampus obtained from different-treated rat brain were measured by following the manufacturer's instructions. The levels of interleukin-1 beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) in tissue lysates were assayed with ELISA. Results The water maze test results indicated that chronic treatments with AS-Ⅳ effectively protected the rats from Aß1-42-induced spatial learning and memory impairment. Furthermore, the activities of SOD, GSH-px and CAT decreased by Aß1-42 were also restored by AS-Ⅳ treatment in the hippocampus of rats. In addition, AS-Ⅳ significantly decreased the levels of IL-1ß and TNF-α in the hippocampus of Aß1-42-induced amnesia's rats. Conclusion Our findings suggest that AS-Ⅳ might be a useful chemical in improving the spatial memory and relieving the oxidative stress and neuroinflammation in Alzheimer patients.

High expression of PTBP1 promote invasion of colorectal cancer by alternative splicing of cortactin.

Polypyrimidine tract-binding protein 1 (PTBP1) involving in almost all steps of mRNA regulation including alternative splicing metabolism during tumorigenesis due to its RNA-binding activity. Initially, we found that high expressed PTBP1 and poor prognosis was interrelated in colorectal cancer (CRC) patients with stages II and III CRC, which widely different in prognosis and treatment, by immunohistochemistry. PTBP1 was also upregulated in colon cancer cell lines. In our study, knockdown of PTBP1 by siRNA transfection decreased cell proliferation and invasion in vitro. Denovirus shRNA knockdown of PTBP1 inhibited colorectal cancer growth in vivo. Furthermore, PTBP1 regulates alternative splicing of many target genes involving in tumorgenesis in colon cancer cells. We confirmed that the splicing of cortactin exon 11 which was only contained in cortactin isoform-a, as a PTBP1 target. Knockdown of PTBP1 decreased the expression of cortactin isoform-a by exclusion of exon 11. Also the mRNA levels of PTBP1 and cortactin isoform-a were cooperatively expressed in colorectal cancer tissues. Knocking down cortactin isoform-a significantly decreased cell migration and invasion in colorectal cancer cells. Overexpression of cortactin isoform-a could rescue PTBP1-knockdown effect of cell motility. In summary the study revealed that PTBP1 facilitates colorectal cancer migration and invasion activities by inclusion of cortactin exon 11.

Functions of miR-9 and miR-9* during Aging in SAMP8 Mice and Their Possible Mechanisms.

OBJECTIVE: To explore the functions of miR-9 and miR-9(*) in SAMP8 mice during the aging and their possible mechanisms. METHODS: SAMP8 mice(4-,8-,12-month old,respectively)were selected,three age-matched SAMR1 mice were used as the control group with three mice in each group. The brains were collected and then sectioned for in situ hybridization of miR-9 and miR-9(*). Mimics or inhibitors of miR-9 and miR-9(*) were transfected into N2a cells,and the effects of overexpression or knockdown of the microRNAs on the cell cycle were detected by flow cytometry. Target genes were predicted by bioinformatic analysis and confirmed by dual luciferase assay. RESULTS: Expressions of miR-9 and miR-9(*) in hippocampus of SAMP8 mice were lower than those of SAMR1 mice. Knockdown of miR-9 and miR-9(*) induced a prolonged G1 phase and a shortened S phase in N2a cells;in contrast,miR-9 and miR-9(*) overexpression showed opposite effects. The predicted target genes of miR-9 were PSEN1,SCN2B,MAP3K3,and BACE1,and that of miR-9(*) was CDKn1c. Dual luciferase reporter gene assay showed that miR-9 targeted MAP3K3 while miR-9(*) targeted CDKn1c. CONCLUSION: miR-9 and miR-9(*) play an important role during aging via the target genes MAP3K3 and CDKn1c in the SAMP8 mice.

Ribotrap analysis of proteins associated with FHL3 3'untranslated region in glioma cells.

