The mGluR5-mediated Arc activation protects against experimental traumatic brain injury in rats.

INTRODUCTION: Traumatic brain injury (TBI) is a complex pathophysiological process, and increasing attention has been paid to the important role of post-synaptic density (PSD) proteins, such as glutamate receptors. Our previous study showed that a PSD protein Arc/Arg3.1 (Arc) regulates endoplasmic reticulum (ER) stress and neuronal necroptosis in traumatic injury in vitro. AIM: In this study, we investigated the expression, regulation and biological function of Arc in both in vivo and in vitro experimental TBI models. RESULTS: Traumatic neuronal injury (TNI) induced a temporal upregulation of Arc in cortical neurons, while TBI resulted in sustained increase in Arc expression up to 24 h in rats. The increased expression of Arc was mediated by the activity of metabotropic glutamate receptor 5 (mGluR5), but not dependent on the intracellular calcium (Ca2+) release. By using inhibitors and antagonists, we found that TNI regulates Arc expression via Gq protein and protein turnover. In addition, overexpression of Arc protects against TBI-induced neuronal injury and motor dysfunction both in vivo and in vitro, whereas the long-term cognitive function was not altered. To determine the role of Arc in mGluR5-induced protection, lentivirus-mediated short hairpin RNA (shRNA) transfection was performed to knockdown Arc expression. The mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG)-induced protection against TBI was partially prevented by Arc knockdown. Furthermore, the CHPG-induced attenuation of Ca2+ influx after TNI was dependent on Arc activation and followed regulation of AMPAR subunits. The results of Co-IP and Ca2+ imaging showed that the Arc-Homer1 interaction contributes to the CHPG-induced regulation of intracellular Ca2+ release. CONCLUSION: In summary, the present data indicate that the mGluR5-mediated Arc activation is a protective mechanism that attenuates neurotoxicity following TBI through the regulation of intracellular Ca2+ hemostasis. The AMPAR-associated Ca2+ influx and ER Ca2+ release induced by Homer1-IP3R pathway might be involved in this protection.

Real-time visualization of mRNA synthesis during memory formation in live mice.

Memories are thought to be encoded in populations of neurons called memory trace or engram cells. However, little is known about the dynamics of these cells because of the difficulty in real-time monitoring of them over long periods of time in vivo. To overcome this limitation, we present a genetically encoded RNA indicator (GERI) mouse for intravital chronic imaging of endogenous Arc messenger RNA (mRNA)-a popular marker for memory trace cells. We used our GERI to identify Arc-positive neurons in real time without the delay associated with reporter protein expression in conventional approaches. We found that the Arc-positive neuronal populations rapidly turned over within 2 d in the hippocampal CA1 region, whereas ∼4% of neurons in the retrosplenial cortex consistently expressed Arc following contextual fear conditioning and repeated memory retrievals. Dual imaging of GERI and a calcium indicator in CA1 of mice navigating a virtual reality environment revealed that only the population of neurons expressing Arc during both encoding and retrieval exhibited relatively high calcium activity in a context-specific manner. This in vivo RNA-imaging approach opens the possibility of unraveling the dynamics of the neuronal population underlying various learning and memory processes.

CRISPR/Cas9-mediated SARM1 knockout and epitope-tagged mice reveal that SARM1 does not regulate nuclear transcription, but is expressed in macrophages.

SARM1 is a toll/interleukin-1 receptor -domain containing protein, with roles proposed in both innate immunity and neuronal degeneration. Murine SARM1 has been reported to regulate the transcription of chemokines in both neurons and macrophages; however, the extent to which SARM1 contributes to transcription regulation remains to be fully understood. Here, we identify differential gene expression in bone-marrow-derived macrophages (BMDMs) from C57BL/6 congenic 129 ES cell-derived Sarm1-/- mice compared with wild type (WT). However, we found that passenger genes, which are derived from the 129 donor strain of mice that flank the Sarm1 locus, confound interpretation of the results, since many of the identified differentially regulated genes come from this region. To re-examine the transcriptional role of SARM1 in the absence of passenger genes, here we generated three Sarm1-/- mice using CRISPR/Cas9. Treatment of neurons from these mice with vincristine, a chemotherapeutic drug causing axonal degeneration, confirmed SARM1's function in that process; however, these mice also showed that lack of SARM1 has no impact on transcription of genes previously shown to be affected such as chemokines. To gain further insight into SARM1 function, we generated an epitope-tagged SARM1 mouse. In these mice, we observed high SARM1 protein expression in the brain and brainstem and lower but detectable levels in macrophages. Overall, the generation of these SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages yet has no general role in macrophage transcriptional regulation and has provided important new models to further explore SARM1 function.

