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High-Throughput Screen Detects Calcium Signaling Dysfunction in Hutchinson-Gilford Progeria Syndrome

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High-Throughput Screen Detects Calcium Signaling Dysfunction in Hutchinson-Gilford Progeria Syndrome International Journal of Molecular Sciences Article High-Throughput Screen Detects Calcium Signaling Dysfunction in Hutchinson-Gilford Progeria Syndrome Juan A. Fafián-Labora , Miriam Morente-López, Fco. Javier de Toro and María C. Arufe * Grupo de Terapia Celular y Medicina Regenerativa, Departamento de Fisioterapia, Ciencias Biomédicas y Medicina, Universdidade da Coruña, Agrupación Estratégica INIBIC-CICA, 15006 A Coruña, Spain; [email protected] (J.A.F.-L.); [email protected] (M.M.-L.); [email protected] (F.J.d.T.) * Correspondence: [email protected] Abstract: Hutchinson–Gilford progeria syndrome (HGPS) is a deadly childhood disorder, which is considered a very rare disease. It is caused by an autosomal dominant mutation on the LMNA gene, and it is characterized by accelerated aging. Human cell lines from HGPS patients and healthy parental controls were studied in parallel using next-generation sequencing (NGS) to unravel new non-previously altered molecular pathways. Nine hundred and eleven transcripts were differentially expressed when comparing healthy versus HGPS cell lines from a total of 21,872 transcripts; ITPR1, ITPR3, CACNA2D1, and CAMK2N1 stood out among them due to their links with calcium signaling, and these were validated by Western blot analysis. It was observed that the basal concentration of intracellular Ca2+ was statistically higher in HGPS cell lines compared to healthy ones. The relationship between genes involved in Ca2+ signaling and mitochondria-associated membranes (MAM) was demonstrated through cytosolic calcium handling by means of an automated fluorescent plate reading system (FlexStation 3, Molecular Devices), and apoptosis and mitochondrial ROS Citation: Fafián-Labora, J.A.; production were examined by means of flow cytometry analysis. Altogether, our data suggest that Morente-López, M.; de Toro, F.J.; the Ca2+ signaling pathway is altered in HGPS at least in part due to the overproduction of reactive Arufe, M.C. High-Throughput Screen oxygen species (ROS). Our results unravel a new therapeutic window for the treatment of this rare Detects Calcium Signaling disease and open new strategies to study pathologies involving both accelerated and healthy aging. Dysfunction in Hutchinson-Gilford Progeria Syndrome. Int. J. Mol. Sci. Keywords: HGPS; Ca2+ signaling; GRP75; GRP78; NAC; ROS 2021, 22, 7327. https://doi.org/ 10.3390/ijms22147327 Academic Editor: Davide Gentilini 1. Introduction Received: 21 April 2021 Hutchinson–Gilford progeria syndrome (HGPS) is a deadly childhood genetic dis- Accepted: 1 July 2021 order caused by an autosomal dominant mutation on the LMNA gene and is a very rare Published: 7 July 2021 disease [1]. HGPS belongs to a group of diseases called laminopathies, all of them sharing mutations in the LMNA gene as the causal agent. The LMNA gene encodes for lamins Publisher’s Note: MDPI stays neutral A and C and these have important structural and regulatory functions in the nuclear with regard to jurisdictional claims in envelope [2]. HGPS is characterized by premature aging of the organism and accelerated published maps and institutional affil- senescence at the cellular level [3], and patients die at a mean age of 13 years old, mainly iations. due to cardiovascular complications. The LMNA point mutation most frequently asso- ciated with HGPS promotes the processing and accumulation of a deleterious lamin A isoform, called progerin or lamin A D50 [4], which remains permanently farnesylated [5]. In recent years, therapeutic strategies have focused on reverting the action of progerin, mainly Copyright: © 2021 by the authors. through the use of farnesyltransferase inhibitors [6]. Almost a decade ago, Viteri et al. Licensee MDPI, Basel, Switzerland. demonstrated that progerin promotes the accumulation of oxidized proteins in fibroblast This article is an open access article cell lines derived from HGPS patients [7]. More recently our group demonstrated that distributed under the terms and the deregulation of lamin A alters the oxidative stress balance in mesenchymal stem cells conditions of the Creative Commons (MSCs), modifying their migratory properties and inducing defects in their capacity to Attribution (CC BY) license (https:// differentiate into chondrocytes in our in vitro model. Furthermore, our group has reported creativecommons.org/licenses/by/ that progerin expression induces mitochondrial dysfunction and ROS overproduction [8]. 