OBJECTIVE: To screen the proteins associated with four-and-a-half LIM domains 3 (FHL3) 3' untranslated region (3'UTR) in glioma cells. METHODS: Western blot was adopted to detect the regulatory effect of poly(C)-binding protein 2 (PCBP2) on FHL3. Biotin pull-down and sliver staining were employed to screen and verify the candidate binding proteins of FHL3 3'UTR. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecule annotation system were used to identify and analyze the candidate binding proteins. Immuno- precipitation was conducted to study the interaction between PCBP2 and polypyrimidine tract-binding protein 1 (PTBP1), a binding protein identified by LC-MS/MS. RESULTS: PCBP2 could bind to FHL3 mRNA 3'UTR-A and inhibited the expression of FHL3 in T98G glioms cells. 22 candidate binding proteins were identified. Among them, there were 11 RNA binding proteins, including PCBP2. PTBP1 associated with FHL3 mRNA 3'UTR and interacted with PCBP2 protein. CONCLUSIONS: PCBP2 and PTBP1 can both associate with FHL3 mRNA 3'UTR through forming a protein complex.

The stability of NR2B in the nucleus accumbens controls behavioral and synaptic adaptations to chronic stress.

BACKGROUND: The nucleus accumbens (NAc) is closely correlated with depression. It has been demonstrated that the glutamatergic system in NAc plays an important role in the reward pathway, dysfunction of which would cause anhedonia, a core symptom of depression. We therefore tested whether N-methyl-D-aspartate receptors and the synaptic plasticity in the NAc are regulated by chronic stress and the relevance to depression. METHODS: We applied behavioral tests (n = 12, each group) of social interaction and sucrose preference tests to identify the susceptibility of mice to chronic social defeat stress. We then tested N-methyl-D-aspartate receptor-long-term depression at cortico-accumbal synapse to determine the relationship between the susceptibility and changes in synaptic plasticity (n = 8, each group). We further investigated whether restoration of these changes could produce antidepressant effects (n = 10). RESULTS: We found that chronic stress induced selective downregulation of N-methyl-D-aspartate receptor NR2B subunits in the confined surface membrane pool of NAc neurons. Remarkably, the loss of synaptic NR2B was a long-lived event and further translated to the significant modulation of synaptic plasticity in the form of long-term depression. We further observed that the stress-induced changes were restored by fluoxetine and that resilient mice-those resistant to chronic stress-showed patterns of molecular regulation in the NAc that overlapped dramatically with those seen with fluoxetine treatment. Behaviorally, restoration of NR2B loss prevented the behavioral sensitization of mice to chronic stress. CONCLUSIONS: Our results identify NR2B in the NAc as a key regulator in the modulation of persistent psychomotor plasticity in response to chronic stress.

Screening of substrates of protein arginine methyltransferase 1 in glioma.

OBJECTIVE: To screen the asymmetric dimethyl arginines (ADMA)-containing proteins which could combine with protein arginine methyltransferase 1 (PRMT1). METHODS: Western blot was adopted to identify the expression of PRMT1 and the proteins with ADMA in glioma cell lines and normal brain tissues, and then to detect the changes of ADMA level after knock-down of PRMT1 with RNAi transfection in U87MG cells. Co-Immunoprecipitation (Co-IP), western blot, and sliver staining were employed to screen the candidate binding proteins of PRMT1. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the binding proteins of PRMT1. RESULTS: The expression of PRMT1 and some levels of ADMA were higher in glioma cell lines than in normal brain tissues. After knocking down PRMT1, some ADMA levels were found declined. After screening the binding proteins of PRMT1 with Co-IP and LC-MS/MS, 26 candidate binding proteins were identified. Among them, 6 candidate proteins had higher ions scores (> 38) and bioinformation analysis predicted that SEC23-IP, ANKHD1-EIF4EBP3 protein, and 1-phosphatidylinositol-3-phosphate 5-kinase isoform 2 had possible methylated aginine sites. CONCLUSIONS: The high expression of PRMT1 in glioma may induce the change of ADMA levels. Altogether 26 candidate proteins were identified, which contain ADMA and specifically bind with PRMT1.