NOS-dependent effects of plantar mechanical stimulation on mechanical characteristics and cytoskeletal proteins in rat soleus muscle during hindlimb suspension.

The study was aimed at investigating the mechanisms and structures which determine mechanical properties of skeletal muscles under gravitational unloading and plantar mechanical stimulation (PMS). We hypothesized that PMS would increase NO production and prevent an unloading-induced reduction in skeletal muscle passive stiffness. Wistar rats were hindlimb suspended and subjected to a daily PMS and one group of stimulated animals was also treated with nitric oxide synthase (NOS) inhibitor (L-NAME). Animals received mechanical stimulation of the feet for 4 h a day throughout 7-day hindlimb suspension (HS) according to a scheme that mimics the normal walking of the animal. Seven-day HS led to a significant reduction in soleus muscle weight by 25%. However, PMS did not prevent the atrophic effect induced by HS. Gravitational unloading led to a significant decrease in maximum isometric force and passive stiffness by 38% and 31%, respectively. The use of PMS prevented a decrease in the maximum isometric strength of the soleus muscle. At the same time, the passive stiffness of the soleus in the PMS group significantly exceeded the control values by 40%. L-NAME (NOS inhibitor) administration attenuated the effect of PMS on passive stiffness and maximum force of the soleus muscle. The content of the studied cytoskeletal proteins (α-actinin-2, α-actinin-3, desmin, titin, nebulin) decreased after 7-day HS, but this decrease was successfully prevented by PMS in a NOS-dependent manner. We also observed significant decreases in mRNA expression levels of α-actinin-2, desmin, and titin after HS, which was prevented by PMS. The study also revealed a significant NOS-dependent effect of PMS on the content of collagen-1a, but not collagen-3a. Thus, PMS during mechanical unloading is able to maintain soleus muscle passive tension and force as well as mRNA transcription and protein contents of cytoskeletal proteins in a NOS-dependent manner.

Axon morphogenesis and maintenance require an evolutionary conserved safeguard function of Wnk kinases antagonizing Sarm and Axed.

The molecular and cellular mechanisms underlying complex axon morphogenesis are still poorly understood. We report a novel, evolutionary conserved function for the Drosophila Wnk kinase (dWnk) and its mammalian orthologs, WNK1 and 2, in axon branching. We uncover that dWnk, together with the neuroprotective factor Nmnat, antagonizes the axon-destabilizing factors D-Sarm and Axundead (Axed) during axon branch growth, revealing a developmental function for these proteins. Overexpression of D-Sarm or Axed results in axon branching defects, which can be blocked by overexpression of dWnk or Nmnat. Surprisingly, Wnk kinases are also required for axon maintenance of adult Drosophila and mouse cortical pyramidal neurons. Requirement of Wnk for axon maintenance is independent of its developmental function. Inactivation of dWnk or mouse Wnk1/2 in mature neurons leads to axon degeneration in the adult brain. Therefore, Wnk kinases are novel signaling components that provide a safeguard function in both developing and adult axons.

TSGA10 as a Potential Key Factor in the Process of Spermatid Differentiation/Maturation: Deciphering Its Association with Autophagy Pathway.

Testis-specific gene antigen 10 (TSGA10) plays an important role in spermatogenesis. However, the exact TSGA10 role and its relationship with the autophagy pathway in the process of spermatids differentiation/maturation is still not clear. Therefore, the present study evaluates the role of TSGA10 gene in the spermatid differentiation/maturation through its effect on autophagy and explores possible underlying pathway(s). Sperm samples from patients with teratospermia were collected. The mRNA and protein level of TSGA10 in these samples were assessed by real-time PCR and western blotting. Using the ingenuity pathway analysis (IPA) software, the gene network and interactions of TSGA10 involved in sperm maturation and autophagy were investigated. Based on these analyses, the expression levels of identified genes in patient's samples and healthy controls were further evaluated. Moreover, using flow cytometry analysis, the levels of reactive oxygen species (ROS( production in teratospermic sperm samples were evaluated. The results showed that the expression levels of TSGA10 mRNA and protein decreased significantly in the teratospermic patients compared to controls (P < 0.05). Moreover, a significant reduction in the expression of the important genes involved in sperm maturation and autophagy was observed (P < 0.05). Also, the levels of ROS production in teratospermic sperm samples were shown to be significantly higher compared to those in normal sperms (P < 0.05). Our findings provide new evidence that simultaneous decrease in TSGA10 and autophagy beside the increased level of ROS production in sperm cells might be associated with the abnormalities in the spermatids differentiation/maturation and the formation of sperms with abnormal morphology.

