EMBO/soda-nel huggingface dataset:
Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat (2010)
Figure 3

Regulation of ft/Hpo levels is functionally relevant to neurodegeneration by Atrophins. Statistical significance is marked as follows: ***P<0.001, **P<0.01; *P<0.05 in two-tailed t-test. All qPCR results here and in all figures are the average of three reactions of independent biological replicas. (A) qPCR analysis of the fold changes±s.d. of ft transcription. Downregulation starts at 2 days and progresses at 14 days. (B) qPCR analysis of the enrichment±s.d. of different regions of the ft regulatory elements in ChIP for Atro from BG3 neuronal cell extracts. Enrichment is calculated as the percentage of DNA immunoprecipitated from extracts of cells in which Atro expression has been induced, with respect to the amount immunoprecipitated from uninduced cell extracts according to the (Atro-No Ab)induced/(Atro−No Ab)uninduced formula. The DNA region from IV chromosome has been previously shown not to be immunoprecipitated in ChIP for Atro (Haecker et al, 2007) and has been used as a negative control. (C) Histograms showing the number of ommatidia with a full complement (7) of PR in flies expressing Atro75QN with the Rhodopsin1 driver in either a control (w1118) or different mutant backgrounds and aged at 29°C for 28 days. Heterozygosis for two independent ft alleles and a sav allele significantly enhances the loss of PR. Mild overexpression of Wts via a GMR-wts transgene, which does not display any strong phenotype per se, significantly suppresses the loss of PR. No interaction was detected in this assay with wtsx1 and ykiB5 alleles in heterozygosity. N=430-963 from at least four eyes. (D) Tangential eye sections and histograms showing the degeneration of GMR>Atro75QN, Ubi-Gal80ts flies in combination with different UAS transgenes and aged 14 days as in Figure 1A. UAS-EGFP is used as a negative control. Either Ft or Wts overexpression strongly suppresses loss of PR caused by Atro75QN. Lower panels are provided as examples of quantification. A more modest but still significant rescue is observed by overexpressing a Yki RNAi contruct or Hpo; however, in this case it is to be considered that the overexpression of Hpo per se brings about the loss of at least one PR in ∼5% of ommatidia (data not shown). No effect is detected with overexpression of Sav. Two-tailed t-test: *P<0.05; **P<0.01. The wtsEPG4808 is a previously uncharacterised EP element insertion at the 5′ of the wts gene. Rare homozygous escapers display larger eyes, whereas in combination with GMR-Gal4 this line gives rise to smaller rough eyes, and, finally, qPCR analysis of GMR>wtsEPG4808 indicates a 10-fold increase in the head content of wts mRNA (data not shown).
Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat (2010)
Figure 4

Neurodegeneration by mutations in ft. (A) Tangential eye sections through ftfd clones aged 1, 7 and 14 days, of ftfd,dDGC13 double mutant clones and of ftfd clones in a ykiB5 heterozygous background aged 14 days. Clones are marked by the absence of yellow pigment. Arrows point at degenerating photoreceptors, arrowheads point at intact wt photoreceptors in mosaic ommatidia. The circled ommatidium is genetically wt but non‐autonomously flipped in its polarity and has not degenerated. Lower panels are masks that show clonal borders and quantification of PR number per ommatidia. After 14 days, almost all ftfd cells have degenerated, whereas almost all ftfd,dDGC13 and some ftfd,ykiB5/+ neuronal photoreceptors have not. (B) Histograms showing the quantification of degeneration in ftfd, ftfddDGC13 and ftfdykiB5/+ clones. Loss of ft leads to statistically significant loss of neurons and this is suppressed at 14 days by ykiB5and much more dramatically by dDGC13. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test. (C) Degeneration in mosaic ommatidia in ftfd clones. At this stage of degeneration approximately half of the non‐pigmented (genotypically mutant) PR cells display a degenerative phenotype, whereas virtually all pigmented (genotypically wt) PR cells are normal. χ2‐test: 82.38; P0.001. Total PR N=310 (pigmented) and 327 (not pigmented). (D) Tangential eye sections through MARCM clone mutants for ftfd that express either no transgene or an RNAi construct against yki (UAS-ykiIR) with Tub-Gal4 and aged 14 days at 29°C. Because the transgene UAS-ykiIR is on chromosomal arm 2 L, the same as ft, clones generated with this system carry two copies of UAS-ykiIR and therefore downregulate yki very effectively. No other cell outside the clones expresses the construct and is therefore wt for yki. Mask panel is presented on the right of each section. A quantification of photoreceptor numbers (far right) displays a very significant increase in the number of wt ommatidia (N=210 and 219 from four eyes). ***P0.001 in two‐tailed t‐test. If all classes of ommatidia are considered, χ2‐test=232.30; P0.001 for 3 degrees of freedom.
Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat (2010)
Figure 5

Neurodegeneration by mutations in Hippo pathway genes. All Hippo pathway mutant clones display a remarkable increase in interommatidial space as a consequence of failure in lattice programmed cell death and these lattice cells also degenerate with age (see also Supplementary Figure 12). Arrows point at degenerating neuronal photoreceptor cells. (A) Tangential eye sections through wtsX1 mutant fly eyes. In clones for the null wtsX1 allele, degeneration inside the clones is at severe stages at 14 days. (B) Tangential eye sections through sav3 clones. In sav3 after enclosure many photoreceptors are intact but after 14 days at 29°C most sav mutant photoreceptors have degenerated. (C) Histograms showing the quantification of ommatidia with the full complement of photoreceptors in hpo, wts and sav mutant clones. The progression of loss of photoreceptors is evident and statistically significant in all cases. Mutants for hpoBF33 do not survive for 14 days at 29oC and corresponding sections are shown in Supplementary Figure 12. N=195-268. ***P<0.001, **P<0.01; *P<0.05 in two-tailed t-test.
Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat (2010)
Figure 6

Uncoupling of neurodegeneration and overgrowth. (A) Regression analysis of the overgrowth versus the neurodegeneration observed in loss of function mutants (left) and overexpression of transgenes (right). Overgrowth quantification is shown in Supplementary Figure 14. For loss of function mutant, the neurodegeneration was quantified at 14 days in clones (as shown in Figures 4 and ​and5).5). For UAS-overexpression transgenes neurodegeneration was quantified at 14 days when expressed with GMRGal4; UbiGal80ts. For CycD+Cdk4, these specific data are missing and have not been plotted; however, Supplementary Figure 13 shows that these mutants do not cause neurodegeneration in a different setup in which they are expressed during both development and adult life. In both cases the regression coefficient r2 is extremely low (0.21 and 0.03), indicating an absence of correlation between the two processes. (B) Tangential eye sections through the eyes of flies expressing with GMR-Gal4, Ubi-Gal80ts either UAS-EGFP or UAS-Yki or UAS-YkiS111A,S168A,S250A and aged 1 or 28 days. Arrows point at missing or degenerated photoreceptors. The UAS-YkiS111A,S168A,S250A is so effective that even the very low expression leaking out with this system is enough to affect development and generate mild eye roughness and overproliferation of lattice cells. Histograms showing the quantification of cell loss in eyes shown in A and also eyes from flies aged at the intermediate 14-day stage. Mild but significant degeneration is observed for both Yki forms, with respect to the negative control; however, no statistical difference is observed between the two Yki proteins that have dramatically different effects on overgrowth. ***P<0.001, **P<0.01; *P<0.05 in two-tailed t-test.
Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat (2010)
Figure 7

Neurodegeneration by polyQ Atro partially requires dachs. Tangential eye sections through dGC13 clones (marked by the absence of yellow pigment) either in control flies or those expressing Atro75QN with the Rhodopsin1 driver and aged 28 days. Mask panels below each section exemplify clonal boundaries and PR no. for the ommatidia. Early signs of degeneration indicated by reduced complement of photoreceptors are detected specifically in the pigmented area (arrowhead). At this stage, inside d mutant clones all ommatidia are unaffected and display a wt arrangement (arrows). (B) Histograms showing the quantification of cell loss in eyes shown in (A). A statistically significant rescue is obtained both for the number of wt ommatidia (*P0.05 in two‐tailed t‐test) and for all other categories of ommatidia (χ2=44.76; P0.001 for 2 degrees of freedom).
Polyglutamine Atrophin provokes neurodegeneration in Drosophila by repressing fat (2010)
Figure 8