4.0/). Int. J. Mol. Sci. 2021, 22, 7327. https://doi.org/10.3390/ijms22147327 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 7327 2 of 14 These changes, at least in part, increase protein folding and autophagic proteolysis, indicat- ing an overall loss of proteostasis that would lead to the process of premature aging [9]. The endoplasmic reticulum (ER) is an intracellular organelle essential for protein synthesis, folding, and modification, and it also maintains calcium homeostasis within the cell. Cel- lular function and survival are potentially regulated by Ca entry through store-operated Ca entry (SOCE) channels. These are essential for maintaining intracellular Ca stores [10]. The Ca release from ER stores is controlled by the inositol 1,4,5-trisphosphate receptors (IP3R). Cell survival depends on the ability of cells to respond to ER stress since chronic stress leads to apoptosis [11]. Mitochondria respond to cellular stress by releasing pro- teins, for example, cytochrome C from the space between their membranes, controlling the intrinsic pathway. Cellular stress is manifested as heat, infection, hypoxia, or calcium and nutrient deprivation. These deregulations are associated with the development of diseases such as cancer and tissue damage after increased apoptosis. Apoptosis is the highly controlled process of programmed cell death used by multicellular organisms to clean damaged cells and to maintain cell numbers in the functional organs [12]. The membrane contact site between mitochondria and the endoplasmic reticulum is known as mitochondria-associated membrane (MAM) [13]. This contact site is crucial for various biological events, such as lipid transfer, sterol exchange, Ca transfer, apoptosis, and the suppression of neurological disorders [14]. Previously, we have identified several calcium signaling-related proteins as being significantly modulated in an in vitro model of progerin over-expression [9]. Chen et al. [15] have demonstrated the involvement of ROS and/or altered Ca signaling in HGPS. Our goal in this study was to deepen the understanding of the alteration in calcium signaling in premature aging in human cells from HGPS patients, focusing on the contribution of MAM to the underlying mechanism. 2. Results 2.1. RNAseq Analysis of HGPS and Healthy Control Cell Lines Genomic datasets were deposited in the Gene Expression Omnibus (GEO-NCBI) repos- itory (http://www.ncbi.nlm.nih.gov/geo/, accessed on 30 April 2018). The raw files are public and freely accessible (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE113648, accessed on 30 April 2018). This is an analysis of a previously published at PLoS ONE [16] data set carried out by our group. From a total of 21,872 transcripts measured, 911 were detected as differentially expressed when comparing healthy versus HGPS cell lines with a q-value less than 0.05. A summary of the NGS showing the quantified transcripts is presented in Figure1A. The list of significantly modulated transcripts was imported into String 10.1 software for pathway analysis (Figure1B–D). Transcripts involved in Ca 2+ signaling are indicated in Figure1B and Table A1. The proteins associated with those tran- scripts were used for the network (Figure1C) and cellular component analysis (Figure1D), indicating that most of the proteins are located in membranes and extracellular regions. 2.2. Orthogonal Validation of Ca2+ Flux-Related Proteins and Transcripts Orthogonal validation of NGS results was performed using a battery of Ca2+ trans- porters localized in MAM (Tables A2 and A3). Western blot analysis demonstrated that IP3R1 was significantly (p < 0.05) higher in HGPS vs. healthy cell lines and IP3R3 was significantly (p < 0.05) lower in HGPS vs. healthy cell lines. In addition, the level of GRP78 was statistically significantly lower in HGPS than in healthy cell lines (Figure2A,B). The qRT-PCR analysis demonstrated that ITPR1, CACNA2D1, CDH13, and ACAN expression were significantly (p < 0.05) higher in HGPS vs. healthy cell lines and ITPR3, CAMK2N1, SULF2, and TENM2 expression were significantly (p < 0.05) lower in HGPS vs. healthy cell lines. All of these results validated the results obtained by shotgun analysis (Figure2C). Int. J. Mol. Sci. 2021, 22, 7327 3 of 14 Figure 1 A) B) 12 10 527 384 8 up down in in HGPS HGPS 6 log2 Control value Controllog2 4 20961 non-modulated 911 modulated 2 (q-value ≤ 0.05) 0 -10−10 −5-5 0 5 10 15 −2-2 log2 HGPS value Total # of genes: 21872 −4-4 −6-6 C) D) focal 0.001 p-val adhesion CellularCellular Compartment golgi apparatus elastic fiber plasma membrane extracellular space cell perifery basement membrane extracellular exosome extracellular matrix membrane-bound vesicle extracellular region 0 2 4 6 8 10 12 -log10 FDR − log10 FDR Figure 1. Summary of the massive genomic analysis (RNAseq). (A) Summary of the NGS showing the 21,872 different quantified transcripts. Of these, 911 were considered to be significantly modulated, with q-values ≤ 0.05. (B) Representation of the values for the 911 significantly modulated transcripts and those coding for Ca2+ flux-related proteins (in red). (C) String 10.1 pathway analysis for the Ca2+ flux-related transcripts showing the interaction networks. (D) Statistical analysis shows that most of those transcripts codify for extracellular and membrane-bound (vesicle/exosome) proteins. 2.3. Calcium Dynamics Measurement Basal calcium measurement by means of luminescence spectrometry revealed that HGPS cell lines had a statistically significant basal calcium level that was higher than that of the healthy cell lines (Figure2D). The release of Ca 2+ from HGPS cell lines and the healthy ones was not affected by ATP in a statistically significant way (p < 0.05) (Figure3A).
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