Journal: Human Molecular Genetics

Article Title: Viral vector-mediated SLC9A6 gene replacement reduces cerebellar motor and molecular abnormalities in the shaker rat model of Christianson syndrome

doi: 10.1093/hmg/ddag021

Figure Lengend Snippet: AAV9-CAG-hSLC9A6 AAV increased cerebellar NHE6 and CALB1 abundances determined by western blotting and decreased motor ataxia. (A-D) Mirroring findings with PHP.eB-L7-Slc9a6-GFP AAV, administration of AAV9-CAG-hSLC9A6 AAV at ~ 5 weeks of age minimally increased NHE6 expression (A, C), but this minimal increase led to proportionally greater increases in CALB1 expression (B, D). (E) AAV9-CAG-hSLC9A6 AAV administration at 5 weeks of age significantly improved motor ataxia at 14 weeks of age compared to 7 untreated shaker rats (5 administered a control AAV and 2 uninjected). (F) Rats treated with AAV9-CAG-hSLC9A6 trended towards a decrease in tremor at 14 weeks of age, albeit with substantial variability. Note that each data point is an individual rat, and all n-values for statistics are calculated as number of rats, not technical replicates.

Article Snippet: To generate hSLC9A6 AAV expression plasmid, pAAV-CAG-GFP plasmid (Addgene plasmid #37825, gifted from Edward Boyden) was modified by deleting GFP with BamHI and EcoRI restriction enzymes.

Techniques: Western Blot, Expressing, Control

Journal: Human Molecular Genetics

Article Title: Viral vector-mediated SLC9A6 gene replacement reduces cerebellar motor and molecular abnormalities in the shaker rat model of Christianson syndrome

doi: 10.1093/hmg/ddag021

Figure Lengend Snippet: Following AAV9-CAG-hSLC9A6 AAV administration, ataxia, NHE6, and CALB1, are interrelated, but NHE6 and CALB1 are not correlated with tremor at 14 weeks of age. (A) NHE6 abundance following viral administration of AAV9-CAG-hSLC9A6 AAV positively correlates with CALB1 expression, suggesting a rescue of Purkinje cells. (B) NHE6 abundance is likewise negatively correlated with gait ataxia. (C) CALB1 is highly negatively correlated with gait ataxia. (D-E) While NHE6 and CALB1 abundance were correlated with ataxia and ataxia is correlated with tremor, NHE6 and CALB1 are not significantly correlated with tremor. Grey shaded regions represents 95% confidence intervals.

Article Snippet: To generate hSLC9A6 AAV expression plasmid, pAAV-CAG-GFP plasmid (Addgene plasmid #37825, gifted from Edward Boyden) was modified by deleting GFP with BamHI and EcoRI restriction enzymes.

Techniques: Expressing

Journal: Human Molecular Genetics

Article Title: Viral vector-mediated SLC9A6 gene replacement reduces cerebellar motor and molecular abnormalities in the shaker rat model of Christianson syndrome

doi: 10.1093/hmg/ddag021

Figure Lengend Snippet: (A) In the context of AAV9-CAG-hSLC9A6 AAV experiments, ataxia is strongly correlated with tremor at 14 weeks. (B-C) In the context of PHP.eB-Slc9a6-GFP AAV experiments, the correlation between peak tremor and late-stage ataxia is stronger than the correlation between tremor and ataxia at any given timepoint. (B) Ataxia and tremor are initially strongly related following symptom onset, but this correlation significantly decreases over time. (C) Peak tremor occurred prior to 20 weeks in all but 2 animals; however, the correlation between peak ataxia and late-stage ataxia (21–25 weeks) was higher than the correlation between tremor and ataxia at any individual timepoint. (D) Endpoint NHE6 expression and ataxia were negatively correlated at all timepoints. This negative correlation became stronger over time. (E) NHE6 and tremor were negatively correlated at all timepoints; however, the correlation between NHE6 and tremor did not strengthen over time. Grey shaded regions represents 95% confidence intervals.

Article Snippet: To generate hSLC9A6 AAV expression plasmid, pAAV-CAG-GFP plasmid (Addgene plasmid #37825, gifted from Edward Boyden) was modified by deleting GFP with BamHI and EcoRI restriction enzymes.