Autophagic modifications in ft mutants. (A) EM scan of a wt (left) and ftfd mutant ommatidium (right) of a ftfd clone after 14 days. Scale bar: 1 μm. High‐magnification panels (far right, top to bottom) display autophagosomes with undigested debris, damaged mitochondria and forming phagophores (arrowheads), found in ftfd mutant cells. Scale bar: 0.2 μm for zoom‐in panels. (B) Graphs of the quantification of autophagic vesicles (AV) per photoreceptor found in EM sections of 7‐day (left)‐ and 14‐day (right)‐old ftfd clones. Significant accumulation of AV is found in mutant cells (not pigmented) with respect to genotypically wt (pigmented) cells. **P0.01 and ***P0.001 in one‐tailed t‐test. N=12 pigmented cells versus 20 non‐pigmented cells for the 7‐day graph and 13 pigmented cells versus 21 non‐pigmented cells for the 14‐day graph. (C) Confocal pictures of whole‐mount retinae of a ftfd clone aged 7 days and expressing GFP∷Atg8a ubiquitously with Tub-Gal4. Red is phalloidin marking rhabdomeres, green is GFP and the clone is marked by the absence of β‐gal staining (blue). Small GFP∷Atg8a dots accumulate specifically inside ft mutant cells (arrow). (D) Confocal pictures of whole‐mount retinae of a ftfd clone aged 7 days (left) and 14 days (right). Red is phalloidin marking rhabdomeres, blue is p62 and the clone is marked by the absence of GFP staining (green). p62 starts to gather in small dots specifically inside ft mutant cells (arrow) and then accumulates massively (arrow) in the ft mutant clones as many cells degenerate.
Combined Metabolic Activators Therapy Ameliorates Liver Fat in Nonalcoholic Fatty Liver Disease Patients (2021)
Figure 1

Figure 1. CMA improves liver fat and clinical parameters

(A) Study design for testing the effects of CMA in NAFLD patients. Differences in clinical variables including (B) liver fat, plasma levels of (C) ALT, (D) AST, (E) uric acid and (F) creatinine are presented in the CMA and placebo groups on Days 0, 14 and 70 after weight loss adjustment. Adj.P indicates p value after weight loss adjustment. Statistical significance is defined based on paired Student's t test. p< 0.05. The boxes show the distribution of the clinical parameters in different groups. The bottom and top of the boxes represent the 25th and 75th percentiles. The central band represents the median value. The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values. The sample sizes on Days 0, 14 or 70 were marked in each boxplot.
Combined Metabolic Activators Therapy Ameliorates Liver Fat in Nonalcoholic Fatty Liver Disease Patients (2021)
Figure 4

Figure 4. CMA decreases plasma levels of inflammatory proteins

A) Heatmap shows log2FC based alterations between the significantly different inflammation related proteins on Day 70 vs Day 0 in the CMA and placebo groups. Asterisks indicate statistical significance based on paired Student's t test. p < 0.05; B) Heatmap shows the correlation between the plasma levels of all inflammation related proteins and plasma levels of the individual metabolic activators including serine, cysteine, carnitine and nicotinamide. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Heatmap shows the associations between the significantly different inflammation related proteins (CD8A, CSF-1, CCL23, FGF-21 and OSM) C) with the 10 most significantly correlated plasma metabolites, D) with the abundance of the species in the gut microbiome and E) with the abundance of the species in the oral microbiome. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Cor.Coeff: Correlation coefficient; Log2FC: log2(fold change).
Bi-allelic VPS16 variants limit HOPS/CORVET levels and cause a mucopolysaccharidosis-like disease (2021)
Figure 3

Figure 3. The c.2272-18C>A mutation impairs VPS16 splicing and leads to reduced levels of VPS16 and HOPS/CORVET components.

(A) The c.2272-18C>A variant generates a new splice acceptor sequence 16 base pairs upstream of the normal exon 23 border, resulting in a frame-shift and premature stop codon.

(B) Levels of VPS16 transcripts in fibroblasts by qPCR. The probe of the exon 22-24 PCR spans the misspliced intron-exon border. Data was normalized to levels of POLR2A using the ∆∆Ct method.

(C) Quantifications of VPS16 in fibroblast lysates by immunoblotting. Representative immunoblots (bottom) and summary quantifications (top) of the levels of VPS16 in patient A (n=4) and patient B (n=3), normalized to levels of actin (ACTB) and expressed as % of controls.

(D) Schematic illustration of HOPS and CORVET complexes showing their subunit compositions. The Rab5-binding CORVET subunits VPS3 and VPS8 replace the Rab7-binding VPS39 and VPS41 of HOPS. Models adapted from Bröcker et al (2012).

(E-G) Quantifications of HOPS/CORVET subunits in fibroblast lysates (top) and representative immunoblots (bottom), analyzed as in (C). (E) VPS33A, (F) VPS11, (G) VPS18.

(H) Analysis of VPS33A and VPS11 mRNA levels in fibroblasts by qPCR. Data was normalized to levels of POLR2A using the ∆∆Ct method.

(I) Immunoblots (left) and quantifications normalized to control (right) of VPS16, VPS33A and VPS11 in lysates from fibroblasts transduced with a lentivirus to express VPS16 or control (empty vector).

Data information: All immunoblots were acquired using fluorescent secondaries and pseudocolored to greyscale. Stars denote unspecific bands. All data shown as mean ±SEM of 3-4 biological replicates. Statistical comparisons were made by two-tailed unpaired student's t-tests [panels B, C (right), E (right), F, G and I] or one-way ANOVA with Dunnett's multiple comparisons tests [panels C (left) and E(left)]. * p < 0.05; ** p < 0.01; *** p < 0.001.
Bi-allelic VPS16 variants limit HOPS/CORVET levels and cause a mucopolysaccharidosis-like disease (2021)
Figure 4

Figure 4. Analysis of transferrin uptake and trafficking in fibroblasts.

(A-B) Analysis of the early endosome marker EEA1 in fibroblasts transduced with control or VPS16-expressing lentivirus by confocal microscopy. (A) Representative confocal micrographs. Scale bar, 20 μm. (B) Quantification of EEA1-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates).

(C) Analysis of transferrin uptake in fibroblasts transduced with the indicated lentiviruses. Representative confocal micrographs of fibroblasts fed with Alexa488-conjugated Transferrin for 30 min. Scale bar, 10 μm.

(D) Quantifications of the number and fluorescence intensities of intracellular Transferrin puncta (n=28-30 fields from 3 independent experiments). Colored horizontal bars indicate median values and whiskers 5 and 95 percentiles.

(E) Immunoblot for Transferrin receptor (TfR) levels in the indicated fibroblasts under basal and serum-starved conditions.

(F) Summary quantification of Transferrin receptor levels in non-starved fibroblasts transduced with the indicated lentiviruses, normalized to actin (ACTB) and expressed as % of controls (n=6 biological replicates).

(G-H) Analysis of intracellular Transferrin trafficking. Fibroblasts fed with fluorescently labeled Transferrin were co-stained with LysoTracker (G) or an antibody against LAMP2 (H). Representative images (left) of fibroblasts transduced with control virus, with magnified areas demonstrating co-localized puncta (arrowheads). Scale bars 10 μm or 2 μm (inserts). Quantifications (right) of the co-localization between Transferrin and LysoTracker or LAMP2, respectively, expressed as Pearson correlation coefficients. Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles (n=10 optical sections from 3 independent experiments).

Data information: Bar graphs represent data as mean ±SEM. Statistical comparisons between the indicated groups by one-way ANOVA with Holm-Sidak's multiple comparisons tests. * p < 0.05; ** p < 0.01; *** p < 0.001.
Bi-allelic VPS16 variants limit HOPS/CORVET levels and cause a mucopolysaccharidosis-like disease (2021)
Figure 5

Figure 5. Analysis of lysosomal distribution and function in fibroblasts.

(A) Confocal micrographs of fibroblasts transduced with control or VPS16-expressing lentivirus and labeled with LysoTracker. Scale bar, 20 μm.

(B) Quantification of LysoTracker-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates).

(C-D) Representative immunoblots (bottom) and summary quantifications (top) of the levels of LAMP1 (C) and LAMP2 (D) in fibroblasts, normalized to levels of actin (ACTB) and expressed as % of controls (n=3 biological replicates).

(E) Analysis of lysosomal Cathepsin D (CtsD) processing by immunoblotting. Immunoblot to detect CtsD isoforms (left) and quantification (right) of processing, calculated as the percentage of CtsD divided by the sum of all isoforms (n=3 biological replicates).

Data information: Bar graphs represent data as mean ±SEM. Statistical comparisons between the indicated groups by two-tailed unpaired student's t-tests (panel E) or one-way ANOVA with Holm-Sidak's multiple comparisons tests (panel B). * p < 0.05; ** p < 0.01.
Bi-allelic VPS16 variants limit HOPS/CORVET levels and cause a mucopolysaccharidosis-like disease (2021)
Figure 6

Figure 6. Analysis of autophagosome formation and turnover in fibroblasts.

(A) Analysis of p62/SQSTM1 levels in patient and control fibroblasts transduced with control or VPS16-expressing lentivirus under basal conditions and after 2 hours of serum-starvation.

