Journal: International Journal of Molecular Sciences

Article Title: Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

doi: 10.3390/ijms22073653

Figure Lengend Snippet: miR-483-5p predicted binding to mRNAs from ERK1 ( MAPK3 ) and ERK2 ( MAPK1 ). ( A ) Top 8 out of 1818 predicted targets of miR-483-5p from TargetScan 7.2 (based on context++ score) and the overlap with four experimentally-validated targets of miR-483-5p in miRTarBase 7.0. ( B ) Common targets predicted from TargetScan 7.2 for miR-483-5p and the neuroprotective miR-132-3p. ( C ) Table of predicted miRNA affinities for TAU ( MAPT ), ERK1 ( MAPK3 ) and ERK2 ( MAPK1 ) mRNAs in AD literature. LA = Luciferase assay, RT-qPCR = Reverse transcriptase quantitative PCR, IMB = Immunoblotting, N/A = Not available. The superscripted numbers indicate references where these interactions were verified.

Article Snippet: Quantitative PCR was performed according to manufacturer’s protocol using a TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA) with a thermal profile of 95 °C for 20 s; 95 °C for 3 s and 60 °C for 30 s in 40 cycles on a CFX96 Touch Real time PCR (Bio-Rad, Hercules, CA, USA). miR-191-5p was used as reference for miR-483-5p quantification and GAPDH was used as reference for mRNA quantification.

Techniques: Binding Assay, Luciferase, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

doi: 10.3390/ijms22073653

Figure Lengend Snippet: miR-483-5p does not directly bind to TAU mRNA or impact TAU levels. ( A ) Scheme represents the predicted binding site for miR-483-5p in the 3′-UTR of the TAU mRNA. ( B ) Prediction indicates human-specific miR-483-5p binding site in 3′-untranslated region (3′-UTR) of the TAU mRNA among different species. ( C ) Quantitative reporter assay for luciferase activity in HEK293 cells. Reporter constructs carrying a single binding site (CTR, WT, MUT, PM) for miR-483-5p were tested. The miRNA activity on four pmirGLO constructs was measured simultaneously: an empty pmirGLO vector (CTR), a wild-type potential binding site for miR-483-5p (WT), a mutated binding site (MUT), and a site with full complementarity (PM). Interaction scheme of WT and MUT binding site of 3′-UTR of the TAU mRNA with miR-483-5p are shown below. PM (not shown here) is designed to perfectly match the miR-483-5p. Firefly luciferase activity was normalized against renilla luciferase activity. Data are shown as mean ± standard error from four independent experiments. One way ANOVA with post-hoc Tukey HSD was used for statistical analysis. ( D ) RT-qPCR analysis of TAU mRNA levels upon miR-483-5p mimic transfection in SK-N-MC cells. GAPDH was used as a reference. Data are shown as mean ± standard error from three biological replicates; Unpaired Student’s t -test was used for statistical analysis. In all cases, annotations were made on plots only where significant differences ( p < 0.05) were found.

Article Snippet: Quantitative PCR was performed according to manufacturer’s protocol using a TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA) with a thermal profile of 95 °C for 20 s; 95 °C for 3 s and 60 °C for 30 s in 40 cycles on a CFX96 Touch Real time PCR (Bio-Rad, Hercules, CA, USA). miR-191-5p was used as reference for miR-483-5p quantification and GAPDH was used as reference for mRNA quantification.

Techniques: Binding Assay, Reporter Assay, Luciferase, Activity Assay, Construct, Plasmid Preparation, Quantitative RT-PCR, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

doi: 10.3390/ijms22073653

Figure Lengend Snippet: miR-483-5p downregulates ERK1 at both the mRNA and protein level in HEK293 cells. ( A ) Scheme representing the predicted binding site for miR-483-5p in ERK1 and ERK2 3′-UTRs. ( B ) RT-qPCR analysis of ERK1 mRNA levels upon transfection with 10 nM and 50 nM of miR-483-5p mimic. GAPDH was used for normalization. ( C ) Immunoblot quantitative analysis of ERK1 protein levels upon transfection with miR-483-5p mimic at different concentrations. β-actin levels were used for normalization. ( D ) Representative immunoblot for ERK1/2 and pERK1/2 protein levels upon transfection with scramble and miR-483-5p mimic at 100 nM. ( E , F ) Immunoblot densitometry analysis of ERK1/2 and pERK1/2 protein levels upon transfection with scramble and miR-483-5p mimic at 100 nM. Data are shown as mean ± standard error from three independent experiments; Unpaired Student’s t -test was used for statistical analysis.

