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International Journal of Molecular... May 2024Astrocytes actively participate in neurotransmitter homeostasis by bidirectional communication with neuronal cells, a concept named the tripartite synapse, yet their...
Astrocytes actively participate in neurotransmitter homeostasis by bidirectional communication with neuronal cells, a concept named the tripartite synapse, yet their role in dopamine (DA) homeostasis remains understudied. In the present study, we investigated the kinetic and molecular mechanisms of DA transport in cultured striatal astrocytes of adult rats. Kinetic uptake experiments were performed using radiolabeled [H]-DA, whereas mRNA expression of the dopamine, norepinephrine, organic cation and plasma membrane monoamine transporters (DAT, NET, OCTs and PMAT) and DA receptors D1 and D2 was determined by qPCR. Additionally, astrocyte cultures were subjected to a 24 h treatment with the DA receptor agonist apomorphine, the DA receptor antagonist haloperidol and the DA precursor L-DOPA. [H]-DA uptake exhibited temperature, concentration and sodium dependence, with potent inhibition by desipramine, nortriptyline and decynium-22, suggesting the involvement of multiple transporters. qPCR revealed prominent mRNA expression of the NET, the PMAT and OCT1, alongside lower levels of mRNA for OCT2, OCT3 and the DAT. Notably, apomorphine significantly altered NET, PMAT and D1 mRNA expression, while haloperidol and L-DOPA had a modest impact. Our findings demonstrate that striatal astrocytes aid in DA clearance by multiple transporters, which are influenced by dopaminergic drugs. Our study enhances the understanding of regional DA uptake, paving the way for targeted therapeutic interventions in dopaminergic disorders.
Topics: Animals; Astrocytes; Dopamine; Rats; Corpus Striatum; Haloperidol; Kinetics; Dopamine Plasma Membrane Transport Proteins; Apomorphine; Cells, Cultured; Male; Receptors, Dopamine D1; Biological Transport; Levodopa
PubMed: 38791173
DOI: 10.3390/ijms25105135 -
Genes May 2024Primordial germ cells (PGCs) are the precursors of functional gametes and the only cell type capable of transmitting genetic and epigenetic information from generation...
Primordial germ cells (PGCs) are the precursors of functional gametes and the only cell type capable of transmitting genetic and epigenetic information from generation to generation. These cells offer valuable starting material for cell-based genetic engineering and genetic preservation, as well as epigenetic studies. While chicken PGCs have demonstrated resilience in maintaining their germness characteristics during both culturing and cryopreservation, their handling remains a complex challenge requiring further refinement. Herein, the study aimed to compare the effects of different conditions (freezing-thawing and in vitro cultivation) on the expression of PGC-specific marker genes. Embryonic blood containing circulating PGCs was isolated from purebred Green-legged Partridgelike chicken embryos at 14-16 Hamburger-Hamilton (HH) embryonic development stage. The blood was pooled separately for males and females following sex determination. The conditions applied to the blood containing PGCs were as follows: (1) fresh isolation; (2) cryopreservation for a short term (2 days); and (3) in vitro culture (3 months) with long-term cryopreservation of purified PGCs (~2 years). To characterize PGCs, RNA isolation was carried out, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of specific germ cell markers (, , and ), as well as pluripotency markers ( and ). The investigated genes exhibited consistent expression among PGCs maintained under diverse conditions, with no discernible differences observed between males and females. Notably, the analyzed markers demonstrated higher expression levels in PGCs when subjected to freezing than in their freshly isolated counterparts.
Topics: Animals; Cryopreservation; Germ Cells; Chickens; Male; Female; Chick Embryo; Cells, Cultured; Biomarkers
PubMed: 38790253
DOI: 10.3390/genes15050624 -
Genes Apr 2024Cervical cancer is among the highest-ranking types of cancer worldwide, with human papillomavirus (HPV) as the agent driving the malignant process. One aspect of the...
