STIM1 Deficiency Leads to Specific Down-Regulation of ITPR3 in SH-SY5Y Cells

STIM1 is an endoplasmic reticulum (ER) protein that modulates the activity of a number of Ca2+ transport systems. By direct physical interaction with ORAI1, a plasma membrane Ca2+ channel, STIM1 activates the ICRAC current, whereas the binding with the voltage-operated Ca2+ channel CaV1.2 inhibits the current through this latter channel. In this way, STIM1 is a key regulator of Ca2+ signaling in excitable and non-excitable cells, and altered STIM1 levels have been reported to underlie several pathologies, including immunodeficiency, neurodegenerative diseases, and cancer. In both sporadic and familial Alzheimer’s disease, a decrease of STIM1 protein levels accounts for the alteration of Ca2+ handling that compromises neuronal cell viability. Using SH-SY5Y cells edited by CRISPR/Cas9 to knockout STIM1 gene expression, this work evaluated the molecular mechanisms underlying the cell death triggered by the deficiency of STIM1, demonstrating that STIM1 is a positive regulator of ITPR3 gene expression. ITPR3 (or IP3R3) is a Ca2+ channel enriched at ER-mitochondria contact sites where it provides Ca2+ for transport into the mitochondria. Thus, STIM1 deficiency leads to a strong reduction of ITPR3 transcript and ITPR3 protein levels, a consequent decrease of the mitochondria free Ca2+ concentration ([Ca2+]mit), reduction of mitochondrial oxygen consumption rate, and decrease in ATP synthesis rate. All these values were normalized by ectopic expression of ITPR3 in STIM1-KO cells, providing strong evidence for a new mode of regulation of [Ca2+]mit mediated by the STIM1-ITPR3 axis.

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Nuclear Expression of a Mitochondrial DNA Gene: Mitochondrial Targeting of Allotopically Expressed Mutant ATP6 in Transgenic Mice

Journal of Biomedicine and Biotechnology ◽

10.1155/2012/541245 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 11

Author(s):

David A. Dunn ◽

Carl A. Pinkert

Keyword(s):

Mitochondrial Dna ◽

Atp Synthesis ◽

Serum Lactate ◽

Superior Performance ◽

Synthesis Rate ◽

Mitochondrial Targeting ◽

Biochemical Tests ◽

Balance Beam ◽

Protein Levels

Nuclear encoding of mitochondrial DNA transgenes followed by mitochondrial targeting of the expressed proteins (allotopic expression; AE) represents a potentially powerful strategy for creating animal models of mtDNA disease. Mice were created that allotopically express either a mutant (A6M) or wildtype (A6W)mt-Atp6transgene. Compared to non-transgenic controls, A6M mice displayed neuromuscular and motor deficiencies (wire hang, pole, and balance beam analyses;P<0.05), no locomotor differences (gait analysis;P<0.05) and enhanced endurance in Rota-Rod evaluations (P<0.05). A6W mice exhibited inferior muscle strength (wire hang test;P<0.05), no difference in balance beam footsteps, accelerating Rota-Rod, pole test and gait analyses; (P<0.05) and superior performance in balance beam time-to-cross and constant velocity Rota-Rod analyses (P<0.05) in comparison to non-transgenic control mice. Mice of both transgenic lines did not differ from non-transgenic controls in a number of bioenergetic and biochemical tests including measurements of serum lactate and mitochondrial MnSOD protein levels, ATP synthesis rate, and oxygen consumption (P>0.05). This study illustrates a mouse model capable of circumventingin vivomitochondrial mutations. Moreover, it provides evidence supporting AE as a tool for mtDNA disease research with implications in development of DNA-based therapeutics.

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Silencing of Plasma Membrane Ca2+-ATPase Isoforms 2 and 3 Impairs Energy Metabolism in Differentiating PC12 Cells

BioMed Research International ◽

10.1155/2014/735106 ◽

2014 ◽

Vol 2014 ◽

pp. 1-13 ◽

Cited By ~ 5

Author(s):

Tomasz Boczek ◽

Malwina Lisek ◽

Bozena Ferenc ◽

Antoni Kowalski ◽

Magdalena Wiktorska ◽

...

Keyword(s):

Plasma Membrane ◽

Pc12 Cells ◽

Molecular Mechanisms ◽

Atp Synthesis ◽

Mitochondrial Activity ◽

Excitable Cells ◽

Close Link ◽

Atp Level ◽

Atp Generation

A close link between Ca2+, ATP level, and neurogenesis is apparent; however, the molecular mechanisms of this relationship have not been completely elucidated. Transient elevations of cytosolic Ca2+may boost ATP synthesis, but ATP is also consumed by ion pumps to maintain a low Ca2+in cytosol. In differentiation process plasma membrane Ca2+ATPase (PMCA) is considered as one of the major players for Ca2+homeostasis. From four PMCA isoforms, the fastest PMCA2 and PMCA3 are expressed predominantly in excitable cells. In the present study we assessed whether PMCA isoform composition may affect energy balance in differentiating PC12 cells. We found that PMCA2-downregulated cells showed higher basal O2consumption, lower NAD(P)H level, and increased activity of ETC. These changes associated with higher[Ca2+]cresulted in elevated ATP level. Since PMCA2-reduced cells demonstrated greatest sensitivity to ETC inhibition, we suppose that the main source of energy for PMCA isoforms 1, 3, and 4 was oxidative phosphorylation. Contrary, cells with unchanged PMCA2 expression exhibited prevalence of glycolysis in ATP generation. Our results with PMCA2- or PMCA3-downregulated lines provide an evidence of a novel role of PMCA isoforms in regulation of bioenergetic pathways, and mitochondrial activity and maintenance of ATP level during PC12 cells differentiation.

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Arylsulfatase A, a genetic modifier of Parkinson’s disease, is an α-synuclein chaperone

Brain ◽

10.1093/brain/awz205 ◽

2019 ◽

Vol 142 (9) ◽

pp. 2845-2859 ◽

Cited By ~ 16

Author(s):

Jun Sung Lee ◽

Kazuaki Kanai ◽

Mari Suzuki ◽

Woojin S Kim ◽

Han Soo Yoo ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Genetic Modifier ◽

Ectopic Expression ◽

Physical Interaction ◽

Dependent Manner ◽

Arylsulfatase A ◽

Lysosomal Storage ◽

Protein Levels

AbstractMutations in lysosomal genes increase the risk of neurodegenerative diseases, as is the case for Parkinson’s disease. Here, we found that pathogenic and protective mutations in arylsulfatase A (ARSA), a gene responsible for metachromatic leukodystrophy, a lysosomal storage disorder, are linked to Parkinson’s disease. Plasma ARSA protein levels were changed in Parkinson’s disease patients. ARSA deficiency caused increases in α-synuclein aggregation and secretion, and increases in α-synuclein propagation in cells and nematodes. Despite being a lysosomal protein, ARSA directly interacts with α-synuclein in the cytosol. The interaction was more extensive with protective ARSA variant and less with pathogenic ARSA variant than wild-type. ARSA inhibited the in vitro fibrillation of α-synuclein in a dose-dependent manner. Ectopic expression of ARSA reversed the α-synuclein phenotypes in both cell and fly models of synucleinopathy, the effects correlating with the extent of the physical interaction between these molecules. Collectively, these results suggest that ARSA is a genetic modifier of Parkinson’s disease pathogenesis, acting as a molecular chaperone for α-synuclein.

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Changes in Serum Iron and Leukocyte mRNA Levels of Genes Involved in Iron Metabolism in Amateur Marathon Runners—Effect of the Running Pace

Genes ◽

10.3390/genes10060460 ◽

2019 ◽

Vol 10 (6) ◽

pp. 460 ◽

Cited By ~ 3

Author(s):

Agata Grzybkowska ◽

Katarzyna Anczykowska ◽

Wojciech Ratkowski ◽

Piotr Aschenbrenner ◽

Jędrzej Antosiewicz ◽

...

Keyword(s):

Gene Expression ◽

Iron Metabolism ◽

Serum Iron ◽

Molecular Mechanisms ◽

Mrna Levels ◽

Marathon Runners ◽

Time Points ◽

Protein Levels ◽

Reactive Protein ◽

Blood Morphology

Iron is essential for physical activity due to its role in energy production pathways and oxygen transportation via hemoglobin and myoglobin. Changes in iron-related biochemical parameters after physical exercise in athletes are of substantial research interest, but molecular mechanisms such as gene expression are still rarely tested in sports. In this paper, we evaluated the mRNA levels of genes related to iron metabolism (PCBP1, PCBP2, FTL, FTH, and TFRC) in leukocytes of 24 amateur runners at four time points: before, immediately after, 3 h after, and 24 h after a marathon. We measured blood morphology as well as serum concentrations of iron, ferritin, and C-reactive protein (CRP). Our results showed significant changes in gene expression (except for TFRC), serum iron, CRP, and morphology after the marathon. However, the alterations in mRNA and protein levels occurred at different time points (immediately and 3 h post-run, respectively). The levels of circulating ferritin remained stable, whereas the number of transcripts in leukocytes differed significantly. We also showed that running pace might influence mRNA expression. Our results indicated that changes in the mRNA of genes involved in iron metabolism occurred independently of serum iron and ferritin concentrations.

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HPV E7-mediated NCAPH ectopic expression regulates the carcinogenesis of cervical carcinoma via PI3K/AKT/SGK pathway

Cell Death and Disease ◽

10.1038/s41419-020-03244-9 ◽

2020 ◽

Vol 11 (12) ◽

Author(s):

Meng Wang ◽

Xiaowen Qiao ◽

Tamara Cooper ◽

Wei Pan ◽

Liang Liu ◽

...

Keyword(s):

Gene Expression ◽

Cervical Cancer ◽

Cancer Cells ◽

Molecular Mechanisms ◽

Ectopic Expression ◽

Depth Of Invasion ◽

Mesenchymal Transition ◽

Hpv E7

AbstractCervical cancer is one of the most common gynecological tumors in the world, and human papillomavirus (HPV) infection is its causative agent. However, the molecular mechanisms involved in the carcinogenesis of cervical cancer still require clarification. Here we found that knockdown of Non-SMC (Structural Maintenance of Chromosomes) condensin I complex subunit H (NCAPH) gene expression significantly inhibited the proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) of cervical cancer cells in vitro, and restrained xenograft tumor formation in vivo. Intriguingly, HPV E7 could form a positive feedback loop with NCAPH. E7 upregulated NCAPH gene expression via E2F1 which initiated NCAPH transcription by binding to its promoter directly. Silencing of NCAPH reduced E7 transcription via promoting the transition of AP-1 heterodimer from c-Fos/c-Jun to Fra-1/c-Jun. Moreover, the E7-mediated NCAPH overexpression was involved in the activation of the PI3K/AKT/SGK signaling pathway. In vivo, NCAPH expression in cervical cancer tissues was significantly higher than which in normal cervix and high-grade squamous intraepithelial lesion (HSIL) tissues, and its expression was significantly correlated with tumor size, depth of invasion and lymph node metastasis. Patients with high NCAPH expression had a significantly better survival outcomes than those with low-expression, suggesting that NCAPH-induced cell proliferation might sensitize cancer cells to adjuvant therapy. In conclusion, our results revealed the role of NCAPH in the carcinogenesis of cervical cancer in vitro and in vivo. The interaction between E7 and NCAPH expands the mechanism of HPV induced tumorigenesis and that of host genes regulating HPV E7.

