Fig 2: Glial activation in retinas of Vps13b?Ex3/?Ex3 eyes with and without cataract. (A–D) Representative images of retinal sections (n = 3 per group) from Vps13b+/+ eyes (left panels) and Vps13b?Ex3/?Ex3 eyes with hypermature cataract (right panels) at 3 months of age stained with H&E (A) or antibodies against GFAP (B) or Iba1 (C). Cataractous eyes (HC) showed large and multiple regions with retinal folds and edemas in which astroglial and microglial proliferations were prominent along with activated Müller cells. The arrow in A (lower panel) highlights a fibrotic area in the vitreous humor near an edema. VH, vitreous humor; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; ED, edema. Scale bars: 10 µm.

Fig 3: Expression of the microglial marker Iba1 in the dentate gyrus of mice from (a, b) Furnas and (c, d) Rabo de Peixe. Scale bar: 50 µm. Magnification of a section of the total microglia in the dentate gyrus of (c) Furnas and (d) Rabo de Peixe. GL: granular layer; PL: polymorphic layer. Scale bar: 20 µm.

Fig 4: Inflammatory and proliferative profiles of Vps13b?Ex3/?Ex3 retinas based on protein expression levels. (A) Western blot analysis of Iba1 and GFAP expression levels, as well as Stat3 and Erk1/2 phosphorylation levels, on protein extracts from Vps13b+/+, cataract-free Vps13b?Ex3/?Ex3, and nuclear sclerotic Vps13b?Ex3/?Ex3 eyes, as well as Vps13b?Ex3/?Ex3 eyes with HC, at 2 months of age. GAPDH was used as loading control. (B–F) Relative protein levels of Iba1, GFAP, Stat3, Phospho-Stat3/Stat3, and Phospho-Erk2/Erk2 from western blot analysis presented in (A). Protein levels were normalized to GAPDH levels and the average Vps13b+/+ value. The data are presented as a boxplot showing minimum, maximum, and quartile values for each group (n = 3). Although some Vps13b?Ex3/?Ex3 retinas of eyes without HC presented with increased Iba1 and GFAP levels representative of glial activation and proliferation, this increase became systematic in eyes with HC. Iba1 was increased by 11-fold (B) and GFAP by 5-fold (C) compared to controls. In addition, the pro-inflammatory protein Stat3 was also more prominent in retinas of eyes with HC. Comparison of the Phospho-Stat3/Stat3 ratio (D) and Stat3/GAPDH ratio (E) shows that increased levels of Phospho-Stat3 in cases of HC were mostly due to the overexpression of Stat3. Interestingly, Vps13b?Ex3/?Ex3 retinas of eyes without HC also displayed a moderately elevated expression of Stat3 that did not translate into increased phosphorylation of the protein. Last, the key regulator of cell proliferation Erk was only over-activated in eyes with HC (F).

Fig 5: Quantification of Iba1+ cells/mm2 in both dentate gyrus layers: (a) granular and (b) polymorphic layers of mice from the two study locations. Data reported in the bar graph is represented as mean ± SEM. The statistical analysis was performed using Student's t-test, ***p < 0.001.