[Role of extracellular signal-regulated protein kinase 5 in the biosynthesis of follicle-stimulating hormone-stimulated progesterone in primary granulosa cells].

OBJECTIVE: To study the role of extracellular signal-regulated protein kinase 5 (ERK5) during the biosynthesis of follicle-stimulating hormone (FSH)-mediated progesterone in primary granulosa cells. METHODS: The expressions of phosphorylated and general forms of ERKS in primary granulosa cells after the treatment of FSH were detected by Western blot analysis. The subcellular localization of ERK5 was observed by confocal microscopy. The effect of ERK5 on FSH-mediated progesterone biosynthesis in primary granulosa cells was analyzed using recombinant adenovirus vectors. RESULTS: ERK5 activation was induced by FSH in a time-dependent manner in primary cultured granulosa cells, although the general ERK5 protein level decreased also in a time-dependent manner. The treatment of FSH showed no remarkable effect on the subcellular distribution of endogenous ERK5, which was mainly in the cytoplasm of granulosa cells. The co-infection of Ad-caMEK5 and Ad-wtERK5 increased the progesterone production and StAR expression in primary cultured granulosa cells, whereas inhibition of ERK5 activation suppressed the FSH-stimulated progesterone production. CONCLUSION: ERK5 may stimulate FSH-mediated progesterone production in primary cultured granulosa cells.

[Screening and identification of natural antisense transcript in mouse cerebral cortex].

OBJECTIVE: To screen and identify the possible existence of natural antisense transcript (NAT) within the mouse neocortex. METHODS: Sixty-three cerebral cortex layer-specific genes were screened by bioinformatics prediction in mice, among which 31 mice with potential NATs were screened. NAT was identified using reverse transcription polymerase chain reaction (RT-PCR) and then cloned in pGEM-T Vector System for sequencing. RESULTS: Among 31 genes predicted using bioinformatics, 8 were proved to be NAT positive by RT-PCR. CONCLUSIONS: NATs exist in the mouse neocortex tissue during the development of cerebral cortex. NATs may influence mouse cortical development by regulating the related coding genes.

[A population-based epidemiologic study of bowel habits in Guangdong province].

OBJECTIVE: To determine the bowel habits and its perceptions in the general population of Guangdong province. METHODS: Random clustered sampling involving permanent inhabitants aged 18 - 80 year was carried out under stratification of urban and suburban areas in Guangdong province. Questionnaire included the items on the characteristics of people being selected and their bowel habits. RESULTS: A total of 4103 residents (male 1878, female 2225) were investigated. Mean age among the responders was 42.81 ± 14.13 year. Among 4056 subjects (missing = 47), 2972 subjects (73.3%) reported daily defecation, and 3951 subjects (97.4%) reported stool frequency between 3 times per week and three times per day. Two hundred and seventy subjects (6.6%) reported abnormal bowel habits by themselves. The stool frequency (OR = 2.03, 95%CI: 1.54 - 2.67), forms of stool (OR = 2.75, 95%CI: 2.35 - 3.22) and straining (OR = 3.56, 95%CI: 2.49 - 5.11) were significantly associated with self-reported abnormal bowel habits. Among 3949 subjects (missing = 154), 644 (16.3%) were defined as having abnormal bowel habits according to Rome II criteria. There was poor agreement between self-reported abnormal bowel habits and that defined by Rome II criteria (Kappa = 0.312). CONCLUSION: It seemed to be appropriate that the normal stool frequency was defined as bowel movements between 3 times per week and three times per day in the general population. The prevalence of self-reported abnormal bowel habits was lower than that defined by Rome II criteria and the agreement between these two definitions was poor.

D-Tyr-tRNA(Tyr) deacylase, a new role in Alzheimer's-associated disease in SAMP8 mice.

