Cultured CSH had a cell density between 30 and 70% for the assays 24 hours after seeding, which corresponded to approximately 2x104 cells per well in a 24 well plate (Corning™, Life Sciences, USA). With 500 µl of MCS, the CRISPR Cas9 assay was performed, in which the assembly of the ribonucleoprotein complex (RNP) was carried out in a 1.3: 1 ratio of personalized unique guide RNA containing a targeting sequence (sgRNA). GeneArt Platinum Cas9 nuclease (Life Technologies, USA), Lipofectamine™ CRISPRMAX™ Cas9 (Invitrogen, USA) was also used following the manufacturer's instructions. The sgRNA (GACCGUGGCCGAAGU) (Synthego, USA) was a 20-nucleotide sequence that is homologous to the exon 2 region of the ARSA gene and that directs the activity of the Cas9 nuclease. Additionally, the sgRNA sequence was used to align two or more sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with chromosome 22 of Homo Sapiens (NC_000022.11), for analysis. Positioning of Guide RNA Sequences and / or Primers.
The cells present in the 24-well plate were trypsinized, 50 µl of the prepared mixture of sgRNA, reduced serum medium Opti-MEM™, Lipofectamine™ Cas9 Plus Reagent, GeneArt™ Platinum™ Cas9 nuclease, and Lipofectamine™ CRISPRMAX were added to the well. TM transfection reagent according to the manufacturer's instructions (13). This was incubated at 37 ° C in a 5% CO2 atmosphere for 3 days. After incubation, cell clonal expansion was performed by limiting the dilution, incubated at 37 ° C in an atmosphere of 5% CO2 until a confluence of 80%, and transferred to a 60 mm3 Petri plate, for subsequent performance of the essays.
Reverse transcriptase polymerase chain reaction in real time (RT-qPCR)
To quantify the expression of the ARSA gene in Schwann cells, before and after transfection with CRISPR Cas9. RNA extraction was performed using the TRIzol® method (Ambion, USA). The concentration and quality of the RNA were measured by spectrophotometry in NanoDrop 2000 (Thermo Fisher Scientific, USA), considering the ratio A260 / A280 1.75. RT-qPCR assays were performed with 2X Luna Universal Probe One-Step RT-qPCR (New England Biolabs, USA), 0.4 µM Forward / reverse predesigned primers of TaqMan® gene expression assays (Life Technologies, USA) + fluorophore-labeled TaqMan probe FAM, 20X Luna WarmStart RT enzyme mix, and 100 ng / µl sample RNA for a final volume of 20 µL. Thermal cycling was carried out at 55 ° C for 20 min for reverse transcription, followed by 95 ° C for 5 min and then 45 cycles of 95 ° C for 15 s, 60 ° C for 45 s, and a final elongation of 60 ° C. for 5 min, for a 69 bp amplicon. The analysis of the results was carried out with the Bio-Rad CFX Manager 3.1 software version 3.1.1517.0823.
Analysis was carried out to determine the number of copies expressed in the ARSA gene of transfected cells compared to those without transfection, the relative quantification method was carried out, the endogenous gene used as a control was Glyceraldehyde-3-phosphate dehydrogenase (GADPH), with the formula:
Sequencing and bioinformatic analysis
Before sequencing exon 2 of the ARSA gene, which allowed analysis of mutation in transfected cells, conventional PCR amplification was carried out in a final volume of 50 µL, 2X Master Mix Go Taq® Green (PROMEGA, USA), 10 nM Primer F 5'CCTACCTGGTCGTGGGAGTA3 ', Primer R 5'TGTCCCGCAGGGCCG3' and 100 ng of DNA. The thermal cycle was carried out at 94 ° C for 5 min, followed by 35 cycles of 94 ° C for 30 s, 60.5 ° C 40 s, then 72 ° C for 30 s and 72 ° C for 5 min. Electrophoresis was performed with a 1.0% agarose gel, at 80 volts for 45 minutes, to obtain a band of 256 bp. The PCR product was sequenced by Capillary Electrophoresis Sequencing at Macrogen Inc. (South Korea). Once the sequence was obtained, the assembly was performed with SeqMan Ultra-LaserGene Version 17 software (DNASTAR, USA) and its homology with exon 2 of the ARSA gene (NG_009260.2) was analyzed. With the observed differences, a high resolution three-dimensional theoretical structural model of the protein was built using the Swiss model server structure assessment tool (https://swissmodel.expasy.org/assess) using the human arylsulfatase protein sequence as a template. A (PDB ID: 1AUK).