Techniques: Expressing

Journal: bioRxiv

Article Title: Regulation of Nuclear Transcription by Mitochondrial RNA

doi: 10.1101/2022.12.10.519922

Figure Lengend Snippet: ( A-J ) ECs were transfected with Scr or LNA-S and exposed to HT for 72 hrs in biological replicates and then subjected to scRNA and snRNA-seq. UMAP embedding colored by cells (scRNA) or nuclei (snRNA) in ( A ) or by unsupervised clustering ( B ). ( C ) Fraction of cells (scRNA) or nuclei (snRNA) in each cluster from (B). ( D, E ) GO terms enriched by Snc-KD in snRNA-seq ( D ) or scRNA-seq ( E ). ( F ) Venn diagrams showing the number of common down or up-regulated DEGs between sc- and snRNA-seq. ( G ) Venn diagram showing the number of DEGs commonly down-regulated by Snc-KD in three RNA-seq datasets. Top ranked DEGs involved in innate immune and inflammatory response among the 43 intersecting genes are shown. ( H, I ) Correlation plot between Snc-KD-caused fold change (FC) in sn- and scRNA-seq of the commonly detected genes excluding the 43 intersecting genes (H) or the 43 intersecting genes ( I ). SCC are indicated. ( J ) Nascent RNA levels of ICAM1 and VCAM1 were quantified by RT-qPCR in the same batches of ECs used for sc- and snRNA-seq. ( K , L ) ECs were infected with control AAV (Ctrl) or AAV-driven SncmtRNA overexpression (Snc-OE) for 48 hrs. ( K ) RT-qPCR of respective transcripts. ( L ) Enriched pathways (GO terms) by Snc-OE identified from bulk RNA-seq. ( M ) Quantification of the activity of luciferase reporter driven by promoter containing IRF3/7 binding sites upon 293T cells infected by AAV (Ctrl) or Snc-OE.

Article Snippet: The control AAV vector, pAAV-CAG-tdTomato (Plasmid #59462) was a gift from Edward Boyden and obtained from Addgene (Addgene plasmid # 59462).

Techniques: Transfection, RNA Sequencing, Quantitative RT-PCR, Infection, Control, Over Expression, Activity Assay, Luciferase, Binding Assay

Journal: bioRxiv

Article Title: Repetitive sensory stimulation potentiates and recruits sensory-evoked cortical population activity

doi: 10.1101/2024.08.06.605968

Figure Lengend Snippet: (A) Left, examples of averaged baseline & stimulus-related raw iOS images, evoked by one train of whisker deflections. Right, example barrel map overlayed over a brightfield image of the blood vessels. Green dots represent the location of GCaMP6s-expressing cells in the C2 barrel column. (B) Average 2PLSM image of GCaMP6s-expressing neurons. (C) Experimental design: the PW, which corresponds to the barrel column containing the GCaMP6s-expressing cells, is always used to read out the sensory stimulus-evoked response. The PW for PRWS (blue) or a control far-away whisker (CW) for CRWS (orange) is stimulated during rhythmic whisker stimulation (RWS, 8Hz, 10min). (D) Experimental protocol: the PW is stimulated at 0.1 Hz for 10 min pre- and post-RWS. RWS (8 Hz, 10 min) is performed on either the PW (PRWS, blue) or a far-away CW (CRWS, orange). Whisker movement index during the stimulus protocol can be found in - . (E & F) Left, example trace of the GCaMP6s fluorescence, in response to PW stimulation (0.1 Hz, 10 min) pre- and post-PRWS ( E ) or CRWS (F) . The signals in E are from the cell circled in B . Right, the PW-evoked response strength (RS, amplitude X whisker-evoked signal probability, (ΔF/F 0 )/Nstim) pre- and post-PRWS ( E , n=1099 cells, **P=0.002, N=11 mice, P=0.5, full descriptive statistics can be found in Table 1-1) or CRWS ( F , n=829 cells, P=0.4; N=11 mice, P=0.6). Grey lines, paired responses. Violin plots depict median (solid) and quartiles (dotted) bars. Squares, the mean over cells (±SEM). Circles, the mean over mice (±SEM). (G) Pre- versus post-RWS RS ((ΔF/F 0 )/Nstim) with the simple linear regression for PRWS (blue, n=1099 cells) and CRWS (orange, n=829 cells). Comparing slopes (PRWS=0.62±0.014, CRWS=0.85±0.019, F=92.9, DFn=1, DFd=1924, ****P<0.0001). (H) Frequency distribution of the pre-RWS RS for PRWS & CRWS, bin size 0.01(ΔF/F 0 )/Nstim. High responders (resp) were identified as outliers (dots above, Iterative Grubb’s outlier test, α=0.0001). (I & J) Violin plot of the RS pre- and post-PRWS and CRWS, for low & moderate responders (I , PRWS, n=1058 cells, ****P<0.0001; N=11 mice, P=0.006; CRWS, n=792 cells, P=0.3; N=11 mice, P=0.7), and for high responders ( J , PRWS, paired t-test, n=41 cells, ***P=0.0008; N=11 mice, P=0.04; CRWS, n=37 cells, ****P<0.0001; N=11 mice, P=0.0001). (K) The pre-RWS/post-RWS ratio (in %) for PRWS and CRWS low & moderate, and high responders (mixed effects model, N=11 mice, P=0.0004; multiple comparisons: PRWS low & moderate vs high, ***P=0.0004; CRWS low & moderate vs high, P=0.1; Low & moderate PRWS vs CRWS, *P=0.018; High PRWS vs CRWS, P=0.7).

Article Snippet: AAV2-CAG-GCaMP6s-WPRE-SV40 (U Penn Vector Core, RRID: Addgene_100844, 100 nl) or AAV1-hSyn-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA (addgene, RRID: Addgene_50942; 100 nl) was delivered to L2/3 of the right barrel cortex at the approximate location of the C2 barrel column (1.4mm posterior, 3.5mm lateral from bregma, 300mm below the pia) ( ).

Techniques: Whisker Assay, Expressing, Control, Fluorescence

Journal: bioRxiv

Article Title: Repetitive sensory stimulation potentiates and recruits sensory-evoked cortical population activity

doi: 10.1101/2024.08.06.605968

Figure Lengend Snippet: (A) Left, example trace of GCaMP6s fluorescence from a neuron showing persistent PW-evoked responses (0.1 Hz, 10 min) pre- and post-PRWS. Right, violin and pairwise representation of pre- and post-PRWS (n=465 cells, ****P<0.0001; N=11 mice, P=0.003) or CRWS (Paired t test, n=279 cells, P=0.06; N=11 mice, P=0.2). (B&C) Left, example trace of GCaMP6s fluorescence from neurons of which responses were recruited (B) or suppressed (C) post-PRWS. (B) Right, the mean PW-evoked response strength (RS) of recruited neurons, post-PRWS (n=307 cells) & CRWS (Unpaired t test, n=131 cells, P=0.1; N=11 mice, P=0.5). (C) Right, the mean response strength of suppressed neurons pre-PRWS (n=205 cells) & CRWS (Unpaired t test, n=258 cells, ****P<0.0001; N=11 mice P=0.037). (D) Pie charts with the percentages (%) of persistent (grey), recruited (red), suppressed (blue), no response (white), and high (pink) responders for PRWS (n=1099 cells) & CRWS (n=829 cells, Chi-square=19.8, DF=4, ***P<0.0001).

Article Snippet: AAV2-CAG-GCaMP6s-WPRE-SV40 (U Penn Vector Core, RRID: Addgene_100844, 100 nl) or AAV1-hSyn-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA (addgene, RRID: Addgene_50942; 100 nl) was delivered to L2/3 of the right barrel cortex at the approximate location of the C2 barrel column (1.4mm posterior, 3.5mm lateral from bregma, 300mm below the pia) ( ).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Repetitive sensory stimulation potentiates and recruits sensory-evoked cortical population activity