OBJECTIVE: To assess the expression level of D-Tyr-tRNA(Tyr) deacylase (DTD) in SAMP8 mice and speculate the function of DTD in disorders associated with Alzheimer's disease (AD). METHODS: Altogether 12 SAMP8 mice and 12 SAMR1 mice were used in this study. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were performed to detect the mRNA and protein levels of DTD in the mice. Purified DTD protein was injected into lateral ventricle to investigate the function of DTD in SAMP mice. The behavior of the mice was tested by using a Step-through Test System. RESULTS: Both mRNA and protein levels of DTD were found to be significantly lower in SAMP8 mice compared with those in SAMR1 mice (P<0.05). In vivo injection of DTD protein did not lead to an obvious change in behavior of SAM mice. CONCLUSIONS: DTD might function in the process of AD-associated pathology and could possibly participate in physiology process in a long-term manner to orchestrate with other regulators in order to maintain the balance of organism.

Nectin-like molecule 1 inhibits the migration and invasion of U251 glioma cells by regulating the expression of an extracellular matrix protein osteopontin.

OBJECTIVE: To investigate the molecular mechanism of nectin-like molecule 1 (NECL1) inhibiting the migration and invasion of U251 glioma cells. METHODS: We infected U251 glioma cells with adeno-nectin-like molecule 1 (Ad-NECL1) or empty adenovirus (Ad). Transwell and wound healing assays were performed to observe the migration of U251 cells incubated with the cell supernatant from Ad-NECL1 or Ad infected U251 cells. DNA microarray was applied to screen the gene expression profile after the restoration of NECL1 in U251 glioma cell lines. The differential expression of osteopontin (OPN), a gene related to migration and invasion, was further analyzed with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. RESULTS: The restoration of NECL1 inhibited migration of U251 cells significantly (P<0.05). Altogether 195 genes were found differentially expressed by microarray, in which 175 were up-regulated and 20 down-regulated, including 9 extracellular matrix proteins involved in the migration of cells. Both mRNA and protein expressions of OPN, the most markedly reduced extracellular matrix protein, were found decreased in U251 cells after restoration of NECL1. Immunohistochemical assay also detected an increase of OPN in glioma tissues, related with the progressing of malignant grade. CONCLUSION: A link might exist between NECL1 and the extracellular matrix protein OPN in inhibiting the migration and invasion of U251 glioma cells.

Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin-related kinase C receptor, is involved in neurotrophin 3-mediated neurite outgrowth in mouse cortex neurons.

BACKGROUND: The downstream of tyrosine kinase/docking protein (Dok) adaptor protein family has seven members, Dok1 to Dok7, that act as substrates of multiple receptor tyrosine kinase and non-receptor tyrosine kinase. The tropomyosin-related kinase (Trk) receptor family, which has three members (TrkA, TrkB and TrkC), are receptor tyrosine kinases that play pivotal roles in many stages of nervous system development, such as differentiation, migration, axon and dendrite projection and neuron patterning. Upon related neurotrophin growth factor stimulation, dimerisation and autophosphorylation of Trk receptors can occur, recruiting adaptor proteins to mediate signal transduction. RESULTS: In this report, by using yeast two-hybrid assays, glutathione S-transferase (GST) precipitation assays and coimmunoprecipitation (Co-IP) experiments, we demonstrate that Dok6 selectively binds to the NPQY motif of TrkC through its phosphotyrosine-binding (PTB) domain in a kinase activity-dependent manner. We further confirmed their interaction by coimmunoprecipitation and colocalisation in E18.5 mouse cortex neurons, which provided more in vivo evidence. Next, we demonstrated that Dok6 is involved in neurite outgrowth in mouse cortex neurons via the RNAi method. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon neurotrophin 3 (NT-3) stimulation. CONCLUSIONS: We conclude that Dok6 interacts with the NPQY motif of the TrkC receptor through its PTB domain in a kinase activity-dependent manner, and works as a novel substrate of the TrkC receptor involved in NT-3-mediated neurite outgrowth in mouse cortex neurons.