Changes in concentrations of NMDA receptor subunit GluN2B, Arc and syntaxin-1 in dorsal hippocampus Schaffer collateral synapses in a rat learned helplessness model of depression.

Major depressive disorder involves changes in synaptic structure and function, but the molecular underpinnings of these changes are still not established. In an initial pilot experiment, whole-brain synaptosome screening with quantitative western blotting was performed to identify synaptic proteins that may show concentration changes in a congenital rat learned helplessness model of depression. We found that the N-methyl-d-aspartate receptor (NMDAR) subunits GluN2A/GluN2B, activity-regulated cytoskeleton-associated protein (Arc) and syntaxin-1 showed significant concentration differences between congenitally learned helpless (LH) and nonlearned helpless (NLH) rats. Having identified these three proteins, we then performed more elaborate quantitative immunogold electron microscopic analyses of the proteins in a specific synapse type in the dorsal hippocampus: the Schaffer collateral synapse in the CA1 region. We expanded the setup to include also unstressed wild-type (WT) rats. The concentrations of the proteins in the LH and NLH groups were compared to WT animals. In this specific synapse, we found that the concentration of NMDARs was increased in postsynaptic spines in both LH and NLH rats. The concentration of Arc was significantly increased in postsynaptic densities in LH animals as well as in presynaptic cytoplasm of NLH rats. The concentration of syntaxin-1 was significantly increased in both presynaptic terminals and postsynaptic spines in LH animals, while pre- and postsynaptic syntaxin-1 concentrations were significantly decreased in NLH animals. These protein changes suggest pathways by which synaptic plasticity may be increased in dorsal hippocampal Schaffer collateral synapses during depression, corresponding to decreased synaptic stability.

Effects of CMYA1 overexpression on cardiac structure and function in mice.

CMYA1 (cardiomyopathy-associated protein 1, also termed Xin) localizes to the intercalated disks (ICDs) of the myocardium and functions to maintain ICD structural integrity and support signal transduction among cardiomyocytes. Our previous study showed that CMYA1 overexpression impairs the function of gap junction intercellular communication processes. Successful model generation was verified based on PCR, western blot analysis, immunohistochemistry, and immunofluorescence analysis. Myocardial CMYA1 expression was confirmed at both the mRNA and the protein levels in the CMYA1-OE transgenic mice. Masson's trichrome staining and electron microscopy revealed myocardial fibrosis and uneven bead width or the interruption of ICDs in the hearts of the CMYA1-OE transgenic mice. Furthermore, the Cx43 protein level was reduced in the CMYA1-OE mice, and co-immunoprecipitation assays of heart tissue protein extracts revealed a physical interaction between CMYA1 and Cx43. Electrocardiogram analysis enabled the detection of an obvious ventricular bigeminy for the CMYA1-OE mice. In summary, analysis of our mouse model indicates that elevated CMYA1 levels may induce myocardial fibrosis, impair ICDs, and downregulate the expression of Cx43. The observed ventricular bigeminy in the CMYA1-OE mice may be mediated by the reduced Cx43 protein level.

CircRNA SEPT9 contributes to malignant behaviors of glioma cells via miR-432-5p-mediated regulation of LASP1.