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Telomere associated gene expression as well as TERT protein level and telomerase activity are altered in the ovarian follicles of aged mice

Scientific Reports ◽

10.1038/s41598-021-95239-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Esra Gozde Kosebent ◽

Saffet Ozturk

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Structural Integrity ◽

Cell Types ◽

Telomerase Activity ◽

Ovarian Follicles ◽

Biological Aging ◽

Aged Mice ◽

Protein Levels ◽

Ovarian Aging

AbstractTelomeres cap the ends of eukaryotic chromosomes to maintain genomic stability and integrity during an organism’s lifespan. The length of telomeres inevitably shortens due to DNA replication, genotoxic agents, and biological aging. A limited number of cell types, e.g., stem cells, germline cells, and early embryos can elongate shortened telomeres via the enzymatic action of telomerase, which is composed of telomerase reverse transcriptase (TERT) and telomerase RNA component (Terc). Additionally, telomere-associated proteins including telomeric repeat binding factor 1 (TRF1) and 2 (TRF2), as well as protection of telomeres 1a (POT1a), bind to telomeres to maintain their structural integrity and length. During ovarian aging in mammals, telomeres progressively shorten, accompanied by fertility loss; however, the molecular mechanism underlying this attrition during follicle development remains unclear. In this study, the primary, secondary, preantral, and antral follicles were obtained either from 6-week-old adult (n = 19) or 52-week-old aged (n = 12) mice. We revealed that the Tert, Terc, Trf1, Trf2, and Pot1a gene expression (P < 0.001) and TERT protein (P < 0.01) levels significantly decreased in certain ovarian follicles of the aged group when compared to those of the adult group. Also, telomerase activity exhibited remarkable changes in the follicles of both groups. Consequently, altered telomere-associated gene expression and reduced TERT protein levels in the follicles of aged mice may be a determinant of telomere shortening during ovarian aging, and infertility appearing in the later decades of reproductive lifespan. Further investigations are required to determine the molecular mechanisms underlying these alterations in the follicles during ovarian aging.

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Research Progress in the Molecular Functions of Plant mTERF Proteins

Cells ◽

10.3390/cells10020205 ◽

2021 ◽

Vol 10 (2) ◽

pp. 205

Author(s):

Pedro Robles ◽

Víctor Quesada

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Current Knowledge ◽

Transcription Termination ◽

Atp Synthesis ◽

Research Progress ◽

Mitochondrial Genomes ◽

Molecular Functions ◽

The Many ◽

New Research

Present-day chloroplast and mitochondrial genomes contain only a few dozen genes involved in ATP synthesis, photosynthesis, and gene expression. The proteins encoded by these genes are only a small fraction of the many hundreds of proteins that act in chloroplasts and mitochondria. Hence, the vast majority, including components of organellar gene expression (OGE) machineries, are encoded by nuclear genes, translated into the cytosol and imported to these organelles. Consequently, the expression of nuclear and organellar genomes has to be very precisely coordinated. Furthermore, OGE regulation is crucial to chloroplast and mitochondria biogenesis, and hence, to plant growth and development. Notwithstanding, the molecular mechanisms governing OGE are still poorly understood. Recent results have revealed the increasing importance of nuclear-encoded modular proteins capable of binding nucleic acids and regulating OGE. Mitochondrial transcription termination factor (mTERF) proteins are a good example of this category of OGE regulators. Plant mTERFs are located in chloroplasts and/or mitochondria, and have been characterized mainly from the isolation and analyses of Arabidopsis and maize mutants. These studies have revealed their fundamental roles in different plant development aspects and responses to abiotic stress. Fourteen mTERFs have been hitherto characterized in land plants, albeit to a different extent. These numbers are limited if we consider that 31 and 35 mTERFs have been, respectively, identified in maize and Arabidopsis. Notwithstanding, remarkable progress has been made in recent years to elucidate the molecular mechanisms by which mTERFs regulate OGE. Consequently, it has been experimentally demonstrated that plant mTERFs are required for the transcription termination of chloroplast genes (mTERF6 and mTERF8), transcriptional pausing and the stabilization of chloroplast transcripts (MDA1/mTERF5), intron splicing in chloroplasts (BSM/RUG2/mTERF4 and Zm-mTERF4) and mitochondria (mTERF15 and ZmSMK3) and very recently, also in the assembly of chloroplast ribosomes and translation (mTERF9). This review aims to provide a detailed update of current knowledge about the molecular functions of plant mTERF proteins. It principally focuses on new research that has made an outstanding contribution to unravel the molecular mechanisms by which plant mTERFs regulate the expression of chloroplast and mitochondrial genomes.