Cell Viability and Cytotoxicity
Cell viability and proliferation were evaluated using the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazo (MTT) bromide method (Alfa Aesar, UK). Cellular cytotoxicity made it possible to determine the concentrations of sulfatides and metformin for subsequent tests. In each trial, MCS was used as a treatment-free control, which served to normalize the results of the treatments.
CSH was seeded in 96-well plates at a density of 2x104 cells and incubated for 24 hours in an atmosphere of 5% CO2 at 37 ° C, then, in rows of triplicate, they were exposed to concentrations of 10, 25, 50 and 100 µM of sulfatides (Matreya, USA), on the same plate in triplicate, they were exposed to 10, 25, 50, 100, 300 and 1000 µM of Metformin (1,1-Dimethylbig Unidos hydrochloride, 97%, Acros Organics, Belgium). After 24 hours of posttreatment incubation, the medium was replaced with MTT dissolved in MCS at a concentration of 1 mg / ml, for 1 hour in a 5% CO2 atmosphere at 37 ° C, after the time the MTT was removed from each well and replaced by 100 µL of ACS grade dimethylsulfoxide (DMSO) (amresco®, USA). The color intensity was immediately measured by spectrophotometry on the Thermo Scientific Varioskan Flash plate reader (Thermo Fisher Scientific, USA), at a wavelength of 570 nm. Additionally, photos were taken on the Eclipse Ti-S microscope, 10X magnification.
In the analyzes, the mean, the standard deviation, and the level of cytotoxicity were determined according to ISO 10993-5 of 2009, which highlights the classification of cytotoxicity according to the percentage of cell viability: 71 - 100%: noncytotoxic, <70% the compound has cytotoxic potential (14).
Cell Death Assessment
Nontransfected and transfected cells were cultured in 96-well plates at a density of 3x104 for 2 hours at 37 ° C in a 5% CO2 atmosphere, for subsequent treatment with different concentrations of sulfatides and metformin dissolved in culture medium. After treatment, they were incubated for 24 hours at 37 ° C in a 5% CO2 atmosphere, on the day of the test cell death was determined by incubating the cells with 0.05 µM of SYTOX™ Green nucleic acid stain (Invitrogen, USA) dissolved in MCS (MCS-SYTOX™), a total of 100 µl of MCS-SYTOX™ was added to each well, the plate was incubated for 15 minutes and subsequently the cells were observed in the Eclipse Ti-S fluorescence microscope (Nikon, Japan) (470 nm excitation, 523 nm emission). Similarly, a Thermo Scientific Varioskan Flash microplate reader (Thermo Fisher Scientific, USA) was used for the determination of fluorescence.
To determine dead cells, the average fluorescence of the wells in triplicate was used to calculate the ratio between the fluorescence of the control wells and the fluorescence of the wells containing lysed cells in the presence of 0.1% TRITON® X -100 (amresco, USA) used as a positive control and as a negative control cell with MCS. Interpreting that, the greater the presence of fluorescence, the greater the cell death.
Apoptosis was evaluated using the caspase 3/7 and Annexin V assay, essential to differentiate between the apoptosis and necrosis processes. The CSHs were seeded until they reached a confluence of 70%. Caspase 3 and 7 activity was determined using the CellEvent ™ Caspase-3 / 7 Green Flow Cytometry Assay Kit (Life Technologies, USA).
CSHs were treated with concentrations of 100 µM of sulfatides and 500 µM Metformin in culture medium, incubated for 24 hours at 37 ° C, at a CO2 concentration of 5%, the positive control used was 4 µM Doxorubicin (Ebewe, Austria), the cytometry tubes each containing 1 ml of cell suspension in phosphate buffered saline (PBS) were treated according to the manufacturer's instructions (15). Finally, the samples were analyzed using a 488 nm excitation filter and a 530/30 emission filter (green) for the CellEvent ™ reagent and a 690/50 (red) filter for SYTOX ™ AADvanced ™. This was read on FACScan III flow cytometry equipment (Becton Dickinson, USA). In which a minimum capture of 2,000 events was made.