doi: 10.1101/2024.08.06.605968

Figure Lengend Snippet: (A) Raster plot of GCaMP6s fluorescence intensity (ΔF/F 0 ) for PRWS at each acquisition for pre-PRWS (-10 min) and post-PRWS (10, 60, 120, & 180 min). Neurons sorted from top to bottom by decreasing response strength pre- vs post-PRWS (n=410 cells). Arrowheads, examples of the five subpopulations: persistent (persist.), recruited (recruit.), suppressed (suppr.), no response (no resp.), and high (hi resp.) responders. (B) Top, example 2PLSM images of neurons expressing AAV1-hSyn-mRubyGSG-P2A-GCaMP6s across the longitudinal experimental protocol -10 min pre-PRWS, & 10, 60, 120, & 180 min post-PRWS. mRuby (red) serves as an activity-independent marker, whereas GCaMP6s (green) reports Ca 2+ signals upon PW-stimulation. The lower images represent high magnifications of the cells in the square inset on top. (B) Bottom, PW-evoked RS pre-PRWS (-10 min) or CRWS and post-PRWS or CRWS (10, 60, 120, & 180 min) (PRWS n=382 cells, or CRWS n=304 cells; Two-way RM ANOVA, ***P=0.0006; N=6 mice, P=0.026). Multiple comparisons for PRWS (Dunnett’s, -10 min vs, 10 ****P<0.0001; or 60 ***P=0.0001). (C) PW-evoked Ca 2+ signal probability (P S [#events/Nstim]) (PRWS n=382 cells, CRWS n=304 cells; Two-way RM ANOVA, ****P<0.0001; N=6 mice, P=0.026). Multiple comparisons for PRWS (Dunnett’s, -10 min vs, 10****P<0.0001; or 60 **P=0.001; or 120 P=0.7; or 180 P=0.99) & CRWS (-10 min vs, 10 P=0.99; or 60, P=0.3; or 120 P=0.2; or 180 P=0.3). (D) PW-evoked Ca 2+ signal amplitudes (Ā S [ΔF/F 0 ]) (PRWS n=382 cells, CRWS n=304 cells; Two-way RM ANOVA, ****P<0.0001; N=6, P=0.25). Multiple comparisons for PRWS (Dunnett’s, -10 min vs, 10 *P=0.01; or 60 **P=0.002; or 120 P=0.8; or 180 P=0.4) & CRWS (-10min vs, 10 P=0.2; or 60 P=0.6; or 120 P=0.1; or 180 P=0.5). (E) PW-evoked response strength, pre- and 24 hrs post-PRWS or CRWS (PRWS n=162 cells, CRWS n=134 cells; Two-way RM ANOVA, P=0.36; PRWS 3 mice, CRWS 2 mice, P=0.054).

Article Snippet: AAV2-CAG-GCaMP6s-WPRE-SV40 (U Penn Vector Core, RRID: Addgene_100844, 100 nl) or AAV1-hSyn-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA (addgene, RRID: Addgene_50942; 100 nl) was delivered to L2/3 of the right barrel cortex at the approximate location of the C2 barrel column (1.4mm posterior, 3.5mm lateral from bregma, 300mm below the pia) ( ).

Techniques: Fluorescence, Expressing, Activity Assay, Marker

Journal: bioRxiv

Article Title: Repetitive sensory stimulation potentiates and recruits sensory-evoked cortical population activity

doi: 10.1101/2024.08.06.605968

Figure Lengend Snippet: (A, B, Left) Example traces of GCaMP6s (black) or mRuby fluorescence (red) for a 20-sec baseline before and during PRWS or CRWS. Violin and pairwise representation of fluorescence intensities (integrated over 20s) (violin plot median: white bar, quartiles: dotted bars) baseline vs PRWS (n=290 cells, Paired t test, ****P<0.0001; N=3 mice, P=0.027) or CRWS (n=115 cells, Paired t test, P=0.6; N=3 mice, P=0.8). (C) Fluorescence intensity (integrated over 20s) during PRWS vs PW-evoked response strength change (post/pre), (n=115 cells, Pearson r correlation, r=0.0002, P=1.0). Inset, pre- and post-PRWS response strength (n=115 cells, pre=0.027±0.002, post=0.031±0.002, Paired t test, P=0.027). (D) Fluorescence intensity during PRWS (integrated over 20s) vs pre-PRWS. Pink line simple linear regression, black dotted lines 95% confidence intervals. (n=115 cells, Pearson r correlation, r=-0.35, ***P=0.0001; simple linear regression, slope=-0.008, non-zero? P=0.0001).

