control virus  (Addgene inc)

Journal: bioRxiv

Article Title: C1q and immunoglobulins mediate activity-dependent synapse loss in the adult brain

doi: 10.1101/2024.12.18.629085

Figure Lengend Snippet: (A) Schematic of WT experimental paradigm. Created in https://BioRender.com . (B) Representative widefield images of c-Fos labeling at contralateral and ipsilateral MEC of hM3Dq+CNO WT mouse, scale bar = 50 μm. (C) Quantification of c-Fos immunoreactive area between hemispheres in each mouse group. n = 6 EGFP+Veh mice (contra = 45 ROIs, ipsi = 45 ROIs), n = 5 EGFP+CNO mice (contra = 45 ROIs, ipsi = 45 ROIs), n = 8 hM3Dq+Veh mice (contra = 63 ROIs, ipsi = 63 ROIs) and n = 7 hM3Dq+CNO mice (contra = 66 ROIs, ipsi = 66 ROIs), 2-3 brain sections per hemisphere, 3 ROIs analysed per section. p-values from Bonferroni post-hoc test after 2-way mixed ANOVA with group as between- and hemisphere as within-subject factor (interaction p = 0.008 after log 10 transformation). (D) Representative Airyscan images of C1q puncta at contralateral and ipsilateral DGML of hM3Dq+CNO WT mouse, scale bar = 2 μm. (E) Quantification of C1q puncta density between hemispheres in each mouse group. n = 6 EGFP+Veh mice (contra = 33 ROIs, ipsi = 33 ROIs), n = 6 EGFP+CNO mice (contra = 48 ROIs, ipsi = 48 ROIs), n = 9 hM3Dq+Veh mice (contra = 48 ROIs, ipsi = 48 ROIs) and n = 7 hM3Dq+CNO mice (contra = 54 ROIs, ipsi = 54 ROIs), 1-3 brain sections per hemisphere, 3 ROIs analysed per section. p-values from Bonferroni post-hoc test after 2-way mixed ANOVA with group as between- and hemisphere as within-subject factor (interaction p = 0.004). (F) Schematic of J20 experimental paradigm. Created in https://BioRender.com . (G) Representative widefield images of pPDH labeling at contralateral and ipsilateral MEC of hM4Di+CNO J20 mouse, scale bar = 50 μm. (H) Quantification of pPDH immunoreactive area between hemispheres in each mouse group. n = 3 EGFP+Veh mice (contra = 18 ROIs, ipsi = 18 ROIs), n = 3 EGFP+CNO mice (contra = 25 ROIs, ipsi = 26 ROIs), n = 3 hM4Di+Veh mice (contra = 22 ROIs, ipsi = 27 ROIs) and n = 6 hM4Di+CNO mice (contra = 54 ROIs, ipsi = 52 ROIs), 2-3 brain sections per hemisphere, 3 ROIs analysed per section. p-values from Bonferroni post-hoc test after 2-way mixed ANOVA with group as between- and hemisphere as within-subject factor (interaction p = 0.020). (I) Representative Airyscan images of C1q puncta at contralateral and ipsilateral DGML of hM4Di+CNO J20 mouse, scale bar = 2 μm. (J) Quantification of C1q puncta density between hemispheres in hM4Di+CNO J20 mouse. n = 3 EGFP+Veh mice (contra = 18 ROIs, ipsi = 18 ROIs), n = 3 EGFP+CNO mice (contra = 18 ROIs, ipsi = 18 ROIs), n = 3 hM4Di+Veh mice (contra = 18 ROIs, ipsi = 18 ROIs) and n = 6 hM4Di+CNO mice (contra = 33 ROIs, ipsi = 33 ROIs), 2 brain sections per hemisphere, 3 ROIs analysed per section. p-values from Bonferroni post-hoc test after 2-way mixed ANOVA with group as between- and hemisphere as within-subject factor (interaction p = 0.025). Throughout, square points represent males and circular points represent females, linked points indicate data from the same mouse brain, where points are linked 1 point = 1 hemisphere average = average of brain sections. Data shown as mean ± SEM. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Pulled long-shaft borosilicate pipettes (3.5” tubes, #3-000-203-G/X, Drummond Scientific) were backfilled with mineral oil (330779, Sigma-Aldrich) before being loaded with either DREADD virus (AAV8- CaMKIIa-HA-hM3Dq-IRES-mCitrine, #50466-AAV8, Addgene; titre = 1.6×10 13 gc/mL or AAV8-CaMKIIa-HA-hM4Di-IRES-mCitrine, #50467-AAV8, Addgene; titre = 1.6×10 13 gc/mL) or control EGFP virus (AAV8-CaMKIIa-EGFP, #50469-AAV8, Addgene; titre = 2.3×10 13 gc/mL).