BACKGROUND
Cervical cancer is among the highest-ranking types of cancer worldwide, with human papillomavirus (HPV) as the agent driving the malignant process. One aspect of the infection's evolution is given by epigenetic modifications, mainly DNA methylation and chromatin alteration. These processes are guided by several chromatin remodeling complexes, including NuRD. The purpose of this study was to evaluate the genome-wide binding patterns of the NuRD complex components (MBD2 and MBD3) in the presence of active HPV16 E6 and E7 oncogenes and to determine the potential of identified genes through an experimental model to differentiate between cervical precursor lesions, with the aim of establishing their utility as biomarkers.
METHODS
The experimental model was built using the CaSki cell line and shRNA for E6 and E7 HPV16 silencing, ChIP-seq, qRT-PCR, and Western blot analyses. Selected genes' expression was also assessed in patients.
RESULTS
Several genes have been identified to exhibit altered transcriptional activity due to the influence of HPV16 E6/E7 viral oncogenes acting through the MBD2/MBD3 NuRD complex, linking them to viral infection and cervical oncogenesis.
CONCLUSIONS
The impacted genes primarily play roles in governing gene transcription, mRNA processing, and regulation of translation. Understanding these mechanisms offers valuable insights into the process of HPV-induced oncogenesis.
Topics: Female; Humans; Carcinogenesis; Cell Line, Tumor; Chromatin Assembly and Disassembly; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Human papillomavirus 16; Mi-2 Nucleosome Remodeling and Deacetylase Complex; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Papillomavirus Infections; Repressor Proteins; Uterine Cervical Neoplasms
PubMed: 38790189
DOI: 10.3390/genes15050560 -
Molecular Neurobiology May 2024Alzheimer's disease (AD) is a common progressive degenerative disease of the central nervous system in aging populations. This study aimed to investigate the effects of...
Combined Catalpol and Tetramethylpyrazine Promote Axonal Plasticity in Alzheimer's Disease by Inducing Astrocytes to Secrete Exosomes Carrying CDK5 mRNA and Regulating STAT3 Phosphorylation.
Alzheimer's disease (AD) is a common progressive degenerative disease of the central nervous system in aging populations. This study aimed to investigate the effects of combined catalpol and tetramethylpyrazine (CT) in promoting axonal plasticity in AD and the potential underlying mechanism. Astrocytes were treated with different concentrations of compatible CT. Exosomes were collected and subjected to sequencing analysis, which was followed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes. Amyloid precursor protein/presenilin 1 (APP/PS1) double-transfected male mice were used as the in vivo AD models. Astrocyte-derived exosomes that were transfected with cyclin-dependent kinase 5 (CDK5) or CT treatment were injected into the tail vein of mice. The levels of CDK5, synaptic plasticity marker protein neurofilament 200 (NF200), and growth-associated protein 43 (GAP-43) in the hippocampus of mice were compared in each group. Immunofluorescence staining was used to detect the localization of STAT3 and to visualize synaptic morphology via β-tubulin-III (TUBB3). Astrocyte-derived exosomes transfected with siCDK5 or treated with CT were co-cultured with HT-22 cells, which were untransfected or silenced for signal transducer and activator of transcription 3 (STAT3). Amyloid β-protein (Aβ)1-42 was induced in the in vitro AD models. The viability, apoptosis, and expression levels of NF200 and GAP-43 proteins in the hippocampal neurons of each group were compared. In total, 166 differentially expressed genes in CT-induced astrocyte-derived exosomes were included in the KEGG analysis, and they were found to be enriched in 12 pathways, mainly in axon guidance. CT treatment significantly increased the level of CDK5 mRNA in astrocyte-derived exosomes-these exosomes restored CDK5 mRNA and protein levels in the hippocampus of the in vivo AD model mice and the in vitro AD model; promoted p-STAT3 (Ser727), NF200 and GAP-43 proteins; and promoted the regeneration and extension of neuronal synapses. Silencing of CDK5 blocked both neuronal protection as well as induction of axonal plasticity in AD by CT-treated exosomes in vitro and in vivo. Moreover, silencing of STAT3 blocked both neuronal protection as well as induction of axonal plasticity in AD caused by CDK5 overexpression or CT-treated astrocyte-induced exosomes. CT promotes axonal plasticity in AD by inducing astrocytes to secrete exosomes carrying CDK5 mRNA and regulating STAT3 (Ser727) phosphorylation.
PubMed: 38789892
DOI: 10.1007/s12035-024-04251-z -
ENeuro Jun 2024Studies on genetic robustness recently revealed transcriptional adaptation (TA) as a mechanism by which an organism can compensate for genetic mutations through...
Studies on genetic robustness recently revealed transcriptional adaptation (TA) as a mechanism by which an organism can compensate for genetic mutations through activation of homologous genes. Here, we discovered that genetic mutations, introducing a premature termination codon (PTC) in the amyloid precursor protein-b () gene, activated TA of two other family members, and amyloid precursor-like protein-2 (), in zebrafish. The observed transcriptional response of and required degradation of mutant mRNA and did not depend on Appb protein level. Furthermore, TA between amyloid precursor protein (APP) family members was observed in human neuronal progenitor cells; however, compensation was only present during early neuronal differentiation and could not be detected in a more differentiated neuronal stage or adult zebrafish brain. Using knockdown and chemical inhibition, we showed that nonsense-mediated mRNA decay (NMD) is involved in degradation of mutant mRNA and that Upf1 and Upf2, key proteins in the NMD pathway, regulate the endogenous transcript levels of , , , and In conclusion, our results suggest that the expression level of App family members is regulated by the NMD pathway and that mutations destabilizing / mRNA can induce genetic compensation by other family members through TA in both zebrafish and human neuronal progenitors.
Topics: Zebrafish; Animals; Amyloid beta-Protein Precursor; Nonsense Mediated mRNA Decay; Humans; Zebrafish Proteins; RNA, Messenger; Neural Stem Cells; Mutation; Animals, Genetically Modified
PubMed: 38789273
DOI: 10.1523/ENEURO.0034-24.2024 -
Cells May 2024Our study focused on assessing the effects of three newly identified BRCA1 exon 11 variants (c.1019T>C, c.2363T>G, and c.3192T>C) on breast cancer susceptibility. Using...
Our study focused on assessing the effects of three newly identified BRCA1 exon 11 variants (c.1019T>C, c.2363T>G, and c.3192T>C) on breast cancer susceptibility. Using computational predictions and experimental splicing assays, we evaluated their potential as pathogenic mutations. Our in silico analyses suggested that the c.2363T>G and c.3192T>C variants could impact both splicing and protein function, resulting in the V340A and V788G mutations, respectively. We further examined their splicing effects using minigene assays in MCF7 and SKBR3 breast cancer cell lines. Interestingly, we found that the c.2363T>G variant significantly altered splicing patterns in MCF7 cells but not in SKBR3 cells. This finding suggests a potential influence of cellular context on the variant's effects. While attempts to correlate in silico predictions with RNA binding factors were inconclusive, this observation underscores the complexity of splicing regulation. Splicing is governed by various factors, including cellular contexts and protein interactions, making it challenging to predict outcomes accurately. Further research is needed to fully understand the functional consequences of the c.2363T>G variant in breast cancer pathogenesis. Integrating computational predictions with experimental data will provide valuable insights into the role of alternative splicing regulation in different breast cancer types and stages.
Topics: Humans; Exons; BRCA1 Protein; RNA Precursors; RNA Splicing; Breast Neoplasms; Female; Cell Line, Tumor; Mutation; MCF-7 Cells; Alternative Splicing; Genetic Predisposition to Disease
PubMed: 38786046
DOI: 10.3390/cells13100824 -
Biomolecules May 2024One of the hallmarks of cancer is metabolic reprogramming in tumor cells, and aerobic glycolysis is the primary mechanism by which glucose is quickly transformed into... (Review)
Review
One of the hallmarks of cancer is metabolic reprogramming in tumor cells, and aerobic glycolysis is the primary mechanism by which glucose is quickly transformed into lactate. As one of the primary rate-limiting enzymes, pyruvate kinase (PK) M is engaged in the last phase of aerobic glycolysis. Alternative splicing is a crucial mechanism for protein diversity, and it promotes precursor mRNA splicing to produce dominance, resulting in low expression. Specific splicing isoforms are produced in various tissues or illness situations, and the post-translational modifications are linked to numerous disorders, including cancers. hnRNPs are one of the main components of the splicing factor families. However, there have been no comprehensive studies on hnRNPs regulating alternative splicing. Therefore, this review focuses on the regulatory network of hnRNPs on pre-mRNA alternative splicing in tumors and clinical drug research. We elucidate the role of alternative splicing in tumor progression, prognosis, and the potential mechanism of abnormal RNA splicing. We also summarize the drug targets retarding tumorous splicing events, which may be critical to improving the specificity and effectiveness of current therapeutic interventions.
Topics: Humans; Alternative Splicing; Neoplasms; Heterogeneous-Nuclear Ribonucleoproteins; Pyruvate Kinase; Disease Progression; Gene Expression Regulation, Neoplastic; Animals
PubMed: 38785973
DOI: 10.3390/biom14050566 -
Microbiology Spectrum Jul 2024Sweet orange () is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic...
Sweet orange () is one of the most important fruit crops worldwide. Virus infections in this crop can interfere with cellular processes, causing dramatic economic losses. By performing RT-qPCR analyses, we demonstrated that citrus psorosis virus (CPsV)-infected orange plants exhibited higher levels of unprocessed microRNA (miRNA) precursors than healthy plants. This result correlated with the reported reduction of mature miRNAs species. The protein 24K, the CPsV suppressor of RNA silencing (VSR), interacts with miRNA precursors . Thus, this protein becomes a candidate responsible for the increased accumulation of unprocessed miRNAs. We analyzed 24K RNA-binding and protein-protein interaction domains and described patterns of its subcellular localization. We also showed that 24K colocalizes within nuclear D-bodies with the miRNA biogenesis proteins DICER-LIKE 1 (DCL1), HYPONASTIC LEAVES 1 (HYL1), and SERRATE (SE). According to the results of bimolecular fluorescence complementation and co-immunoprecipitation assays, the 24K protein interacts with HYL1 and SE. Thus, 24K may inhibit miRNA processing in CPsV-infected citrus plants by direct interaction with the miRNA processing complex. This work contributes to the understanding of how a virus can alter the regulatory mechanisms of the host, particularly miRNA biogenesis and function.IMPORTANCESweet oranges can suffer from disease symptoms induced by virus infections, thus resulting in drastic economic losses. In sweet orange plants, CPsV alters the accumulation of some precursors from the regulatory molecules called miRNAs. This alteration leads to a decreased level of mature miRNA species. This misregulation may be due to a direct association of one of the viral proteins (24K) with miRNA precursors. On the other hand, 24K may act with components of the cell miRNA processing machinery through a series of predicted RNA-binding and protein-protein interaction domains.
Topics: MicroRNAs; Plant Diseases; Viral Proteins; Citrus sinensis; Plant Viruses; Plant Proteins; RNA-Binding Proteins; RNA Processing, Post-Transcriptional; Citrus; RNA Precursors
PubMed: 38785434
DOI: 10.1128/spectrum.03513-23 -
Environmental Toxicology and Chemistry Jul 2024Numerous pharmaceutical and industrial chemicals are classified as endocrine-disrupting chemicals (EDCs) that interfere with hormonal homeostasis, leading to...