Furthermore, the translocation of phosphatidyl-serine from the inner to the outer face of the membrane as a differentiator of apoptosis and mitochondrial membrane potential for living cells was evaluated using the MitoTracker® Red Kit and Alexa® Fluorine 488 Annexin V (Invitrogen, UK), for flow cytometry using FACScan III equipment (Becton Dickinson, USA) with a capture of 2,000 events, following the manufacturer's instructions (16). This assay is based on phosphatidylserine translocation and changes in mitochondrial membrane potential.
Apoptosis was induced in CSHs by treating them with concentrations of 10, 25, 50 and 100 µM of sulfatides and 500 µM of Metformin and treated with 100 µM of sulfatides and 500 µM of Metformin, simultaneously. A negative control was prepared by incubating cells in the absence of the inducing agent and as a positive control for necrosis, cells were incubated with 4 µM Doxorubicin (Ebewe, Austria).
Apoptotic cells show green fluorescence with decreased red fluorescence, and living cells show very little green fluorescence and high bright red fluorescence. These populations could be easily distinguished using a flow cytometer by measuring fluorescence in an emission spectrum at 530 and 585 nm.
Standardization of carbonylcyanuro-p-trifluoromethoxyphenylhydrazone the number of cells of carbonylcyanuro-p-trifluoromethoxyphenylhydrazone and the concentration of carbonylcyanuro-p-trifluoromethoxyphenylhydrazone (FCCP, ChemScene, USA) was carried out, against the response to this uncoupler, as recommended in the Mito Stress Test Kit protocol (Agilent Technologies, USA) (17), to guarantee optimal and reproducible culture conditions for comparability between the data and scientific results obtained with CSH; for this reason, the selected population was 50,000 cells.
Nontransfected and transfected CSH were cultured for 2 hours in 24-well Seahorse plates (Agilent Technologies, USA) at a density of 5x104 in 100 µL of MCS, treated with a volume of 150 µL with concentrations of 10, 25, 50 and 100 µM of sulfatides and 500 µM of Metformin for 24 hours at 37 ° C in 5% CO2. Mitochondrial bioenergetic function was determined with the XF Cell Mito Stress Test kit to measure mitochondrial metabolism (Agilent Technologies, USA) in extracellular flux analysis with the Seahorse XFe24 Analyzer (Agilent Technologies, USA), following the manufacturer's instructions (17).
At the end of each assay, the protein concentration was determined using the Bradford method; To normalize the assay, the proteins were read at a wavelength of 590 nm, using a Thermo Scientific Varioskan Flash plate reader (Thermo Fisher Scientific, USES).
One hour before starting the experiments, the CSHs were washed and changed to DMEM culture medium (Caisson Labs, USA) without buffers, which was supplemented with 1 mM pyruvate, 10 mM glutamine, and 5.5 mM glucose, and the medium was adjusted to pH 7.4. After establishing the baseline of the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR), metabolic changes were measured with the addition of inhibitors, 1.5 µM Oligomycin (ChemScene, USA) and 1 µM of both FCCP and Antimycin A / Rotenone (ChemScene, USA).
Mitochondrial Reactive Oxygen Species (ROS)
Mitochondrial ROS levels were detected using the MitoSOX ™ Red fluorescent probe (Invitrogen, USA), following the manufacturer's instructions (18), the fluorescent intensity was measured by an Eclipse Ti-S inverted microscope (Nikon, Japan) with a fluorescence lamp emission / excitation of 510/580 nm, respectively.
Statistical analyzes were performed with Graphpad Prism 8 (Graphpad Software, USA). All data are presented as mean ± SEM. Statistical differences between two groups were analyzed using the t test for unpaired data. If more than two groups were evaluated, the groups were analyzed using one-way analysis of variance (ANOVA) with the Bonferroni test. A value of p *: p <0.05, **: p <0.01, ***: p <0.001 was considered statistically significant.