BACKGROUND: Circular RNA (circRNA) septin 9 (circSEPT9; hsa_circ_0005320) has been reported to be abnormally up-regulated in glioma. However, the exact role and working mechanism of circSEPT9 in glioma progression are barely known. METHODS: RNA and protein levels were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay, respectively. Cell proliferation was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay and flow cytometry. Cell apoptosis was evaluated by flow cytometry. Cell motility was analyzed by transwell assays. Cell glycolytic metabolism was analyzed using commercial kits. Dual-luciferase reporter assay, RNA-pull down assay and RNA immunoprecipitation (RIP) assay were conducted to verify the intermolecular interactions. Xenograft mice model was utilized to assess the role of circSEPT9 in vivo. RESULTS: CircSEPT9 was highly expressed in glioma tissues and cell lines. CircSEPT9 interference inhibited the proliferation, migration, invasion and glycolytic metabolism and triggered the apoptosis of glioma cells. MicroRNA-432-5p (miR-432-5p) was a target of circSEPT9, and circSEPT9 silencing-mediated effects in glioma cells were largely alleviated by the addition of anti-miR-432-5p. MiR-432-5p bound to the 3' untranslated region (3'UTR) of LIM and SH3 protein 1 (LASP1), and LASP1 overexpression largely overturned miR-432-5p-induced effects in glioma cells. CircSEPT9 up-regulated LASP1 expression by acting as miR-432-5p sponge. CircSEPT9 silencing suppressed xenograft tumor growth in vivo. CONCLUSION: CircSEPT9 exerted an oncogenic role to enhance the malignant behaviors of glioma cells by binding to miR-432-5p to induce LASP1 expression.

Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated hemizygous deletion.

Leishmania parasites cycle between sand-fly vectors and mammalian hosts adapting to alternating environments by stage-differentiation accompanied by changes in the proteome profiles. Translation regulation plays a central role in driving the differential program of gene expression since control of gene regulation in Leishmania is mostly post-transcriptional. The Leishmania genome encodes six eIF4E paralogs, some of which bind a dedicated eIF4G candidate, and each eIF4E is assumed to have specific functions with perhaps some overlaps. However, LeishIF4E2 does not bind any known eIF4G ortholog and was previously shown to comigrate with the polysomal fractions of sucrose gradients in contrast to the other initiation factors that usually comigrate with pre-initiation and initiation complexes. Here we deleted one of the two LeishIF4E2 gene copies using the CRISPR-Cas9 methodology. The deletion caused severe alterations in the morphology of the mutant cells that became round, small, and equipped with a very short flagellum that did not protrude from its pocket. Reduced expression of LeishIF4E2 had no global effect on translation and growth, unlike other LeishIF4Es; however, there was a change in the proteome profile of the LeishIF4E2(+/-) cells. Upregulated proteins were related mainly to general metabolic processes including enzymes involved in fatty acid metabolism, DNA repair and replication, signaling, and cellular motor activity. The downregulated proteins included flagellar rod and cytoskeletal proteins, as well as surface antigens involved in virulence. Moreover, the LeishIF4E2(+/-) cells were impaired in their ability to infect cultured macrophages. Overall, LeishIF4E2 does not behave like a general translation factor and its function remains elusive. Our results also suggest that the individual LeishIF4Es perform unique functions.

Bile duct ligation causes opposite impacts on the expression and function of BCRP and P-gp in rat brain partly via affecting membrane expression of ezrin/radixin/moesin proteins.

Breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) are co-located at blood-brain barrier (BBB) cells, preventing their substrates from entering brain. Accumulating evidence demonstrates that liver failure impairs P-gp and BCRP expression and function in the brain. In the current study, we investigated how liver failure influenced the expression and function of brain BCRP and P-gp in rats subjected to bile duct ligation (BDL). The function of BCRP, P-gp and BBB integrity was assessed using distribution of prazosin, rhodamine 123 and fluorescein, respectively. We showed that BDL significantly decreased BCRP function, but increased P-gp function without affecting BBB integrity. Furthermore, we found that BDL significantly downregulated the expression of membrane BCRP and upregulated the expression of membrane P-gp protein in the cortex and hippocampus. In human cerebral microvascular endothelial cells, NH4Cl plus unconjugated bilirubin significantly decreased BCRP function and expression of membrane BCRP protein, but upregulated P-gp function and expression of membrane P-gp protein. The decreased expression of membrane BCRP protein was linked to the decreased expression of membrane radixin protein, while the increased expression of membrane P-gp protein was related to the increased location of membrane ezrin protein. Silencing ezrin impaired membrane location of P-gp, whereas silencing radixin impaired membrane location of BCRP protein. BDL rats showed the increased expression of membrane ezrin protein and decreased expression of membrane radixin protein in the brain. We conclude that BDL causes opposite effects on the expression and function of brain BCRP and P-gp, attributing to the altered expression of membrane radixin and ezrin protein, respectively, due to hyperbilirubinemia and hyperammonemia.

