Molecular cloning of the gene promoter encoding the human CaVγ2/Stargazin divergent transcript (CACNG2-DT): characterization and regulation by the cAMP-PKA/CREB signaling pathway

CaVγ2 (Stargazin or TARPγ2) is a protein expressed in various types of neurons whose function was initially associated with a decrease in the functional expression of voltage-gated presynaptic Ca2+ channels (CaV) and which is now known to promote the trafficking of the postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) towards the cell membrane. Alterations in CaVγ2 expression has been associated with several neurological disorders, such as absence epilepsy. However, its regulation at the transcriptional level has not been intensively addressed. It has been reported that the promoter of the Cacng2 gene, encoding the rat CaVγ2, is bidirectional and regulates the transcription of a long non-coding RNA (lncRNA) in the antisense direction. Here, we investigate the proximal promoter region of the human CACNG2 gene in the antisense direction and show that this region includes two functional cAMP response elements that regulate the expression of a lncRNA called CACNG2-DT. The activity of these sites is significantly enhanced by forskolin, an adenylate cyclase activator, and inhibited by H89, a protein kinase A (PKA) antagonist. Therefore, this regulatory mechanism implies the activation of G protein-coupled receptors and downstream phosphorylation. Interestingly, we also found that the expression of CACNG2-DT may increase the levels of the CaVγ2 subunit. Together, these data provide novel information on the organization of the human CACNG2-DT gene promoter, describe modulatory domains and mechanisms that can mediate various regulatory inputs, and provide initial information on the molecular mechanisms that regulate the functional expression of the CaVγ2 protein

Gene Therapy Corrects Mitochondrial Dysfunction in Hematopoietic Progenitor Cells and Fibroblasts from Coq9R239X Mice

This study has been submitted to the patent's offices at the "University of Granada" and "Fundación Progreso y Salud". Please note that the results of this manuscript have been submitted to patent protection (application number P201630630; title: “Uses of Coenzyme Q biosynthetic proteins”; date:05/16/2016).Recent clinical trials have shown that in vivo and ex vivo gene therapy strategies can be an option for the treatment of several neurological disorders. Both strategies require efficient and safe vectors to 1) deliver the therapeutic gene directly into the CNS or 2) to genetically modify stem cells that will be used as Trojan horses for the systemic delivery of the therapeutic protein. A group of target diseases for these therapeutic strategies are mitochondrial encephalopathies due to mutations in nuclear DNA genes. In this study, we have developed a lentiviral vector (CCoq9WP) able to overexpress Coq9 mRNA and COQ9 protein in mouse embryonic fibroblasts (MEFs) and hematopoietic progenitor cells (HPCs) from Coq9R239X mice, an animal model of mitochondrial encephalopathy due to primary Coenzyme Q (CoQ) deficiency. Ectopic over-expression of Coq9 in both cell types restored the CoQ biosynthetic pathway and mitochondrial function, improving the fitness of the transduced cells. These results show the potential of the CCoq9WP lentiviral vector as a tool for gene therapy to treat mitochondrial encephalopathies.This work was supported by grants from Ministerio de Economía y Competitividad (Spain) and the European Regional Development Fund (ERDF) from the European Union, to LCL through the research grants SAF2013-47761-R and SAF2015-65786-R; by Fondo de Investigaciones Sanitarias ISCIII (Spain) and the European Regional Development Fund (ERDF) from the European Union through the research grants PI12/01097 and ISCIII Red de Terapia Celular TerCel RD12/0019/0006 to FM; by the Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía-FEDER/Fondo de Cohesion Europeo (FSE) de Andalucía through the research grants P10-CTS-6133 to LCL; P09-CTS-04532, PI-57069, PI-0001/2009 and PAIDI-Bio-326 to F.M.; PI-0160/2012 to KB and PI-0407/2012 to MC; by the NIH through the research P01HD080642 to LCL and by the foundation “todos somos raros, todos somos únicos” to LCL. LCL is supported by the ‘Ramón y Cajal’ National Programme, Ministerio de Economía y Competitividad, Spain (RYC-2011-07643)

Effect of α₂δ-1, Sp1, or TLR-4 silencing on the proliferation and migration of U87 cells.