Article Snippet: AAV2-CAG-GCaMP6s-WPRE-SV40 (U Penn Vector Core, RRID: Addgene_100844, 100 nl) or AAV1-hSyn-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA (addgene, RRID: Addgene_50942; 100 nl) was delivered to L2/3 of the right barrel cortex at the approximate location of the C2 barrel column (1.4mm posterior, 3.5mm lateral from bregma, 300mm below the pia) ( ).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Repetitive sensory stimulation potentiates and recruits sensory-evoked cortical population activity

doi: 10.1101/2024.08.06.605968

Figure Lengend Snippet: (A) Left, Example 2PLSM image of flex.mRuby.GCaMP6s-expressing VIP interneurons in the VIP-Cre mouse line. Right, representative confocal image after post-hoc anti-VIP immunocytochemistry on slices of barrel cortex from 2PLSM imaged VIP-Cre mice (green, anti-VIP; red, AAV1.CAG.Flex.mRuby.P2A.GCaMP6s; blue, Hoechst staining). (B) Pre-RWS PW-evoked response strength (RS) of VIP interneurons (n=341 cells, N=7 mice), and low & moderate (n=1058 cells, N=11 mice) and high (n=41, N=11 mice, one-way ANOVA, ****P<0.0001) responding L2/3 neurons. Squares and circles represent the means ± SEM over cells and mice, respectively. (C) Left, example trace of GCaMP6s (black) or mRuby fluorescence from a VIP interneuron, pre- and post-PRWS. Right, pre- and post-PRWS PW-evoked RS of VIP neurons (Paired t test, n=341 cells, P=0.2; N=7 mice, P=0.45). Grey lines, paired responses. Violin plots depict median (solid) and quartiles (dotted) bars. (D, E), Left, example trace of GCaMP6s fluorescence (black) or mRuby (integrated over 20s) from a VIP interneuron before and during PRWS (D) or CRWS (E) . Right , paired response and violin plots of normalized fluorescence intensity (norm.) during baseline, PRWS (Paired t-test, n=341 cells, ****P<0.0001; N=7 mice, P=0.047) or CRWS (Paired t-test, n=231 cells, ****P<0.0001; N=5 mice, P=0.08). (F) VIP interneurons were imaged at two planes in upper layers of S1, plane (P) 1 is closest to the pia and P2 is 100µm below. (G, H) Left, average VIP interneuron GCaMP6s fluorescence for P1 (darker green) and P2 (light green) for baseline (integrated over 20s) and during PRWS (G) or CRWS (H) . Right, normalized integrated fluorescence intensities duirng PRWS (G , P1 vs P2, Paired t test, **P=0.006) or CRWS (H, P1 vs P2, Paired t test, P=0.18). (I) Circuit diagram summarizing the RWS-evoked plasticity model. PRWS (blue) activates first-order thalamocortical (TC; red) as well as higher-order TC and feedback inputs (green), which activate disinhibitory VIP interneurons (grey). These combined inputs drive a potentiation of PW-evoked responses and a recruitment of neuronal responsivity (28%). CRWS (orange) may only activate higher-order TC and feedback inputs, also activating disinhibitory VIP interneurons, but this is not sufficient to drive potentiation and favors suppression of neurons (30%).

Article Snippet: AAV2-CAG-GCaMP6s-WPRE-SV40 (U Penn Vector Core, RRID: Addgene_100844, 100 nl) or AAV1-hSyn-mRuby2-GSG-P2A-GCaMP6s-WPRE-pA (addgene, RRID: Addgene_50942; 100 nl) was delivered to L2/3 of the right barrel cortex at the approximate location of the C2 barrel column (1.4mm posterior, 3.5mm lateral from bregma, 300mm below the pia) ( ).

Techniques: Expressing, Immunocytochemistry, Staining, Fluorescence