Article Snippet: Quantitative PCR was performed according to manufacturer’s protocol using a TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA) with a thermal profile of 95 °C for 20 s; 95 °C for 3 s and 60 °C for 30 s in 40 cycles on a CFX96 Touch Real time PCR (Bio-Rad, Hercules, CA, USA). miR-191-5p was used as reference for miR-483-5p quantification and GAPDH was used as reference for mRNA quantification.

Techniques: Binding Assay, Quantitative RT-PCR, Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

doi: 10.3390/ijms22073653

Figure Lengend Snippet: CRISPR/Cas9-mediated knockout of miR-483-5p. ( A ) Scheme of the CRISPR/Cas9-mediated genomic deletion of miR-483-5p using upstream and downstream sgRNAs (sg1 and sg2). Fragment sizes of the wild-type allele (945 bp) and deleted region (212 bp). ( B ) Agarose gel electrophoresis of PCR amplified fragments (using primers shown in A ) from polyclonal cell populations retrieved upon transfection with empty plasmid without sgRNA (Cas9 only) and Cas9 +/sgRNA expressing plasmid (Cas9 + miR-483-5p sgRNA). 733 bp band corresponds to deleted allele. ( C ) Fold change decrease in miR-483-5p levels in CRISPR/Cas9-deleted cells (Cas9 + miR-483-5p sgRNAmiR-483 KO). miR-191-5p was used for normalization. ( D ) Fold change increase in ERK1 mRNA ( MAPK3 ) levels in CRISPR/Cas9-deleted cells. GAPDH was used for normalization. Data represent the mean ± standard deviation from two independent experiments. Statistical analysis was performed using an unpaired Student’s t -test on log 2 (fold change).

Article Snippet: Quantitative PCR was performed according to manufacturer’s protocol using a TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA) with a thermal profile of 95 °C for 20 s; 95 °C for 3 s and 60 °C for 30 s in 40 cycles on a CFX96 Touch Real time PCR (Bio-Rad, Hercules, CA, USA). miR-191-5p was used as reference for miR-483-5p quantification and GAPDH was used as reference for mRNA quantification.

Techniques: CRISPR, Knock-Out, Agarose Gel Electrophoresis, Amplification, Transfection, Plasmid Preparation, Expressing, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

doi: 10.3390/ijms22073653

Figure Lengend Snippet: miR-483-5p mediated downregulation of total and phosphorylated ERK1/2 correlates with reduced levels of phosphorylated TAU in HEK293 cells expressing EGFP-TAU and upon transfection with scramble and miR-483-5p mimics at 100 nM. ( A ) Representative immunoblots of total and phosphorylated levels of TAU, ERK1 and ERK2 under the different experimental conditions. β-actin was used as loading control. ( B ) Quantitative analysis of the immunoblot levels for TAU, ERK1 and ERK2. ( C ) Quantitative analysis of the immunoblot levels for pTAU, pERK1 and pERK2. Data are shown as mean ± standard error of three independent experiments. One way ANOVA with post-hoc Tukey HSD was used and statistically significant differences are shown with p values.

Article Snippet: Quantitative PCR was performed according to manufacturer’s protocol using a TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA) with a thermal profile of 95 °C for 20 s; 95 °C for 3 s and 60 °C for 30 s in 40 cycles on a CFX96 Touch Real time PCR (Bio-Rad, Hercules, CA, USA). miR-191-5p was used as reference for miR-483-5p quantification and GAPDH was used as reference for mRNA quantification.

Techniques: Expressing, Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Candidate Alzheimer’s Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression

doi: 10.3390/ijms22073653

Figure Lengend Snippet: miR-483-5p-mediated downregulation of phosphorylated ERK1/2 is associated with a trend in decreased levels of phosphorylated TAU in SK-N-MC cells. ( A ) Representative immunoblots of total and phosphorylated levels of TAU, ERK1 and ERK2 in untransfected (mock) or transfected SK-N-MC cells with 100 nM miR-483-5p mimic. C q values for miR-483-5p transfected cells are shown and in mock miR-483-5p was undetermined (Und). β-actin was used as loading control. ( B ) Quantitative analysis of the immunoblot levels for TAU, ERK1 and ERK2. ( C ) Quantitative analysis of the immunoblot levels for pTAU, pERK1 and pERK2. Data are shown as mean ± standard error of the mean from four biological replicates; Unpaired Student’s t -test was used to compare groups and we observed no statistically significant differences.

Article Snippet: Quantitative PCR was performed according to manufacturer’s protocol using a TaqMan ® Fast Advanced Master Mix (Applied Biosystems, Bedford, MA, USA) with a thermal profile of 95 °C for 20 s; 95 °C for 3 s and 60 °C for 30 s in 40 cycles on a CFX96 Touch Real time PCR (Bio-Rad, Hercules, CA, USA). miR-191-5p was used as reference for miR-483-5p quantification and GAPDH was used as reference for mRNA quantification.

Techniques: Western Blot, Transfection