Expression of sterile-α and armadillo motif containing protein (SARM) in rheumatoid arthritis monocytes correlates with TLR2-induced IL-1ß and disease activity.

OBJECTIVE: Cartilage and bone damage in RA are associated with elevated IL-1ß. The effects of IL-1ß can be reduced by biological therapies that target IL-1ß or TNF-α. However, the mechanisms responsible for increased IL-1ß and the effect of anti-TNF-α have not been fully elucidated. Recently, sterile-α and armadillo motif containing protein (SARM) was identified as a negative regulator of toll-like receptor (TLR) induced IL-1ß secretion through an interaction with the inflammasome. This study set out to investigate SARM during TLR-induced IL-1ß secretion in RA peripheral blood monocytes and in patients commencing anti-TNF-α treatment. METHODS: Monocytes were isolated from RA patients and healthy controls; disease activity was measured by DAS28. IL-1ß secretion was measured by ELISA following TLR1/2, TLR4 and TLR7/8 stimulation. The mRNA expression of SARM1, IL-1ß and the components of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome were measured by quantitative PCR. SARM protein expression was measured by western blotting. RESULTS: TLR1/2 activation induced elevated IL-1ß in RA monocytes compared with healthy controls (P = 0.0009), which negatively correlated with SARM1 expression (P = 0.0086). Lower SARM expression also correlated with higher disease activity (P = 0.0246). Additionally, patients responding to anti-TNF-α treatment demonstrated a rapid upregulation of SARM, which was not observed in non-responders. CONCLUSION: Together, these data highlight a potential contribution from SARM to RA pathophysiology where decreased SARM may lead to elevated IL-1ß associated with RA pathogenesis. Furthermore, the data additionally present a potential mechanism by which TNF-α blockade can modify IL-1ß secretion.

Prevalence of MYOC risk variants for glaucoma in different populations.

PURPOSE: To assess the clinical relevance of myocilin (MYOC) gene variants as risk factors for glaucoma in literature and to estimate their prevalence in different populations. METHODS: We reviewed the literature for published MYOC variants in glaucoma patients and estimated their prevalence in general population using gnomAD and BRAVO databases. We used several bioinformatics tools and the criteria of the American College of Medical Genetics and Genomics (ACMG) to assess the pathogenicity of the variants. We evaluated the carrier frequency of the variants in gnomAD, including its subpopulations. RESULTS: We found 13 missense and 5 loss-of-function (LOF) reported variants in MYOC that were both probable pathogenic or risk variants and listed in gnomAD. Six likely pathogenic missense variants were p.(Cys25Arg), p.(Gln48His), p.(Gly326Ser), p.(Thr353Ile), p.(Thr377Met) and p.(Gly399Val). They were most prevalent in East and South Asia (frequency, 0.92% and 0.81%, respectively). The most common missense variants were p.(Thr353Ile) (0.91% in East Asia) and p.(Gln48His) (0.79% in South Asia). Five LOF variants were p.(Arg46Ter), p.(Arg91Ter), p.(Arg272Ter), p.(Gln368Ter) and p.(Tyr453MetfsTer11). We considered these glaucoma risk variants. They were most prevalent in the East Asian and the Finnish population (0.93% and 0.33%, respectively). CONCLUSION: Pathogenic MYOC variants appear to be population-associated. Our results highlight allelic heterogeneity of MYOC variants in open-angle glaucoma. Many of the probable pathogenic variants are over-represented in some of the populations causing doubt of their status as monogenic disease-causing variants.

De novo small deletion affecting transcription start site of short isoform of AUTS2 gene in a patient with syndromic neurodevelopmental defects.

Disruption of the autism susceptibility candidate 2 (AUTS2) gene through genomic rearrangements, copy number variations (CNVs), and intragenic deletions and mutations, has been recurrently involved in syndromic forms of developmental delay and intellectual disability, known as AUTS2 syndrome. The AUTS2 gene plays an important role in regulation of neuronal migration, and when altered, associates with a variable phenotype from severely to mildly affected patients. The more severe phenotypes significantly correlate with the presence of defects affecting the C-terminus part of the gene. This article reports a new patient with a syndromic neurodevelopmental disorder, who presents a deletion of 30 nucleotides in the exon 9 of the AUTS2 gene. Importantly, this deletion includes the transcription start site for the AUTS2 short transcript isoform, which has an important role in brain development. Gene expression analysis of AUTS2 full-length and short isoforms revealed that the deletion found in this patient causes a remarkable reduction in the expression level, not only of the short isoform, but also of the full AUTS2 transcripts. This report adds more evidence for the role of mutated AUTS2 short transcripts in the development of a severe phenotype in the AUTS2 syndrome.

