Journal: The Journal of Biological Chemistry

Article Title: Cytotoxic tau released from lung microvascular endothelial cells upon infection with Pseudomonas aeruginosa promotes neuronal tauopathy

doi: 10.1016/j.jbc.2021.101482

Figure Lengend Snippet: Endothelium-derived tau nucleates neuronal tau. HEK293 FRET reporter cells that stably express the RD-CFP and RD-YFP were used. A , summary of normalized FRET efficiency in cells transfected with the indicated amount of tau P301L plasmids. Each group is plotted as mean ± S.E.M. ∗ p = 0.030 and 0.0048 versus vehicle control for 1 μg and 1.5 μg, respectively (ANOVA, followed by Dunnett’s test). B , representative FRET signal micrographs after plasmid transfection. The dark areas indicate a high FRET signal, whereas lighter areas indicate a low signal. Blue is the background and indicates no cell or FRET signal. Vehicle was OPTI-MEM without DNA plasmid. C , summary of normalized FRET efficiency in cells transfected with endothelium-derived tau. Endothelium-derived tau was prepared from endothelial media after the cells were exposed to the indicated Pseudomonas aeruginosa strains. Each group is plotted as mean ± S.E.M. Asterisk denotes statistical significance versus vehicle control (ANOVA, followed by Dunnett’s test; PA103 p = 0.0013; ExoY p < 0.001; and PA808 p = 0.043). D , representative FRET signal micrographs after transfection. Vehicle HBSS was collected from endothelial medium, but the cells were not infected with P. aeruginosa . The numbers of experimentation (n) are indicated in brackets . Vertical colorimetry shows FRET signal strength; blue = background. The scale bars represent 100 μm. HBSS, Hank’s balanced salt solution.

Article Snippet: Cells grown at ∼70% confluence on 12-well plate containing coverslips were either incubated with 8-pCPT-cAMP (15 μM), 8-pCPT-cGMP (15 μM), okadaic acid (4 nM), or vehicle (DMSO), or were transfected with endothelium-derived tau seeds prepared from the control or bacteria-infected cell supernatant using the Xfect protein transfection reagent (Clontech Lab) according to manufacturer’s instruction.

Techniques: Derivative Assay, Stable Transfection, Transfection, Plasmid Preparation, Infection, Colorimetric Assay

Journal: The Journal of Biological Chemistry

Article Title: Cytotoxic tau released from lung microvascular endothelial cells upon infection with Pseudomonas aeruginosa promotes neuronal tauopathy

doi: 10.1016/j.jbc.2021.101482

Figure Lengend Snippet: Endothelium-derived tau nucleates full-length neuronal tau. A , summary of normalized BiFC signal after the indicated treatments. Left , positive control studies included treatment using a membrane permeable form of cAMP or cGMP and okadaic acid (OA). BiFC signal was normalized to DMSO vehicle control. Statistical comparison was performed using ANOVA and Dunnett’s post hoc tests; the asterisks denote statistical significance versus DMSO (cAMP p = 0.0012, n = 7; cGMP p = 0.016, n = 7; and OA p = 0.018, n = 5). ( right ) Endothelium-derived tau transfection studies were normalized to the vehicle (Veh, water). HBSS cell medium from endothelial cells was not exposed to Pseudomonas aeruginosa . Statistical comparison was performed using ANOVA and Dunnett’s post hoc tests; the asterisks denote significance versus Veh (ExoY p = 0.0016; PA103 p = 0.0078; n = 5). B , representative BiFC fluorescence micrographs. Yellow fluorescence emitted from the Venus protein indicates neuronal tau aggregation. Vertical colorimetry shows BiFC signal strength. The scale bar represents 100 μm. C , summary of BiFC signal after the indicated treatments, normalized to HBSS control. ExoY-infected endothelial supernatant (ExoY) was used as a positive control. ExoY immunodepleted with an IgG antibody (IgG depleted) or the T22 antibody (T22 depleted) and the eluates from the respective antibodies were applied to the BiFC cells. Statistical comparison was performed using ANOVA and Dunnett’s post hoc tests; the asterisks denote significance versus HBSS (ExoY p = 0.030; T22 eluate p = 0.029, n = 4). IgG depleted control showed a nonsignificant trend of elevated BiFC signal ( p = 0.054, n = 4). D , representative BiFC fluorescence micrographs. The strength of yellow fluorescence indicates neuronal tau aggregation. Vertical colorimetry shows BiFC signal strength. The scale bar represents 100 μm. BiFC, biomolecular fluorescence complementation; HBSS, Hank’s balanced salt solution.