Techniques: Labeling, Transformation Assay

Journal: bioRxiv

Article Title: C1q and immunoglobulins mediate activity-dependent synapse loss in the adult brain

doi: 10.1101/2024.12.18.629085

Figure Lengend Snippet: (A) Representative Airyscan images of VGLUT1 puncta at contralateral and ipsilateral DGML of hM3Dq+CNO WT mouse, scale bar = 2 μm. (B) Quantification of VGLUT1 puncta density between hemispheres in each mouse group. n = 6 EGFP+Veh mice (contra = 33 ROIs, ipsi = 33 ROIs), n = 6 EGFP+CNO mice (contra = 48 ROIs, ipsi = 48 ROIs), n = 9 hM3Dq+Veh mice (contra = 36 ROIs, ipsi = 36 ROIs) and n = 7 hM3Dq+CNO mice (contra = 48 ROIs, ipsi = 48 ROIs), 1-3 brain sections per hemisphere, 3 ROIs analysed per section. p-values from Bonferroni post-hoc test after 2-way mixed ANOVA with group as between- and hemisphere as within-subject factor (interaction p = 0.019). (C) Representative Airyscan images of VGLUT2 puncta at contralateral and ipsilateral DGML of hM3Dq+CNO WT mouse, scale bar = 2 μm. (D) Quantification of VGLUT2 puncta density between hemispheres in each mouse group. Data are mean ± SEM, n = 5 EGFP+Veh mice (contra = 36 ROIs, ipsi = 36 ROIs), n = 5 EGFP+CNO mice (contra = 42 ROIs, ipsi = 42 ROIs), n = 5 hM3Dq+Veh mice (contra = 36 ROIs, ipsi = 36 ROIs) and n = 7 hM3Dq+CNO mice (contra = 39 ROIs, ipsi = 39 ROIs), 2-4 brain sections per hemisphere, 2-3 ROIs analysed per section. p-values from Bonferroni post-hoc test after 2-way mixed ANOVA with group as between- and hemisphere as within-subject factor (interaction p = 0.015). (E) Representative Airyscan images of Homer1 puncta at contralateral and ipsilateral DGML of hM4Di+CNO J20 mouse, scale bar = 2 μm. (F) Quantification of Homer1 puncta density between hemispheres in hM4Di+CNO J20 mouse. n = 3 EGFP+Veh mice (contra = 17 ROIs, ipsi = 17 ROIs), n = 3 EGFP+CNO mice (contra = 18 ROIs, ipsi = 18 ROIs), n = 3 hM4Di+Veh mice (contra = 15 ROIs, ipsi = 18 ROIs) and n = 6 hM4Di+CNO mice (contra = 40 ROIs, ipsi = 40 ROIs), 2 brain sections per hemisphere, 2-3 ROIs analysed per section. p-values from Bonferroni post-hoc test after 2-way mixed ANOVA with group as between- and hemisphere as within-subject factor (interaction p = 0.014). (G) Representative images of C1q and VGLUT2 colocalization at the contralateral and ipsilateral DGML, colocalized puncta indicated by yellow arrowheads, scale bar = 2 μm. (H) Quantification of percentage total VGLUT2 puncta colocalizing with C1q at contralateral and ipsilateral DGML of hM3Dq+CNO WT mouse. n = 5 mice (contra = 28 ROIs, ipsi = 27 ROIs), 1 brain section per hemisphere, 3 ROIs analysed per section. p-value from paired t test. (I) Schematic of C1qa KO experimental paradigm. Created in https://BioRender.com . (J) Representative widefield images of c-Fos labeling at contralateral and ipsilateral MEC of hM3Dq+CNO C1qa KO mouse, scale bar = 50 μm. (K) Quantification of c-Fos immunoreactive area between hemispheres in hM3Dq+CNO C1qa KO mouse. n = 5 C1qa KO mice (contra = 42 ROIs, ipsi = 42 ROIs), 2-3 brain sections per hemisphere, 3 ROIs analysed per section. p-values from paired t test. (L) Representative Airyscan images of VGLUT2 puncta at contralateral and ipsilateral DGML of hM3Dq+CNO C1qa KO mouse, scale bar = 2 μm. (M) Quantification of VGLUT2 puncta density between hemispheres in hM3Dq+CNO C1qa KO mouse. n = 5 C1qa KO mice (contra = 39 ROIs, ipsi = 39 ROIs), 2-5 brain sections per hemisphere, 3 ROIs analysed per section. p-values from paired t test. Throughout, square points represent males and circular points represent females, linked points indicate data from the same mouse brain, where points are linked 1 point = 1 hemisphere average = average of brain sections. Data shown as mean ± SEM. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Pulled long-shaft borosilicate pipettes (3.5” tubes, #3-000-203-G/X, Drummond Scientific) were backfilled with mineral oil (330779, Sigma-Aldrich) before being loaded with either DREADD virus (AAV8- CaMKIIa-HA-hM3Dq-IRES-mCitrine, #50466-AAV8, Addgene; titre = 1.6×10 13 gc/mL or AAV8-CaMKIIa-HA-hM4Di-IRES-mCitrine, #50467-AAV8, Addgene; titre = 1.6×10 13 gc/mL) or control EGFP virus (AAV8-CaMKIIa-EGFP, #50469-AAV8, Addgene; titre = 2.3×10 13 gc/mL).