Expression of lipoma preferred partner in mammary and extramammary Paget disease.

BACKGOUND: This study aims to identify the expression of lipoma preferred partner (LPP) in Paget disease (PD) and to further understand the pathogenesis of PD. METHODS: Tissue microarray was used to evaluate the expression of LPP by immunohistochemistry in 40 PD patients. The results of LPP expression were combined with clinical and histopathological characteristics. Patient files were analyzed retrospectively. RESULTS: Twenty-one cases were mammary Paget disease (MPD) and 19 extramammary Paget disease (EMPD) involving the vulva, scrotum, and penis. LPP was expressed in PD and this expression was significantly greater in MPD versus EMPD (P = .031). The expression of LPP in MPD was significantly related with age (P = .009) and expression of Ki-67 (P = .011). No statistically significant differences were observed in LPP expression as related to sex, body location, and time of PD diagnosis. CONCLUSIONS: While LPP is expressed in both MPD and EMPD, the intensity of this expression is greater in MPD. LPP expression is positively correlated with Ki-67 and is more prevalent in middle-aged versus senior MPD patients. Further research is needed to determine its potential role in tumorigenesis and distribution.

Down-Regulation of AHNAK2 Inhibits Cell Proliferation, Migration and Invasion Through Inactivating the MAPK Pathway in Lung Adenocarcinoma.

AHNAK nucleoprotein 2 (AHNAK2) has been emerged as a crucial protein for neuroblast differentiation and cell migration, thereby involving in the development of various cancers. However, the specific molecular mechanism of AHNAK2 in lung adenocarcinoma is inconclusive. By accessing to the Oncomine dataset and GEPIA website, a higher expression level of AHNAK2 was observed in lung adenocarcinoma tissue samples. Overall survival (OS) curve plotted by Kaplan-Meier method showed that up-regulation of AHNAK2 was related with poor prognosis of lung adenocarcinoma patients. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis and western blot were conducted to examine the expression level of genes in lung adenocarcinoma cells. Through functional in vitro experiments, cell proliferation, migration and invasion were all suppressed after AHNAK2 knockdown using Cell counting kit-8 (CCK-8) assay, wound-healing and transwell analysis. Reduction of AHNAK2 decreased the apoptosis rate using flow cytometry analysis. Moreover, the key markers of MAPK pathway, p-MEK, p-ERK and p-P90RSK were decreased due to the transfection of si-AHNAK2 in A549 cells. U0126, a MEK inhibitor, showed the similar effects on MAPK-related protein levels with si-AHNAK2. To sum up, AHNAK2 is significantly increased in lung adenocarcinoma and plays a carcinogenic role by activating the MAPK signaling pathway, providing a novel insight and raising possibility for lung adenocarcinoma treatment.

LINC01503/miR-342-3p facilitates malignancy in non-small-cell lung cancer cells via regulating LASP1.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the major types of lung cancer, which is a prevalent human disease all over the world. LncRNA LINC01503 is a super-enhancer-driven long non-coding RNA that is dysregulated in several types of human cancer. However, its role in NSCLC remains unknown. METHODS: Thirty NSCLC patients were recruited between April 2012 and April 2016. Luciferase reporter assay, qRT-PCR, Cell Counting Kit-8 (CCK-8), Transwell migration assay, RNA pull-down assay, western blotting, 5-ethynyl-29-deoxyuridine (EdU) assays, and flow cytometry were utilized to characterize the roles and relationships among LINC01503, miR-342-3p, and LASP1 in NSCLC. The transplanted mouse model was built to examine their biological functions in vivo. RESULTS: We demonstrated that the expression of lncRNA LINC01503 and LIM and SH3 domain protein 1 (LASP1) were upregulated and miR-342-3p was downregulated in NSCLC samples and cell lines. Functional experiments revealed that inhibiting the expression of LINC01503 or over-expression of miR-342-3p inhibited NSCLC growth and metastasis both in vitro and in vivo. In addition, LINC01503 could bind to miR-342-3p and affect the expression of LASP1. CONCLUSION: These results provide a comprehensive analysis of the roles of LINC01503 as a competing endogenous RNA (ceRNA) in NSCLC progression.