Article Snippet: Cells grown at ∼70% confluence on 12-well plate containing coverslips were either incubated with 8-pCPT-cAMP (15 μM), 8-pCPT-cGMP (15 μM), okadaic acid (4 nM), or vehicle (DMSO), or were transfected with endothelium-derived tau seeds prepared from the control or bacteria-infected cell supernatant using the Xfect protein transfection reagent (Clontech Lab) according to manufacturer’s instruction.

Techniques: Derivative Assay, Positive Control, Transfection, Fluorescence, Colorimetric Assay, Infection

Journal: eLife

Article Title: Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract

doi: 10.7554/eLife.18065

Figure Lengend Snippet: ( A ) General schematic of trans N17 addition experiments to ∆N aggregation reaction to determine how N17 impacts the balance between stable, ∆N oligomers and amyloid fibrillar aggregates. ( B ) 12.5x excess of N17 peptide was added when ∆N aggregation was initiated. Then 60 hr after initiation of aggregation, the fibers were analyzed by cryo-EM. N17-seeded ∆N aggregates become more bundled and more resemble the morphology of Ex1 aggregates . ( C ) 12.5x or 2.5x excess of N17 peptide was added in trans when ∆N aggregation was initiated. The impact of N17 peptide on ∆N oligomers was analyzed by 0.1% SDS-AGE gels and immunoprobed for the C-terminal S-tag. Trans addition of N17 promotes disappearance of these stable ∆N oligomers from the AGE gel. ( D ) 12.5x excess of N17 peptide was added in trans 7 hr after initiation of ∆N aggregation, allowing for the formation of ∆N oligomers before N17 addition. Impact of N17 peptide on ∆N oligomers was analyzed by 0.1% SDS-AGE gels and immunoprobed for the C-terminal S-tag. ( E ) Schematic of in vivo N17 addition experiment: ST14a striatal-derived neurons were transfected with the C-terminally GFP tagged mHtt-Ex1 (mHtt-Ex1-GFP). 10 hr after transfection, when expressed mHtt protein is still completely soluble, cells are protein transfected using the Xfect kit (Clontech) with N17 or a mutant N17 peptide (NA) that inhibits aggregation. After 10 hr, resulting cells containing GFP fluorescent puncta are counted. ( F ) Fluorescence microscopy of ST14a striatal neurons transfected with mHtt-Ex1-GFP and N17 variant peptides (left) and quantification of cells containing puncta (right). Data are mean ± SEM of three independent experiments with at least 200 cells counted in each condition. Scale bar is 20 µm. *p<0.05. DOI: http://dx.doi.org/10.7554/eLife.18065.013

Article Snippet: 10 hr after transfection, when expressed mHtt protein is still completely soluble, cells are protein transfected using the Xfect kit (Clontech) with N17 or a mutant N17 peptide (NA) that inhibits aggregation.

Techniques: Cryo-EM Sample Prep, In Vivo, Derivative Assay, Transfection, Mutagenesis, Fluorescence, Microscopy, Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Selective Activation of CNS and Reference PPARGC1A Promoters Is Associated with Distinct Gene Programs Relevant for Neurodegenerative Diseases

doi: 10.3390/ijms22073296

Figure Lengend Snippet: The CNS and reference gene (RG) promoters are selectively activated by specific single guide RNAs (sgRNAs) designed to target them. ( a ) Schematic representation of the PPARGC1A locus showing the CNS and RG promoters (top), locations of sgRNAs used for transfections; CNS-specific exons B1 , B4 and B5 in color; the structure of the RG is displayed on the right; CNS-specific transcripts and RG transcripts are shown below; * pink or red lines refer to alternatively spliced transcripts encoding stop codons in exon 7A or in an extension of exon 3, respectively. ( b , c ) Selective effects of individual sgRNAs targeting the RG or CNS promoters on transcription initiation; individual sgRNAs or their mixtures were transfected into clonal SH-SY5Y cells expressing CRISPR-associated deactivated (dCas9) protein fused to the tripartite transcriptional activator VPR and levels of E1E2 and B1B4 transcripts selective for RG or CNS promoter activation were measured by qRT-PCR 36 h after transfection. Log-fold levels are expressed relative to transcript levels of the clonal cells transfected with scrambled sgRNA. Interactions of all sgRNA mixtures shown were significant ( p < 0.001) when the use of three, two, or one sgRNA was compared. ( d ) Effects of sgRNA transfections on CNS-specific and RG transcript levels and levels of transcripts encoding truncated isoforms and initiated at either promoter.

Article Snippet: For the stable integration of dCas9-VPR into the genome of cell lines, pLenti-EF1a-dCas9-SAM plasmids (ABM) along with the trans-complementation plasmids pMD2.G expressing VSV-G envelope and phCMV-8.91 expressing GAG/POL were transfected into HEK293T cells using jetPEI or Xfect transfection reagents (Polyplus Transfection T or Clontech, Mountain View, CA, USA) in serum and antibiotic free medium.