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BET bromodomain inhibitors PFI-1 and JQ1 are identified in an epigenetic compound screen to enhance C9ORF72 gene expression and shown to ameliorate C9ORF72-associated pathological and behavioral abnormalities in a C9ALS/FTD model

Clinical Epigenetics ◽

10.1186/s13148-021-01039-z ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Esteban Quezada ◽

Claudio Cappelli ◽

Iván Diaz ◽

Nur Jury ◽

Nicholas Wightman ◽

...

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Luciferase Reporter ◽

Loss Of Function ◽

Protein Levels ◽

Hexanucleotide Repeat ◽

Rna Foci ◽

Behavioral Abnormalities ◽

Bet Inhibitors

Abstract Background An intronic GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), referred to as C9ALS/FTD. No cure or effective treatment exist for C9ALS/FTD. Three major molecular mechanisms have emerged to explain C9ALS/FTD disease mechanisms: (1) C9ORF72 loss-of-function through haploinsufficiency, (2) dipeptide repeat (DPR) proteins mediated toxicity by the translation of the repeat RNAs, and more controversial, (3) RNA-mediated toxicity by bidirectional transcription of the repeats that form intranuclear RNA foci. Recent studies indicate a double-hit pathogenic mechanism in C9ALS/FTD, where reduced C9ORF72 protein levels lead to impaired clearance of toxic DPRs. Here we explored whether pharmacological compounds can revert these pathological hallmarks in vitro and cognitive impairment in a C9ALS/FTD mouse model (C9BAC). We specifically focused our study on small molecule inhibitors targeting chromatin-regulating proteins (epidrugs) with the goal of increasing C9ORF72 gene expression and reduce toxic DPRs. Results We generated luciferase reporter cell lines containing 10 (control) or ≥ 90 (mutant) G4C2 HRE located between exon 1a and 1b of the human C9ORF72 gene. In a screen of 14 different epidrugs targeting bromodomains, chromodomains and histone-modifying enzymes, we found that several bromodomain and extra-terminal domain (BET) inhibitors (BETi), including PFI-1 and JQ1, increased luciferase reporter activity. Using primary cortical cultures from C9BAC mice, we further found that PFI-1 treatment increased the expression of V1-V3 transcripts of the human mutant C9ORF72 gene, reduced poly(GP)-DPR inclusions but enhanced intranuclear RNA foci. We also tested whether JQ1, an BETi previously shown to reach the mouse brain by intraperitoneal (i.p.) injection, can revert behavioral abnormalities in C9BAC mice. Interestingly, it was found that JQ1 administration (daily i.p. administration for 7 days) rescued hippocampal-dependent cognitive deficits in C9BAC mice. Conclusions Our findings place BET bromodomain inhibitors as a potential therapy for C9ALS/FTD by ameliorating C9ORF72-associated pathological and behavioral abnormalities. Our finding that PFI-1 increases accumulation of intranuclear RNA foci is in agreement with recent data in flies suggesting that nuclear RNA foci can be neuroprotective by sequestering repeat transcripts that result in toxic DPRs.

