Journal: Neurotherapeutics

Article Title: Validation in Drosophila of the in silico predicted clomipramine as repurposable for SOD1-ALS

doi: 10.1016/j.neurot.2025.e00793

Figure Lengend Snippet: Clomipramine attenuates DNA damage in hSOD1 -ALS flies . A) Representative images of metaphase chromosomes from non-mutant and ALS third instar Drosophila larval brains, untreated (a, b, c) and 0.5 ​mM clomipramine-treated (d, e, f). Panels a, d, e, and f show normal karyotypes; panel b shows an isochromatid centromere break (arrows); panel c reports a chromatid deletion (arrow). Scale bar 5 ​μm. On the right side, bar graphs indicate the number (%) of chromosome aberrations (CABs) ​± ​SEM. Individual untreated (circles) and clomipramine-treated (squares) data points represent each analyzed larval brain (at least 500 ​cells were scored for each brain). Statistical significance was determined using a two-way ANOVA to evaluate the effects of genotype (F (2, 45) ​= ​14.27, p ​< ​0.0001), treatment (F (1, 45) ​= ​30.99, p ​< ​0.0001) and their interaction (F (2, 45) ​= ​5.059, p ​= ​0.0104). Sidak's multiple comparisons test confirms that clomipramine significantly reduced CABs in both hSOD1 A4V (∗∗∗∗p ​< ​0.0001) and hSOD1 G85R (∗∗∗p ​< ​0.001) larvae. B) Confocal microscopy images of γH2AV foci (green) in untreated and 0.5 ​mM clomipramine-treated non-mutant ( elav-Gal4/+ ) and ALS ( elav-Gal4>UAS-hSOD1 A4V and elav-Gal4>UAS-hSOD1 G85R ) larval brains. DAPI was used to stain nuclei. Scale bar 20 ​μm. Bar graphs indicate the number (%) ​± ​SEM of γH2AV-positive cells. Statistical significance was calculated by two-way ANOVA test with genotype (F (2,54) ​= ​7.682, p ​= ​0.0012) and clomipramine treatment as main factors (F (1,54) ​= ​62.62, p ​< ​0.0001). A significant genotype ​× ​treatment interaction (F (2,54) ​= ​10.00, p ​= ​0.0002) was observed. Sidak's multiple comparisons test confirms that clomipramine significantly reduced the number of γH2AV-positive cells in both hSOD1 A4V and hSOD1 G85R (∗∗∗∗p ​< ​0.0001) larvae. Scored cells: elav-Gal4/+ ​= ​2385; elav-Gal4/+ CLO ​= ​2415; elav-Gal4>UAS-hSOD1 A4V ​= ​2762; elav-Gal4>UAS-hSOD1 A4V CLO ​= ​3968; elav-Gal4>UAS-hSOD1 G85R ​= ​3092; elav-Gal4>UAS-hSOD1 G85R CLO ​= ​2010. C) Western blot analysis of Drosophila γH2AV expression in untreated (circles) and clomipramine-treated (squares) non-mutant ( elav-Gal4/+ ) and ALS ( elav-Gal4>UAS-hSOD1 A4V and elav-Gal4>UAS-hSOD1 G85R ) adult fly heads. Giotto protein was used as loading control. Bar graphs represent the mean ​± ​SEM of the normalized ratio between γH2AV and Giotto obtained by three independent biological replicates, as represented by individual data points. Two-way ANOVA showed a significant main effect of treatment (F (1, 12) ​= ​17.79, ∗∗p ​= ​0.0012), but no significant main effect of genotype (F (2, 12) ​= ​1.495, p ​= ​0.2632) or interaction (F (2, 12) ​= ​2.331, p ​= ​0.1395) was observed. Sidak's multiple comparisons test reported a statistically significant decrease in γH2AV expression specifically in the clomipramine-treated hSOD1 G85R group compared to its untreated control (∗∗p ​< ​0.01). D) Agarose gel image of long (∼8.8 ​kb, LA) and short (∼200 bp, SA) amplicons of the Neurexin gene obtained amplifying genomic DNA isolated from non-mutant ( elav-Gal4/+ ) and ALS ( elav-Gal4>UAS-hSOD1 A4V and elav-Gal4>UAS-hSOD1 G85R ) untreated (circles) and clomipramine-treated (squares) adult fly heads. Bar graphs on the right show the mean ​± ​SEM of relative DNA integrity resulting by LA/SA ratio obtained by three independent biological replicates, as indicated by individual data points. Two-way ANOVA revealed a significant main effect of the clomipramine (F (1, 12) ​= ​15.66, p ​= ​0.0019), while no significant main effect of genotype (F (2, 12) ​= ​0.5204, p ​= ​0.6071) or interaction (F (2, 12) ​= ​3.135, p ​= ​0.0803) was observed. Sidak's multiple comparisons test showed that clomipramine treatment significantly increased the LA/SA ratio in both the hSOD1 A4V (∗p ​< ​0.05) and hSOD1 G85R (∗∗p ​< ​0.01) ALS fly models compared to their respective untreated controls.