Techniques: Labeling

Journal: Nature Communications

Article Title: The mitochondrial mRNA-stabilizing protein SLIRP regulates skeletal muscle mitochondrial structure and respiration by exercise-recoverable mechanisms

doi: 10.1038/s41467-024-54183-4

Figure Lengend Snippet: A SLIRP protein content across different wild-type tissues ( n = 3–4, female C57BL/6 J). B SLIRP and LRPPRC protein content in tibialis anterior muscle of Slirp knockout (KO) and littermate wildtype (WT) mice; WT, n = 26; Slirp KO = 35. C SLIRP and LRPPRC protein content in tibialis anterior muscle of WT mice injected with recombinant adeno‐associated virus serotype 6 encoding SLIRP (rAAV6:SLIRP) or rAAV6:eGFP in the contralateral leg as control ( n = 6). D Confocal microscopy of mitochondrial network structure in flexor digitorum brevis muscle fibers of Slirp KO and WT mice ( n = 4, 7–10 fibers per mouse). Corresponding fragmentation index are presented as super plots ; small symbols, each fiber; large symbols, mean of fibers per mouse and color- and symbol-coded for each sex ( ○ male, ◇ female). E TEM images of Slirp KO and WT gastrocnemius muscle ( n = 4; 7–9 fibers per sample). F , G Quantification of percentage of damaged intramyofibrillar (IMF) mitochondria within total mitochondria and relative volume of mitochondria. Small symbols, damaged or total mitochondria per fiber; large identical symbols, average of fibers per biological replicate (female Slirp KO and WT gastrocnemius muscle, n = 4/group). H Mitochondrial respiration in of Slirp KO and WT gastrocnemius muscle (male/female: WT, n = 4/4; Slirp KO, n = 5/5); ○ (blue) male, ◇ (red) female. I Fatty acid oxidation in isolated Slirp KO and WT soleus muscle at rest and in response to contraction (male/female: WT, n = 3/4; Slirp KO, n = 4/8); ○ (blue) male, ◇ (red) female. J RT-qPCR analysis of mitochondrial transcript levels and corresponding immunoblots in gastrocnemius of male Slirp KO and WT mice (mRNA: WT, n = 6; Slirp KO, n = 6; protein: WT, n = 6; Slirp KO, n = 7). K qPCR analysis of mtDNA levels in male Slirp KO and WT mice (WT, n = 6; Slirp KO, n = 7). L , M Climbing assay and life span of control, SLIRP1 and SLIRP2 knockdown flies ( n = 3–9, 10 flies per sample for climbing assay; n = 10, 10 flies per vial for lifespan assay). Data are shown as mean ± SEM, including individual values, where applicable. Geno, main effect of genotype; substrate, main effect of substrate addition; Substrate X Geno, interaction between genotype and substrate; Contraction, main effect of contraction. * p < 0.05, ** p < 0.01, *** p < 0.001, as per Two-tailed Mann Whitney test ( D , F , G ) on average values, Two-way RM ANOVA with Šídák’s multiple comparisons test ( H , I ), Two-tailed unpaired Student´s t test ( J , K ), ordinary one-way ANOVA with Dunnett’s multiple comparisons test ( L ), Log-rank (Mantel-Cox) test ( M ). Source data are provided as a Source Data file.

Article Snippet: The AAV6 vector for SLIRP overexpression and ultra-purified eGFP control AAV6 virus were manufactured by VectorBuilder Inc. (Shenandoah, Texas, USA; AAV6SP(VB190219-1010jvy)-C).

Techniques: Knock-Out, Injection, Recombinant, Virus, Control, Confocal Microscopy, Isolation, Quantitative RT-PCR, Western Blot, Climbing Assay, Knockdown, Two Tailed Test, MANN-WHITNEY