Calpain inhibition ameliorates depression-like behaviors by reducing inflammation and promoting synaptic protein expression in the hippocampus.

Protease activity correlates with depressive or suicidal behaviors, with calpain activation being especially implicated in depression-like behaviors. However, the role of calpain in depression-like behaviors is currently unknown. In this study, the lipopolysaccharide (LPS) - and chronic unpredictable mild stress (CUMS)-induced depression models were used to evaluate the antidepressant effects of calpain inhibitors. Potential mechanisms were determined using pharmacological and biochemical methods. We found that i. p. injection of a calpain inhibitor, calpeptin, prevented LPS-induced depression-like behaviors, activation of astrocytes, inflammation, and reduction of synaptic protein expression levels. LPS injection (i.p.) promoted calpain activity, which degraded suprachiasmatic nucleus circadian oscillatory protein (SCOP). This led to the activation of ERK and nuclear translocation of nuclear factor kappa-B (NF-κB), the promotion of cytokine release, and the reduction of Arc, and PSD95 expression in the hippocampus. In contrast, i. p. injection of calpeptin blocked these changes. Furthermore, intraventricular injection of calpain inhibitor (PD150606) or an ERK inhibitor ameliorated the LPS-induced depression-like behaviors. Administration of calpeptin also remedied CUMS-induced depression-like behaviors, degradation of SCOP, activation of astrocytes, and reduction of synaptic protein expression levels. Finally, we also demonstrated that memantine, an N-methyl-d-aspartic acid (NMDA) receptor antagonist blocks LPS-induced degradation of SCOP. Together, our results show that calpain inhibition ameliorates depression-like behaviors, probably by reducing inflammation and promoting synaptic protein expression in the hippocampus.

Lactate enhances Arc/arg3.1 expression through hydroxycarboxylic acid receptor 1-ß-arrestin2 pathway in astrocytes.

In recent years, with the discovery and research of lactate-specific receptor HCAR1(hydroxycarboxylic acid receptor 1), lactate is not only as a product of Glycolysis in astrocytes, but also as a signaling molecule which has gradually received attention. Studies have found that lactate can be used as an intercellular signaling molecule involved in synaptic plasticity, and so that peripheral administration of lactate can produce antidepressant effects. Here, we focus on HCAR1 on the most widely distributed astrocytes in the brain, found and verified that lactate could cause Arc/arg3.1 protein overexpression in astrocytes through HCAR1. However, the expression of Arc/arg3.1 does not depend on the Gi protein pathway of HCAR1, and we found that lactate enhanced the expression of Arc/arg3.1 protein through the HCAR1-ß-arrestin2 pathway. In summary, lactate acts on HCAR1 of astrocytes. It enhances the expression of MAPK-dependent Arc through ß-arrestin2, thereby reducing the influx of calcium ions when astrocytes are exposed to glutamate damage, achieving the role of protecting astrocytes and indirectly enhancing the absorption of glutamate by astrocytes. These results also demonstrate that HCAR1 in the brain is a potential therapeutic target in an experimental in vitro model of glutamate damage, which is strongly associated with many neurodegenerative diseases.

Cloning, expression and purification of the low-complexity region of RanBP9 protein.

Recombinant expression and purification of proteins is key for biochemical and biophysical investigations. Although this has become a routine and standard procedure for many proteins, intrinsically disordered ones and those with low complexity sequences pose difficulties. Proteins containing low complexity regions (LCRs) are increasingly becoming significant for their roles in both normal and pathological processes. Here, we report cloning, expression and purification of N-terminal LCR of RanBP9 protein (Nt-RanBP9). RanBP9 is a scaffolding protein present in both cytoplasm and nucleus that is implicated in many cellular processes. Nt-RanBP9 is a poorly understood region of the protein perhaps due to difficulties posed by the LCR. Indeed, conventional methods presented difficulties in Nt-RanBP9 cloning due to its high GC content resulting in insignificant protein expression. These led us to use a different approach of cloning by expressing the protein as a fusion construct containing mCherry or mEGFP using in vivo DNA recombination methods. Our results indicate that expression of mEGFP-tagged Nt-RanBP9 followed by thrombin cleavage of the tag was the most effective method to obtain the protein with >90% purity and good yields. We report and discuss the challenges in obtaining the N-terminal region of RanBP9, a protein with functional implications in multiple biological processes and neurodegenerative diseases.