Journal: Acta neuropathologica

Article Title: SMN regulates GEMIN5 expression and acts as a modifier of GEMIN5-mediated neurodegeneration

doi: 10.1007/s00401-023-02607-8

Figure Lengend Snippet: Smn upregulation suppresses loss of Gemin5 neurodegeneration in vivo. a Schematic representing the experimental to investigating the genetic manipulation of the Drosophila snRNP complex proteins in the background of loss of Gemin5 (Rig) animals in vivo. Drosophila expressing UAS-Rig RNAi were crossed with the GMR-gal4 driver for targeted expression to the Drosophila eye and Tubulin-gal4 driver for ubiquitous expression. b Representative images of female Drosophila eyes expressing GMR-gal4 and Rig RNAi crossed with control (W1118), Luciferase OE, Smn RNAi, and Smn OE. c, d, e Quantification of eye degeneration of control and Rig RNAi flies combined with (c) Luciferase OE, (d) Smn RNAi, and (e) Smn OE (n = 20 Drosophila per group, one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. ****p < 0.0001. NS, not significant. f Quantification of relative eye sizes of control and Rig RNAi flies combined with Smn OE (n = 5 Drosophila per group, one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. *p < 0.05; ****p < 0.001. NS, not significant. g, h qPCR analysis from (n = 4 biological replicates of 6 Drosophila heads per group) confirms significant knockdown of endogenous (g) Rig in the Rig KD groups and significant overexpression of (h) Smn in the Smn OE crosses (one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. *p < 0.05; ****p < 0.0001; NS, not significant. i Representative male images of eclosed Drosophila adults expressing Control, Rig RNAi (pupal lethality), Rig RNAi in combination with over expression of Smn, and Smn OE. j The percentage of eclosed adults for control and Rig RNAi animals combined with Smn OE (n = 100 Drosophila per replicate, one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. ****p < 0.0001

Article Snippet: Western blots were blocked with 2.5% milk solution (BLOT-QuickBlocker reagent, EMB Millipore, #WB57–175GM) and incubated with primary antibody overnight: rabbit Drosophila Rig, 1–1:000 (developed by Bio Boster Biological Technology Co. Ltd, Wuhan, China), rabbit Drosophila Smn (developed by Bio Boster Biological Technology Co. Ltd, Wuhan, China), and mouse anti-tubulin, 1:10,000 (66,031, Protein Tech).

Techniques: In Vivo, Expressing, Control, Luciferase, Knockdown, Over Expression

Journal: Acta neuropathologica

Article Title: SMN regulates GEMIN5 expression and acts as a modifier of GEMIN5-mediated neurodegeneration

doi: 10.1007/s00401-023-02607-8

Figure Lengend Snippet: SMN expression alleviates reduced snRNP biogenesis in GEMIN5-mediated disease. a Confocal images of control and mutant GEMIN5 L1068P neurons transduced with EGFP and lentiviral EGFP-SMN from the same set of neuronal differentiations. The neurons were probed for the snRNP marker Y12 (Sm), GFP, and the neuronal marker microtubule-associated protein 2 (MAP2). The cell nuclei were stained with DAPI. Scale bar = 10 μm. b Quantitative analysis observing a significant decrease in nuclear snRNP expression in L1068P neurons after lentiviral transduction of EGFP. Lentiviral transduction of EGFP-SMN significantly rescues the levels of nuclear snRNP expression in L1068P neurons (n = 75–100 neurons, one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. *p < 0.05; **p < 0.01; ****p < 0.0001. NS, not significant. c Confocal images of control and mutant GEMIN5 H913R neurons transduced with EGFP and lentiviral EGFP-SMN from the same set of neuronal differentiations. The neurons were probed for the small nuclear ribonucleoprotein interacting partner, Coilin (cajal bodies, CBs), GFP, and GEMIN5. The cell nuclei were stained with DAPI. Scale bar = 10 μm. d Quantitative analysis observing the average number of CBs per cell in control and mutant H913R neuronal cells with EGFP and EGFP-SMN lentiviral expression. Only CBs above 0.2 microns were quantified (n = 60–80 neurons, one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. **p < 0.01; NS, not significant. e The percentage of cells with CBs in control and mutant H913R neuronal cells with EGFP and EGFP-SMN expression (n = 60–80 neurons, one-way ANOVA w/ Bonferroni test). Error bars indicate S.E.M. **p < 0.01; NS, not significant. f Representative gel displaying complete in vitro snRNP assembly formation using 3′ Cy3-biotin-labeled U1 snRNA and the cytoplasmic extract from control and L1068P neurons with lentirviral SMN and ASO N1 administration. Untransfected HEK293T cells and HEK293T transfected with GEMIN5 shRNA were used as a positive control for assembly formation. g Representative quantification of 3 biological replicate sample displaying the intensity of the snRNP assembly between groups (n = 3, one-way ANOVA w/ Tukey’s multiple comparisons). Error bars indicate S.E.M. *p < 0.05; **p < 0.01. NS, not significant. h Confocal images of the ventral nerve cord (VNC) of control and ubiquitously expressing GEMIN5 KD Drosophila larvae with and without Smn over expression stained with the snRNP marker Sm (Y12) and nuclei (DAPI). Loss of GEMIN5 in vivo results in a reduction of nuclear Sm which is rescued by upregulation of Smn. Scale bar = 10 μM. i. Confocal images of the ventral nerve cord (VNC) of control and ubiquitously expressing GEMIN5 KD Drosophila larvae with and without Smn over expression stained with the small nuclear ribonucleoprotein interacting partner, dCoilin (CBs) and nuclei (DAPI). Loss of GEMIN5 in vivo results in a drastic reduction of CB formation which is rescued by upregulation of Smn. Scale bar = 10 μM

Article Snippet: Western blots were blocked with 2.5% milk solution (BLOT-QuickBlocker reagent, EMB Millipore, #WB57–175GM) and incubated with primary antibody overnight: rabbit Drosophila Rig, 1–1:000 (developed by Bio Boster Biological Technology Co. Ltd, Wuhan, China), rabbit Drosophila Smn (developed by Bio Boster Biological Technology Co. Ltd, Wuhan, China), and mouse anti-tubulin, 1:10,000 (66,031, Protein Tech).

Techniques: Expressing, Control, Mutagenesis, Transduction, Marker, Staining, In Vitro, Labeling, Transfection, shRNA, Positive Control, Over Expression, In Vivo