(B) Summary quantification of (A), normalized to actin (ACTB) and expressed as % of controls (n=3 biological replicates).

(C) Analysis of LC3 isoforms in serum-starved fibroblasts transduced with control or VPS16-expressing lentivirus and treated with Bafilomycin A1 (Baf A1), as indicated.

(D) Quantification of LC3-II, normalized to levels of LC3-I, actin and expressed as % of controls (n=6 biological replicates).

(E) Autophagic flux, calculated as the relative increase in LC3-II ratios upon addition of Baf A1 (data from D; n=6 biological replicates).

(F-I) Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. (F) Representative confocal micrographs (optical sections) of fibroblasts imaged live under basal or serum-starved conditions. Scale bar, 20 μm. (G-H) Quantification of autophagosomes, defined as green + red puncta (G) and autophago-lysosomes, calculated as the number of red (but not green) puncta divided by the total number of puncta. Colored horizontal bars denote median values and whiskers 5 and 95 percentiles (n=50-113 cells from a total of 3 independent experiments; for exact values see Appendix Table S1). (I) Cell of patient A transfected with mRFP-GFP-LC3, labeled with LysoTracker, and imaged live by confocal microscopy under serum-starved conditions. Insert shows red+blue autolysosomes (arrowheads) and a green+red autophagosome devoid of LysoTracker signal (arrow). Scale bars 10 μm or 2 μm (inserts).

Data information: Bar graphs represent data as mean ±SEM. Statistical comparisons between the indicated groups by one-way ANOVA with Holm-Sidak's multiple comparisons tests. * p < 0.05; *** p < 0.001.
Bi-allelic VPS16 variants limit HOPS/CORVET levels and cause a mucopolysaccharidosis-like disease (2021)
Figure 7

Figure 7. Zebrafish embryos deficient in vps16 demonstrate abnormalities in LROs and myelination.

(A) Schematic representation of the HOPS complex, indicating the high amino acid identity between human and zebrafish for each subunit.

(B) Comparison of control and vps16 embryos at 5 dpf. Compared to control, vps16 embryos have reduced pigmentation in the skin and retinal pigment epithelium and do not develop a swim bladder (arrows). Magnified views show altered melanocyte morphology on the head and pale, translucent eyes.

(C, D) Myelination, as reported using the Tg(mbp:gfp-caax) transgenic, is significantly reduced in vps16 crispants. The yellow box indicates the region assessed in D. Scale bar 100 μm. Bar graphs represent data as mean ±SEM. Statistical comparisons by Mann-Whitney U tests (n=6). ** p < 0.01.
Bi-allelic VPS16 variants limit HOPS/CORVET levels and cause a mucopolysaccharidosis-like disease (2021)
Figure 8

Figure 8. LysoTracker and GFP-Lc3 signal are enhanced in vps16 embryos, suggesting glial dysfunction.

(A, B) LysoTracker-stained acidic compartments in zebrafish larvae brains at 3 and 5 dpf. Total signal was increased across the brains of vps16 crispants at 5 dpf, and abnormal, large puncta observed in the optic tectum from 3 dpf (arrows).

(C) LysoTracker signal colocalizes with the astroglial reporter slc1a2b:citrine and does not appear to overlap with the pan-neuronal reporter elavl3:GCaMP5G.

(D) The puncta indicated in A exist within cells expressing the mpeg1:EGFP transgene, revealing microglia with a grossly altered cellular morphology.

(E) Microglia at 3 dpf contain an increased number of acidic compartments as compared to controls.

(F) Global GFP-Lc3 signal is increased in unstimulated vps16 embryos (left panel), with clusters of puncta present particularly along the midline of the optic tectum (right panel, magnified view) and anterior hindbrain.

(G) Magnified view of the anterior hindbrain showing numerous GFP-Lc3 puncta in the vps16 embryo that do not colocalize with LysoTracker.

Data information: Scale bars 100 μm in (A, F left), 30 μm in (C), 20 μm in (F right) and 10 μm in (D, G). Bar graphs represent data as mean ±SEM. Statistical comparisons by Mann-Whitney U tests. ** p < 0.01; *** p < 0.001. n = 6 each for 3dpf, 3 (ctrl) and 12 (vps16) for 5 dpf in (B). n = 28 (ctrl) and 14 (vps16) in (E). All confocal images representative of a minimum of 6 embryos each.

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