Techniques: Transfection, Expressing, CRISPR, Activation Assay, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Selective Activation of CNS and Reference PPARGC1A Promoters Is Associated with Distinct Gene Programs Relevant for Neurodegenerative Diseases

doi: 10.3390/ijms22073296

Figure Lengend Snippet: RNA sequencing confirms the predicted structure of transcripts generated by activation of the RG or CNS promoters and reveals differentially expressed genes (DEGs) for either promoter activation. ( a ) Sashimi plots of PPARGC1A transcripts generated by transfection of clonal SH-SY5Y cells expressing dCas9-VPR with sgRNAs activating the RG or the CNS promoters or with scrambled sgRNAs, each merged from three biological replicates; read densities across exons are normalized to obtain comparable measures of expression above the x -axis and normalized single-end junction reads are shown as arcs below the x -axis; structure of visualized exons is shown at the bottom. ( b , c ) Volcano plots of DEGs in cells with RG or CNS activated promoters, respectively, in comparison to cells transfected with scrambled sgRNAs. The top 20 most DEGS are highlighted.

Article Snippet: For the stable integration of dCas9-VPR into the genome of cell lines, pLenti-EF1a-dCas9-SAM plasmids (ABM) along with the trans-complementation plasmids pMD2.G expressing VSV-G envelope and phCMV-8.91 expressing GAG/POL were transfected into HEK293T cells using jetPEI or Xfect transfection reagents (Polyplus Transfection T or Clontech, Mountain View, CA, USA) in serum and antibiotic free medium.

Techniques: RNA Sequencing Assay, Generated, Activation Assay, Transfection, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Selective Activation of CNS and Reference PPARGC1A Promoters Is Associated with Distinct Gene Programs Relevant for Neurodegenerative Diseases

doi: 10.3390/ijms22073296

Figure Lengend Snippet: Differentially expressed genes resulting from activation of the RG or CNS promoters reveal promoter selectivity with partial overlap. ( a ) Venn diagram of differentially expressed genes (DEGS) produced by transfection of clonal SH-SY5Y cells expressing dCas9-VPR with sgRNAs activating the RG or the CNS promoters are compared with cells transfected with scrambled sgRNAs. ( b , c ) Top canonical signaling pathways for DEGs after RG or CNS promoter activation, respectively. ( d ) Top nervous system signaling pathways for DEGs after CNS promoter activation; red lines refer to adjusted p -values of 0.05; z -scores > 2.0 or <−2.0 are significant and indicate the direction for the expected entity; n.a, not available; B-H, Bernini-Hochberg adjusted p -values for multiple testing.

Article Snippet: For the stable integration of dCas9-VPR into the genome of cell lines, pLenti-EF1a-dCas9-SAM plasmids (ABM) along with the trans-complementation plasmids pMD2.G expressing VSV-G envelope and phCMV-8.91 expressing GAG/POL were transfected into HEK293T cells using jetPEI or Xfect transfection reagents (Polyplus Transfection T or Clontech, Mountain View, CA, USA) in serum and antibiotic free medium.

Techniques: Activation Assay, Produced, Transfection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Selective Activation of CNS and Reference PPARGC1A Promoters Is Associated with Distinct Gene Programs Relevant for Neurodegenerative Diseases

doi: 10.3390/ijms22073296

Figure Lengend Snippet: CNS-specific and reference PGC-1α proteins affect exon usage via distinct and similar mechanisms. ( a ) Venn diagram showing the number of genes (left) or exons (right) alternatively spliced after activation of the RG or the CNS promoters in comparison to cells transfected with scrambled sgRNAs. Exon usage of SQSTM1 after RG and CNS promoter activation reveals similar changes to transfections of scrambled sgRNAs (control) and additional differences between each other; comparison between ( b ) CNS and RG promoter activation, ( c ) RG promoter activation and control and ( d ) CNS promoter activation and control; different exon usage between pairwise comparisons is indicated by purple boxes representing the affected exon bins; significant differences ( p < 0.0001) are highlighted by stars; * and **, regions different between RG or CNS vs. scrambled; ***, region, different between CNS vs. RG or scrambled. The black lines refer to differences in any comparison.

Article Snippet: For the stable integration of dCas9-VPR into the genome of cell lines, pLenti-EF1a-dCas9-SAM plasmids (ABM) along with the trans-complementation plasmids pMD2.G expressing VSV-G envelope and phCMV-8.91 expressing GAG/POL were transfected into HEK293T cells using jetPEI or Xfect transfection reagents (Polyplus Transfection T or Clontech, Mountain View, CA, USA) in serum and antibiotic free medium.

Techniques: Activation Assay, Comparison, Transfection