A) Comparison of cell number after α2δ-1, Sp1, and TRL-4 knockdown with specific siRNAs of U87 cells as assessed by direct cell counting. Values are expressed as the percentage of untransfected cells (Ctl), and each bar represents the mean ± SEM of triplicate determinations in 3 separate experiments. *P versus untransfected cells. B) Transwell migration assays of control and cells transiently transfected with specific α2δ-1, Sp1, or TLR-4 siRNAs. Representative images from three separate experiments are shown. C) Comparative analysis of the mean ± SEM of 3 transwell independent experiments as in B. *P < 0.05 compared to untransfected cells.</p

Effect of LPS on the proliferation and migration of U87 cells.

A) Comparison of direct and automated cell counting of U87 cells maintained 48 h in culture in the presence and absence of the TLR-4 activator, LPS, as indicated. Cell counts were performed in parallel after treatment with C34, a specific inhibitor of TLR-4, and the vehicle (DMSO) in which it was dissolved. Values are expressed as a percent of control, and each bar represents the mean ± SE of triplicate determinations in 3 separate experiments. B) Cell wound healing assays showing the ability of U87 cells to migrate in culture in the absence and presence of LPS, as indicated. The lower right panel shows a graph of the quantification of the wound area. C) Transwell migration assays of U87 cells in the presence of the activator (LPS) and the inhibitor (C34) of TLR-4, as listed. Typical images from three separate experiments are shown. D, Comparative analysis of transwell migration assay results of U78 cells as in C. Results represent the mean ± SEM of 3 independent experiments. *P < 0.05 compared to untreated cells.</p

Expression of α2δ-1, TLR-4, and Sp1 in human glioblastoma U87 and human neuroblastoma SHSY-5Y (SH) cell lines.

The image shows representative blots of three experiments performed separately. The signal obtained with the β-actin antibody served as the loading control. (TIF)</p

Effect of α₂δ-1, Sp1, or TLR-4 overexpression on the proliferation and migration of U87 cells.

A) Comparison of cell number after transient transfection of U87 cells with α2δ-1 subunit, Sp1, and TRL-4 as assessed by direct cell counting. Values are expressed as the percentage of untransfected cells (Ctl), and each bar represents the mean ± SEM of triplicate determinations in 3 separate experiments. *P versus untransfected cells. B) Transwell migration assays of control and cells transiently transfected with α2δ-1, Sp1, or TLR-4 cDNA clones. Representative images from three independent experiments are shown. C) Comparative analysis of the mean ± SEM of 3 transwell separate experiments as in B. *P < 0.01 compared to untransfected cells.</p

Effect of NF-кB inhibition on Sp1 and α₂δ-1 expression.

A) Western blot assays performed on U87 cell lysates in the control condition and after treatment with the TLR-4 agonist LPS and the transcription factor NF-кB inhibitor PDTC. The image shows a representative assay of three performed separately (upper panel). The signal obtained with the β-actin antibody served as the loading control. The lower panel shows the comparative densitometric analysis of the level of α2δ-1 protein. Data are shown as mean + SEM of 3 independent experiments in triplicate. B) Sp1 Western blot assays performed on U87 cells in the conditions as in A. The image shows a representative assay of three performed separately (upper panel). The signal obtained with the β-actin antibody served as the loading control. The lower panel shows the comparative densitometric analysis of the level of Sp1 protein. Data are shown as mean ± SEM of 3 independent experiments in triplicate. *P < 0.05 compared to untreated cells.</p

S1 Raw images -

(PDF)</p

Effect of gabapentin on U87 cell migration.

A) Representative images from three separate experiments of transwell migration assays in the presence or absence of the TLR-4 activator (LPS) and the antagonist of the α2δ-1 subunit (gabapentin). B) Comparative analysis of the mean ± SEM of 3 transwell independent experiments. *P < 0.05 compared to untreated cells.</p

Upregulation of α₂δ-1 by LPS.

U87 cells were co-transfected with a vector containing the α2δ-1 subunit promoter and the Renilla luciferase coding sequence (as a reporter gene), and the pRSV-βGal vector, which included the RSV promoter and the β-gal coding sequence to correct for differences related to transfection efficiency. Cells were incubated for 48 h in the absence and presence of the TLR-4 activator, LPS. B) Western blot assay performed on U87 cell lysates in the absence and presence of LPS. The image shows a representative assay of three performed separately. C) Densitometric analysis of protein α2δ-1 normalized with respect to the expression of β actin and depicted as a fold change from the control. Asterisks denote statistically significant differences (P < 0.05) with respect to the control (Ctl).</p