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Comprehensive Bioinformatics Analysis Identifies POLR2I as a Key Gene in the Pathogenesis of Hypertensive Nephropathy

Frontiers in Genetics ◽

10.3389/fgene.2021.698570 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shilong You ◽

Jiaqi Xu ◽

Boquan Wu ◽

Shaojun Wu ◽

Ying Zhang ◽

...

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Gene Expression Omnibus ◽

Chronic Hypertension ◽

Hub Genes ◽

Hypertensive Nephropathy ◽

Protein Levels

Hypertensive nephropathy (HN), mainly caused by chronic hypertension, is one of the major causes of end-stage renal disease. However, the pathogenesis of HN remains unclarified, and there is an urgent need for improved treatments. Gene expression profiles for HN and normal tissue were obtained from the Gene Expression Omnibus database. A total of 229 differentially co-expressed genes were identified by weighted gene co-expression network analysis and differential gene expression analysis. These genes were used to construct protein–protein interaction networks to search for hub genes. Following validation in an independent external dataset and in a clinical database, POLR2I, one of the hub genes, was identified as a key gene related to the pathogenesis of HN. The expression level of POLR2I is upregulated in HN, and the up-regulation of POLR2I is positively correlated with renal function in HN. Finally, we verified the protein levels of POLR2I in vivo to confirm the accuracy of our analysis. In conclusion, our study identified POLR2I as a key gene related to the pathogenesis of HN, providing new insights into the molecular mechanisms underlying HN.

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GRN, NOTCH3, FN1, and PINK1 expression in eutopic endometrium – potential biomarkers in the detection of endometriosis – a pilot study

Journal of Assisted Reproduction and Genetics ◽

10.1007/s10815-020-01905-4 ◽

2020 ◽

Vol 37 (11) ◽

pp. 2723-2732

Author(s):

Isabell Holzer ◽

Amanda Machado Weber ◽

Anne Marshall ◽

Alexander Freis ◽

Julia Jauckus ◽

...

Keyword(s):

Gene Expression ◽

Laparoscopic Surgery ◽

Protein Expression ◽

Molecular Mechanisms ◽

Reproductive Age ◽

Tissue Samples ◽

Protein Levels ◽

Clinical Biomarkers ◽

Mrna Gene ◽

Severity Of The Disease

Abstract Purpose Endometriosis (EM) is a common gynecological disease affecting 10–15% of women of reproductive age. However, molecular mechanisms and pathogenesis are still not completely understood. Furthermore, due to the absence of a reliable clinical biomarker, the only viable method for the often-delayed definitive diagnosis is laparoscopic surgery. Our objective was to analyze molecular differences of selected endometrial proteins and genes of women suffering from different stages of EM compared with healthy women to evaluate potential clinical biomarkers. Methods We analyzed eutopic endometrial tissue samples from women undergoing a laparoscopic surgery (n = 58). mRNA gene expression of progranulin (GRN), neurogenic locus notch homolog protein (NOTCH3), fibronectin (FN1), and PTEN-induced kinase 1 (PINK1) was analyzed using qRT-PCR. Protein expression was determined using ELISA and immunohistochemistry. Results Significant differences in gene expression between the different stages of the disease were noted for GRN, NOTCH3, FN1, and PINK1 (p < 0.05). The endometrium of women with minimal EM (ASRM I) showed the highest mRNA expression. Protein levels of GRN and FN1 on the other hand were significantly decreased in the endometrium of women with EM compared with those of healthy controls. Furthermore, for GRN and FN1, we could detect a correlation of protein expression with the severity of the disease. Conclusion Our findings suggest a potential use of GRN and FN1 as clinical biomarkers to detect endometriosis. In addition, GRN, NOTCH3, FN1, and PINK1 could potentially be useful to differentiate between the underlying stages of the disease. However, a validation with a larger study population is needed.

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