Numerous pharmaceutical and industrial chemicals are classified as endocrine-disrupting chemicals (EDCs) that interfere with hormonal homeostasis, leading to developmental disorders and other pathologies. The synthetic estrogen 17α-ethynylestradiol (EE2) is used in oral contraceptives and other hormone therapies. EE2 and other estrogens are inadvertently introduced into aquatic environments through municipal wastewater and agricultural effluents. Exposure of male fish to estrogens increases expression of the egg yolk precursor protein vitellogenin (Vtg), which is used as a molecular marker of exposure to estrogenic EDCs. The mechanisms behind Vtg induction are not fully known, and we hypothesized that it is regulated via DNA methylation. Adult zebrafish were exposed to either dimethyl sulfoxide or 20 ng/L EE2 for 14 days. Messenger RNA (mRNA) expression and DNA methylation were assessed in male zebrafish livers at 0, 0.25, 0.5, 1, 4, 7, and 14 days of exposure; and those of females were assessed at 13 days (n ≥ 4/group/time point). To test the persistence of any changes, we included a recovery group that received EE2 for 7 days and did not receive any for the following 7 days, in the total 14-day study. Methylation of DNA at the vtg1 promoter was assessed with targeted gene bisulfite sequencing in livers of adult male and female zebrafish. A significant increase in vtg1 mRNA was observed in the EE2-exposed male fish as early as 6 h. Interestingly, DNA methylation changes were observed at 4 days. Decreases in the overall methylation of the vtg1 promoter in exposed males resulted in levels comparable to those in female controls, suggesting feminization. Importantly, DNA methylation levels in males remained significantly impacted after 7 days post-EE2 removal, unlike mRNA levels. These data identify an epigenetic mark of feminization that may serve as an indicator of not only estrogenic exposure but also previous exposure to EE2. Environ Toxicol Chem 2024;43:1547-1556. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.
Topics: Animals; Zebrafish; Male; Ethinyl Estradiol; DNA Methylation; Vitellogenins; Promoter Regions, Genetic; CpG Islands; Female; Water Pollutants, Chemical; Zebrafish Proteins; Endocrine Disruptors
PubMed: 38785270
DOI: 10.1002/etc.5879 -
Hepatology (Baltimore, Md.) May 2024Circulating tumor cells (CTCs) are precursors of cancer metastasis. However, how CTCs evade immunosurveillance during hematogenous dissemination remains unclear.
BACKGROUND AND AIMS
Circulating tumor cells (CTCs) are precursors of cancer metastasis. However, how CTCs evade immunosurveillance during hematogenous dissemination remains unclear.
APPROACH AND RESULTS
We identified CTC-platelet adhesions by single-cell RNA sequencing and multiplex immunofluorescence of blood samples from multiple cancer types. Clinically, CTC-platelet aggregates were associated with significantly shorter progression-free survival and overall survival in patients with HCC. In vitro, ex vivo, and in vivo assays demonstrated direct platelet adhesions gifted cancer cells with an evasive ability from NK cell killing by upregulating inhibitory checkpoint CD155 (PVR cell adhesion molecule), therefore facilitating distant metastasis. Mechanistically, CD155 was transcriptionally regulated by the FAK/JNK/c-Jun cascade in a platelet contact-dependent manner. Further competition assays and cytotoxicity experiments revealed that CD155 on CTCs inhibited NK-cell cytotoxicity only by engaging with immune receptor TIGIT, but not CD96 and DNAM1, another 2 receptors for CD155. Interrupting the CD155-TIGIT interactions with a TIGIT antibody restored NK-cell immunosurveillance on CTCs and markedly attenuated tumor metastasis.
CONCLUSIONS
Our results demonstrated CTC evasion from NK-cell-mediated innate immunosurveillance mainly through immune checkpoint CD155-TIGIT, potentially offering an immunotherapeutic strategy for eradicating CTCs.
PubMed: 38779918
DOI: 10.1097/HEP.0000000000000934