Article Snippet: Brains were then squashed and fragmented by a glass slide, frozen in liquid nitrogen, washed twice in PBS 0.1 % Triton (PBST), and incubated overnight at 4 °C with rabbit anti-Histone H2AvD pS137 (γH2AV) primary antibody (1:100 in PBST; cat. 600-401-914, Rockland Immunochemicals, Limerick, PA, USA).

Techniques: Mutagenesis, Confocal Microscopy, Staining, Western Blot, Expressing, Control, Agarose Gel Electrophoresis, Isolation

Journal: PLOS Genetics

Article Title: A novel DNA repair-independent role for Gen nuclease in promoting unscheduled polyploidy cell proliferation

doi: 10.1371/journal.pgen.1011605

Figure Lengend Snippet: (A) Pictures of Ctrl, Gen mut and GenOE brain lobes labeled with antibodies against γH2av (in pink). DNA is shown in cyan. (B) Dot plot graph showing the γH2av index of interphase NBs of the indicated genotypes. (B) Schematic diagram of the experimental set up used to induce replicative stress. Brains were incubated in 50μM of APH for 1h30 and fixed or washed and subsequently released in APH-free medium for 30 min. (D) Pictures of Ctrl, Gen mut and GenOE interphase NBs labelled with antibodies against γH2Av (pink in the merged panels and in grey on the right panels). The white dashed circles surround the nuclei. (E–J) Dot plot graphs showing the γH2Av index of the indicated genotypes in cells treated with APH for 1h30 (E,G. and I) or after release (F, H and J). Bars indicate the mean ± SEM. Statistical significance is shown and determined by Kuskal-Wallis non-parametric test. Experiments were repeated at least two times with a minimum of 10 brain lobes analyzed per condition.

Article Snippet: Rabbit polyclonal anti γH2Av , 1:500 , Rockland immunochemicals , 600-401-914.

Techniques: Labeling, Incubation

Journal: PLOS Genetics

Article Title: A novel DNA repair-independent role for Gen nuclease in promoting unscheduled polyploidy cell proliferation

doi: 10.1371/journal.pgen.1011605

Figure Lengend Snippet: (A) Pictures of polyploid brains (Ctrl) and other combinations of the indicated genotypes showing DNA labeling (grey). The white dashed line surrounds the brain lobe and the pink dashed lines surround polyploid NBs. (B) Dot plot showing cell area of the indicated genotypes. (C) Diagram of the Gen nuclease dead (ND) transgene. Numbers on the top indicate the position of the domains and on the bottom the residues replaced by Alanines. This construct was fused at the C-terminus with a mCherry (mCh) tag. (D) Pictures of polyploid brains (Ctrl) and other combinations of the indicated genotypes labelled with γH2av antibodies (pink in the merged panel and grey in the inset panels on the right). DNA is shown in cyan. The white dashed lines surround brain lobes and polyploid NBs, while the yellow circles surround the NBs magnified in the insets. (E) Dot plot graphs showing the γH2av index of the indicated genotypes. In B and E, bars show the mean ± SEM. Statistical significance is shown and determined by Kuskal-Wallis non-parametric test. Experiments were repeated at least two times with a minimum of 15 brain lobes analyzed per condition.

Article Snippet: Rabbit polyclonal anti γH2Av , 1:500 , Rockland immunochemicals , 600-401-914.

Techniques: DNA Labeling, Construct

Journal: PLOS Genetics

Article Title: A novel DNA repair-independent role for Gen nuclease in promoting unscheduled polyploidy cell proliferation

doi: 10.1371/journal.pgen.1011605

Figure Lengend Snippet: (A) Pictures of Polyploid brains brain lobes labelled with antibodies against PH3 (pink). DNA is shown in cyan. An example of a large polyploid cell showing multinucleated polyploid NBs undergoing mitosis in a synchronous (top) or asynchronous (bottom) manner. On the right, schematic diagrams to illustrate the synchrony/asynchrony. (B) Graph bar showing the percentage of asynchronous polyploid NBs in the indicated genotypes. (C) Pictures of mitotic NBs of the indicated genotypes labelled with antibodies against γH2Av (pink and grey panels on the right). DNA is shown in cyan. (D, E) Dot plot graphs showing the γH2av index of mitotic NBs. Cells were chosen in advanced mitotic stages (but before anaphase), so that all nuclei of a given cell has condensed their chromosomes and progressed through mitosis. (F) Pictures of Polyploid brain lobes of the indicated genotype after a 2h EdU pulse. EdU is shown in pink and grey on the right panels and DNA in cyan. NB insets are highlighted with white dashed squares. The blue lines surround EdU- nuclei. (G) Dot plot graphs showing the Edu signal coverage of the indicated genotypes. In D-E and G, bars show the mean ± SEM. Statistical significance is shown and determined by Kuskal-Wallis non-parametric test. Experiments were repeated at least two times with a minimum of 15 brain lobes analyzed per condition.

Article Snippet: Rabbit polyclonal anti γH2Av , 1:500 , Rockland immunochemicals , 600-401-914.

Techniques: