Lanosterol reverses protein aggregation in cataracts

Journal name:
Nature
Volume:
523,
Pages:
607–611
Date published:
DOI:
doi:10.1038/nature14650
Received
Accepted
Published online

The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people1, and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for cataract prevention and treatment.

At a glance

Figures

  1. Identification of mutations in LSS causing congenital cataracts.
    Figure 1: Identification of mutations in LSS causing congenital cataracts.

    a, Pedigrees of affected families and cataract phenotype. Squares and circles indicate males and females respectively. +, wild-type allele; W581R and G588S are the two mutations. b, Upper panel, DNA sequencing data of an unaffected individual and an affected child (II-1) with a homozygous W581R mutation; lower panel, DNA sequencing data of an unaffected individual and an affected child (IV-1) with a homozygous G588S mutation. The underlined sequence indicates the nucleic acid change. c, Left, colour photograph of patient 1’s right eye in the first pedigree (IV-1) with a total cataract; right, colour photograph of patient 2’s right eye in the same pedigree (IV-3) with a cataract.

  2. LSS mutations abolished the cyclase enzymatic function.
    Figure 2: LSS mutations abolished the cyclase enzymatic function.

    a, Conservation of W581R and G588 in LSS across several species: Homo sapiens, Pan troglodytes, Bos taurus, Mus musculus, Rattus norvegicus, Gallus gallus and Danio rerio. b, Computer modelling of LSS structure and impact of the LSS W581R and G588S mutations. A computer modelling analysis identifies a loop originating from C584 and ending at E578 with the key side chain of W581 at the tip of the loop stabilizing the sterol. The loop is fixed by an S–S bridge and the E578–R639 salt bridge. Amide nitrogen N of G588 interacts with the C584 from the previous helical turn and the Cα hydrogen of G588 is in close proximity to the critical E578, which then forms a strong salt bridge with R639 of the same supporting helix. The mutation G588S causes the side chain of the serine to clash into the E578 residue of the loop and is incompatible with the structure. Arrow indicates the location of the mutant side chain. c, Effect of engineered expression of the wild-type protein (WT LSS) and LSS mutants on sterol content. Wild-type LSS markedly increased lanosterol production, whereas neither W581R nor the G588S mutant exhibited any cyclase activity. n = 3 in each group; ***P < 0.001.

  3. Lanosterol reduced intracellular aggregation of various crystallin mutant proteins.
    Figure 3: Lanosterol reduced intracellular aggregation of various crystallin mutant proteins.

    a, Confocal images of crystallin protein aggregates in human lens progenitor cells. The cataract-causing Y118D mutant of αA-crystallin formed p62-positive intracellular inclusion bodies or aggresomes. Green, eGFP–crystallin proteins; red, p62; blue, nuclei. Cells transfected with peGFP-N1 were used as a control. b, Confocal images of the inhibitory effect of LSS on crystallin aggregates. c, Inhibition of crystallin mutant aggregation by wild-type LSS (WT LSS) and lanosterol, but not mutant LSS or cholesterol. d, Increase in soluble αA-crystallin(Y118D) mutant protein by co-expression of wild-type LSS but not LSS mutants (Y118D co-expressed with pcDNA3.1–N-Flag was used as a control). Quantitative analysis was performed using densitometry of crystallin proteins by western blot analysis of the supernatant or insoluble fraction of cell lysates. n = 3 in each group; representative western blot analysis is shown in Extended Data Fig. 4c; *P < 0.05, **P < 0.01. e, Confocal images of the re-dissolution of preformed crystallin aggregates by lanosterol. Arrows indicate the presence of crystallin aggregation. f, Lanosterol significantly reduced the intracellular aggregation by various cataract-causing mutant crystallin proteins in a concentration-dependent manner. n = 3; P < 1 × 10−4. Cholesterol did not reduce intracellular aggregation. n = 3; P > 0.1. g, Lanosterol increased the soluble fractions of various crystallin mutants in human lens progenitor cells. n = 3; P < 0.001. h, Effects of DMSO, cholesterol or lanosterol on αA-crystallin(Y118D) aggregates in human lens progenitor cells by serial live-cell imaging. Progression of crystallin aggregation dissolution by lanosterol can be observed, as evidenced by decreased green fluorescence following the time-course. i, Effect of lanosterol on dissolution of intracellular crystallin aggregates over time. n = 22 from three biological replicates. The 22 repetitions are shown in open circles distinguished by different colours. The mean ± s.d. values are shown as filled black circles and error bars. The data are best fitted by the single exponential decay process (red line). Scale bars, 10 μm.

  4. Lanosterol re-dissolved pre-formed amyloid-like fibrils of crystallin proteins.
    Figure 4: Lanosterol re-dissolved pre-formed amyloid-like fibrils of crystallin proteins.

    a, Negatively stained TEM photographs of aggregates of αA-crystallin mutant proteins treated by a liposome vehicle, cholesterol or lanosterol in liposomes. Images in the right column of the lanosterol group show a 5× magnification of the image on their left. b, Effect of lanosterol on the re-dissolution of crystallin aggregates by ThT fluorescence (n = 3). Left, β/γ-crystallin mutants; right, α-crystallin mutants. Each bar results from three independent samples.

  5. Lanosterol reduced cataract severity and increased clarity.
    Figure 5: Lanosterol reduced cataract severity and increased clarity.

    a, Photographs of a cataractous rabbit lens treated with lanosterol showing increased lens clarity. Left, before treatment; right, after. b, Boxplot of the quantification of the treatment effect of lanosterol (n = 13). c, Photographs of a cataractous dog lens treated with lanosterol showing increased lens clarity. Left, before treatment; right, after. d, Boxplot of the quantification of the treatment effect of lanosterol (n = 7). Range, median (horizontal line) and mean (circle) are presented. Crosses indicate the maximum and minimum cataract grades measured. Whiskers indicate the standard deviation and the box encompasses a 40% confidence interval.

  6. Genome-wide homozygosity.
    Extended Data Fig. 1: Genome-wide homozygosity.

    a, HomozygosityMapper plots the genome-wide homozygosity as bar charts. To emphasize regions of interest, any score higher than 80% of the maximum score reached in this project is coloured in red. b, The homozygosity scores were plotted against the physical position on chromosome 21, which contains the LSS gene. Red bars indicate regions with highest scores. The right side of the chromosome contains a long continuous homozygous region, where the LSS gene is located.

  7. Representative confocal images of cells co-transfected with Flag-LSS and eGFP.
    Extended Data Fig. 2: Representative confocal images of cells co-transfected with Flag–LSS and eGFP.

    Human lens progenitor cells were co-transfected with either the wild-type or the mutated LSS gene and the eGFP gene for 4 h and cultured for 16 h in fresh culture medium. The cellular distribution of LSS was then visualized using an anti-Flag antibody (purple). The distribution of eGFP (green) was used as a control. The nuclei were stained and visualized by Hoechst 33342 (blue).

  8. Representative confocal images of cells co-transfected with LSS and various cataract-causing crystallin mutants.
    Extended Data Fig. 3: Representative confocal images of cells co-transfected with LSS and various cataract-causing crystallin mutants.

    a, R116C mutant of αA-crystallin. b, R120G mutant of αB-crystallin. c, V187E mutant of βB2-crystallin. c, G129C mutant of γC-crystallin. e, W43R mutant of γD-crystallin. Human lens progenitor cells were co-transfected with either the wild-type or the mutated Flag–LSS gene and the mutant GFP–crystallin gene for 4 h and cultured for 16 h in fresh culture medium. All crystallin mutants formed p62-positive aggregates as indicated by the co-localization of the mutant crystallins and p62. Cells co-transfected with GFP–crystallin and pcDNA3.1-N-Flag were used as controls. The formation of intracellular aggregates of various crystallin proteins was visualized by fluorescence of GFP (green). Wild-type or mutated LSS was detected with an anti-Flag antibody (purple), p62 was stained using an anti-p62 antibody (red), while the nuclei were stained and visualized by Hoechst 33342 staining (blue). Quantitative analysis of cells with aggregates is summarized in Fig. 3c.

  9. Inhibition of crystallin mutant aggregation by wild-type LSS and lanosterol in HLEB-3 cells (a) or HeLa cells (b).
    Extended Data Fig. 4: Inhibition of crystallin mutant aggregation by wild-type LSS and lanosterol in HLEB-3 cells (a) or HeLa cells (b).

    Cells co-transfected with LSS and crystallin mutant constructs were cultured for 24 h before assaying for aggregates. The rescue experiments were performed by addition of 40 μM sterols (lanosterol or cholesterol) to the cell culture medium for 2 h, the sterol medium was then replaced with fresh culture medium and the cells were cultured for a further 12 h. The percentage of cells with crystallin aggregates were calculated from ten randomly selected viewing fields. The values of the wild-type LSS group, mutant group, or mutant plus lanosterol group were calculated. Aggregates were significantly lower in the wild-type LSS and lanosterol groups compared to the control group (P < 1 × 10−4), while aggregates in mutant LSS or cholesterol groups showed no difference to the control group (P > 0.1). c, Human lens progenitor cells were co-transfected with wild-type or mutant LSS plus αA-crystallin(Y118D). αA-crystallin(Y118D) co-expressed with pcDNA3.1-N-Flag was used as a control. After transfection for 4 h and incubation in fresh culture medium for another 24 h, the cells were lysed and centrifuged to separate supernatant and insoluble fractions. LSS and crystallin fusion proteins were detected by antibodies against Flag and GFP, respectively. Red arrows indicate higher crystalline content in the soluble fraction versus in the insoluble fraction in cells containing the WT-LSS. Data were quantified from three independent experiments and summarized in Fig. 3d.

  10. Lanosterol significantly reduced the intracellular aggregation caused by various cataract-causing mutant crystallin proteins in a concentration-dependent manner when assayed in HLEB-3 or HeLa cells.
    Extended Data Fig. 5: Lanosterol significantly reduced the intracellular aggregation caused by various cataract-causing mutant crystallin proteins in a concentration-dependent manner when assayed in HLEB-3 or HeLa cells.

    a, Representative confocal images of HLEB-3 cells transfected with various cataract-causing crystallin mutants. b, Representative confocal images of HeLa cells transfected with various cataract-causing crystallin mutants. Cells were transfected with various crystallin constructs for 4 h and cultured for an additional 24 h in fresh culture medium. Then the cells were treated with 10, 20 and 40 μM lanosterol in 1% (HLEB-3 cells) or 2% DMSO (HeLa cells) for 2 h and cultured for another 12 h. Cells treated with 1% (HLEB-3 cells) or 2% DMSO (HeLa cells) were used as the controls. Formation of intracellular aggregates of various crystallin proteins was visualized by fluorescence of GFP (green) and the nuclei were stained with Hoechst 33342 (blue). Typical intracellular aggregates are indicated by arrows. c, Concentration dependence of the aggregation-dissolving effects of lanosterol when assayed in HLEB-3 cells. d, Concentration dependence of the aggregation-dissolving effects of lanosterol when assayed in HeLa cells.

  11. Treatment by lanosterol, but not cholesterol, increased cataract-causing mutant crystallins in soluble fractions when compared to a control group or a mutant LSS group.
    Extended Data Fig. 6: Treatment by lanosterol, but not cholesterol, increased cataract-causing mutant crystallins in soluble fractions when compared to a control group or a mutant LSS group.

    a, Human lens progenitor cells were transfected with mutant crystallin genes for 4 h, and then incubated in fresh culture medium for another 24 h. The cells were harvested and lysed. Supernatant and insoluble fractions were separated by centrifugation and analysed by western blot analysis. LSS and crystallin fusion proteins were identified by antibodies against Flag and GFP tags, respectively. The lanosterol-treated group is highlighted by red boxes. Cells treated with 1% DMSO were used as a control. β-Actin was used as an internal protein loading control of total cell lysates (TCL). S, supernatant; P, insoluble fraction. b, Effect of DMSO (n = 4) and cholesterol (n = 7) on the size changes of αA-crystallin(Y118D) aggregates in human lens progenitor cells evaluated by single-particle tracking in live-cell imaging. c, Evaluation of the effect of lanosterol on the dissolution of crystallin aggregates by turbidity. Crystallin aggregates were formed by incubating 5 mg ml−1 protein solution at 60 °C for 2 h (α-crystallins) or 37 °C for 48 h (β- and γ-crystallins) in the presence of 1 M guanidine chloride. The preformed aggregates were re-suspended in PBS at a final protein concentration of 0.2 mg ml−1 and were treated with 500 μM sterols in 500 μM DPPC liposome and incubated at 37 °C for 24 h. Aggregates treated with 500 μM DPPC liposome only were used as the controls. d, Concentration-dependent effect of lanosterol on the re-dissolution of amyloid-like fibrils by αA-crystallin mutants evaluated by ThT fluorescence. Aggregates treated with 500 μM DPPC liposome only were used as the controls.

  12. Grading system of cataractous lenses.
    Extended Data Fig. 7: Grading system of cataractous lenses.

    a, Lenses were placed above a grid and photographed. The degree of transparency was scored as 0, a clear lens and absence of opacification (gridlines clearly visible, a′); 1, a blurry lens and a slight degree of opacification (minimal clouding of gridlines, with gridlines still visible, b′); 2, a cloudy lens and presence of diffuse opacification involving almost the entire lens (moderate clouding of gridlines, with main gridlines visible, c′); or 3, an opaque lens and presence of extensive thick opacification involving the entire lens (total clouding of gridlines, with gridlines not seen at all, d′). b, Lanosterol reduced cataract severity and increased clarity in isolated cataractous rabbit lenses. Rabbit lenses (n = 13) were dissected and incubated with lanosterol for 6 days and subsequently assessed for lens clarity and transparency. Pairs of photographs of each cataractous rabbit lens showing before and after treatment with scores underneath are shown. c, Lanosterol reduced cataract severity and increased lens clarity in dogs. Dog eyes with cataracts (n = 7) were treated with lanosterol for 6 weeks and assessed for lens clarity and transparency. A pair of photographs of each study eye before and after treatment is shown with scores underneath. Three control eyes treated with vehicles alone are also presented.

Tables

  1. Exome sequencing and variants
    Extended Data Table 1: Exome sequencing and variants
  2. Treatment effect of lanosterol in cataractous rabbit lenses and dog cataracts.
    Extended Data Table 2: Treatment effect of lanosterol in cataractous rabbit lenses and dog cataracts.
  3. Primers used for sequencing of each exon in the human LSS gene and construction of crystallin mutants
    Extended Data Table 3: Primers used for sequencing of each exon in the human LSS gene and construction of crystallin mutants

References

  1. Pascolini, D. & Mariotti, S. P. Global estimates of visual impairment: 2010. Br. J. Ophthalmol. 96, 614618 (2012)
  2. Bloemendal, H. et al. Ageing and vision: structure, stability and function of lens crystallins. Prog. Biophys. Mol. Biol. 86, 407485 (2004)
  3. Moreau, K. L. & King, J. A. Protein misfolding and aggregation in cataract disease and prospects for prevention. Trends Mol. Med. 18, 273282 (2012)
  4. Huff, M. W. & Telford, D. E. Lord of the rings–the mechanism for oxidosqualene:lanosterol cyclase becomes crystal clear. Trends Pharmacol. Sci. 26, 335340 (2005)
  5. Diehn, J. J., Diehn, M., Marmor, M. F. & Brown, P. O. Differential gene expression in anatomical compartments of the human eye. Genome Biol. 6, R74 (2005)
  6. Mori, M. et al. Lanosterol synthase mutations cause cholesterol deficiency-associated cataracts in the Shumiya cataract rat. J. Clin. Invest. 116, 395404 (2006)
  7. Ng, P. C. & Henikoff, S. Predicting deleterious amino acid substitutions. Genome Res. 11, 863874 (2001)
  8. Adzhubei, I. A. et al. A method and server for predicting damaging missense mutations. Nature Methods 7, 248249 (2010)
  9. Pollard, K. S., Hubisz, M. J., Rosenbloom, K. R. & Siepel, A. Detection of nonneutral substitution rates on mammalian phylogenies. Genome Res. 20, 110121 (2010)
  10. Schwarz, J. M., Cooper, D. N., Schuelke, M. & Seelow, D. MutationTaster2: mutation prediction for the deep-sequencing age. Nature Methods 11, 361362 (2014)
  11. Seelow, D., Schuelke, M., Hildebrandt, F. & Nurnberg, P. HomozygosityMapper–an interactive approach to homozygosity mapping. Nucleic Acids Res. 37, W593W599 (2009)
  12. Thoma, R. et al. Insight into steroid scaffold formation from the structure of human oxidosqualene cyclase. Nature 432, 118122 (2004)
  13. Dobson, C. M. Protein folding and misfolding. Nature 426, 884890 (2003)
  14. Ecroyd, H. & Carver, J. A. Crystallin proteins and amyloid fibrils. Cell. Mol. Life Sci. 66, 6281 (2009)
  15. Braun, N. et al. Multiple molecular architectures of the eye lens chaperone αB-crystallin elucidated by a triple hybrid approach. Proc. Natl Acad. Sci. USA 108, 2049120496 (2011)
  16. Cenedella, R. J. et al. Direct perturbation of lens membrane structure may contribute to cataracts caused by U18666A, an oxidosqualene cyclase inhibitor. J. Lipid Res. 45, 12321241 (2004)
  17. Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26, 589595 (2010)
  18. DePristo, M. A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nature Genet. 43, 491498 (2011)
  19. Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010)
  20. Ruf, A. et al. The monotopic membrane protein human oxidosqualene cyclase is active as monomer. Biochem. Biophys. Res. Commun. 315, 247254 (2004)
  21. Cardozo, T., Totrov, M. & Abagyan, R. Homology modeling by the ICM method. Proteins 23, 403414 (1995)
  22. Abagyan, R. & Argos, P. Optimal protocol and trajectory visualization for conformational searches of peptides and proteins. J. Mol. Biol. 225, 519532 (1992)
  23. Xu, J. et al. The congenital cataract-linked A2V mutation impairs tetramer formation and promotes aggregation of βB2-crystallin. PLoS ONE 7, e51200 (2012)
  24. Wang, B. et al. A novel CRYGD mutation (p.Trp43Arg) causing autosomal dominant congenital cataract in a Chinese family. Hum. Mutat. 32, E1939E1947 (2011)
  25. Gu, F. et al. A novel mutation in AlphaA-crystallin (CRYAA) caused autosomal dominant congenital cataract in a large Chinese family. Hum. Mutat. 29, 769 (2008)
  26. Li, X.-Q. et al. A novel mutation impairing the tertiary structure and stability of γC-crystallin (CRYGC) leads to cataract formation in humans and zebrafish lens. Hum. Mutat. 33, 391401 (2012)
  27. Nagineni, C. N. & Bhat, S. P. Human fetal lens epithelial cells in culture: an in vitro model for the study of crystallin expression and lens differentiation. Curr. Eye Res. 8, 285291 (1989)
  28. Bligh, E. G. & Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911917 (1959)
  29. Wang, S., Leng, X.-Y. & Yan, Y.-B. The benefits of being β-crystallin heteromers: βB1-crystallin protects βA3-crystallin against aggregation during co-refolding. Biochemistry 50, 1045110461 (2011)
  30. Sun, T.-X., Das, B. K. & Liang, J. J. N. Conformational and functional differences between recombinant human lens αA- and αB-crystallin. J. Biol. Chem. 272, 62206225 (1997)
  31. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248254 (1976)
  32. Geraldine, P. et al. Prevention of selenite-induced cataractogenesis by acetyl-L-carnitine: an experimental study. Exp. Eye Res. 83, 13401349 (2006)
  33. Makri, O. E., Ferlemi, A. V., Lamari, F. N. & Georgakopoulos, C. D. Saffron administration prevents selenite-induced cataractogenesis. Mol. Vis. 19, 11881197 (2013)
  34. Zhang, L. et al. Self-assembled lipid–polymer hybrid nanoparticles: a robust drug delivery platform. ACS Nano 2, 16961702 (2008)
  35. La Croix, N. Cataracts: When to refer. Top. Companion Anim. Med. 23, 4650 (2008)

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Author information

  1. Present address: Institute of Molecular Medicine, Peking University, Beijing 100871, China.

    • Ling Zhao
  2. These authors contributed equally to this work.

    • Ling Zhao,
    • Xiang-Jun Chen,
    • Jie Zhu,
    • Yi-Bo Xi,
    • Xu Yang &
    • Li-Dan Hu

Affiliations

  1. Molecular Medicine Research Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China

    • Ling Zhao,
    • Huimin Cai,
    • Gen Li,
    • Guiqun Cao,
    • Xun Hu,
    • Zhiguang Su &
    • Kang Zhang
  2. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China

    • Ling Zhao,
    • Hong Ouyang,
    • Ying Lin,
    • Yandong Wang,
    • Yizhi Liu &
    • Kang Zhang
  3. Shiley Eye Institute and Biomaterials and Tissue Engineering Center, Institute for Engineering in Medicine, University of California San Diego, La Jolla, California 92093, USA

    • Ling Zhao,
    • Jie Zhu,
    • Hong Ouyang,
    • Sherrina H. Patel,
    • Danni Lin,
    • Frances Wu,
    • Ken Flagg,
    • Ying Lin,
    • Daniel Chen,
    • Cindy Wen,
    • Christopher Chung,
    • Austin Qiu,
    • Emily Yeh,
    • Wenqiu Wang,
    • Seanna Grob,
    • Hongrong Luo,
    • Weiwei Gao,
    • David Granet,
    • Liangfang Zhang &
    • Kang Zhang
  4. State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China

    • Xiang-Jun Chen,
    • Yi-Bo Xi,
    • Li-Dan Hu,
    • Harry Christianto Tjondro,
    • Xi-Juan Zhao &
    • Yong-Bin Yan
  5. Department of Ophthalmology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China

    • Jie Zhu
  6. BGI-Shenzhen, Shenzhen 518083, China

    • Xu Yang,
    • Xin Jin,
    • Yingrui Li &
    • Jun Wang
  7. Guangzhou KangRui Biological Pharmaceutical Technology Company, Guangzhou 510005, China

    • Huimin Cai &
    • Rui Hou
  8. CapitalBio Genomics Co., Ltd., Dongguan 523808, China

    • Austin Qiu
  9. Department of Ophthalmology, Shanghai First People's Hospital, School of Medicine, Shanghai JiaoTong University, Shanghai 20080, China

    • Wenqiu Wang &
    • Xiaodong Sun
  10. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California 92093, USA

    • Ruben Abagyan
  11. Department of Biochemistry, University of California Riverside, Riverside, California 92521, USA

    • P. Perry
  12. Department of Nanoengineering, University of California, San Diego, La Jolla, California 92093, USA

    • Weiwei Gao,
    • Liangfang Zhang &
    • Kang Zhang
  13. King Khaled Eye Specialist Hospital, Riyadh, Kingdom of Saudi Arabia

    • Igor Kozak
  14. Veterans Administration Healthcare System, San Diego, California 92093, USA

    • Kang Zhang
  15. Institute of Molecular Medicine, Peking University, Beijing 100871, China

    • Kang Zhang

Contributions

L.Zhao, Y.Liu., Y.-B.Y., L.Zhang and K.Z. designed the study, interpreted data and wrote the manuscript. L.Z., X.-J.C., J.Z., Y.-B.X., X.Y., L.-D.H, H.O., S.H.P., X.J., D.L., F.W., K.F., H.C., G.L., G.C., Y.Li, D.C., C.W., C.C., Y.W., A.Q., E.Y., W.W., X.H., S.G., Z.S., H.C.T., X.-J.Z., H.L., R.H., J.J.P.P., W.G., I.K., D.G., and X.S. performed the experiments; R.A., Y.Li and J.W. contributed to data analysis and interpretation.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: Genome-wide homozygosity. (234 KB)

    a, HomozygosityMapper plots the genome-wide homozygosity as bar charts. To emphasize regions of interest, any score higher than 80% of the maximum score reached in this project is coloured in red. b, The homozygosity scores were plotted against the physical position on chromosome 21, which contains the LSS gene. Red bars indicate regions with highest scores. The right side of the chromosome contains a long continuous homozygous region, where the LSS gene is located.

  2. Extended Data Figure 2: Representative confocal images of cells co-transfected with Flag–LSS and eGFP. (263 KB)

    Human lens progenitor cells were co-transfected with either the wild-type or the mutated LSS gene and the eGFP gene for 4 h and cultured for 16 h in fresh culture medium. The cellular distribution of LSS was then visualized using an anti-Flag antibody (purple). The distribution of eGFP (green) was used as a control. The nuclei were stained and visualized by Hoechst 33342 (blue).

  3. Extended Data Figure 3: Representative confocal images of cells co-transfected with LSS and various cataract-causing crystallin mutants. (527 KB)

    a, R116C mutant of αA-crystallin. b, R120G mutant of αB-crystallin. c, V187E mutant of βB2-crystallin. c, G129C mutant of γC-crystallin. e, W43R mutant of γD-crystallin. Human lens progenitor cells were co-transfected with either the wild-type or the mutated Flag–LSS gene and the mutant GFP–crystallin gene for 4 h and cultured for 16 h in fresh culture medium. All crystallin mutants formed p62-positive aggregates as indicated by the co-localization of the mutant crystallins and p62. Cells co-transfected with GFP–crystallin and pcDNA3.1-N-Flag were used as controls. The formation of intracellular aggregates of various crystallin proteins was visualized by fluorescence of GFP (green). Wild-type or mutated LSS was detected with an anti-Flag antibody (purple), p62 was stained using an anti-p62 antibody (red), while the nuclei were stained and visualized by Hoechst 33342 staining (blue). Quantitative analysis of cells with aggregates is summarized in Fig. 3c.

  4. Extended Data Figure 4: Inhibition of crystallin mutant aggregation by wild-type LSS and lanosterol in HLEB-3 cells (a) or HeLa cells (b). (408 KB)

    Cells co-transfected with LSS and crystallin mutant constructs were cultured for 24 h before assaying for aggregates. The rescue experiments were performed by addition of 40 μM sterols (lanosterol or cholesterol) to the cell culture medium for 2 h, the sterol medium was then replaced with fresh culture medium and the cells were cultured for a further 12 h. The percentage of cells with crystallin aggregates were calculated from ten randomly selected viewing fields. The values of the wild-type LSS group, mutant group, or mutant plus lanosterol group were calculated. Aggregates were significantly lower in the wild-type LSS and lanosterol groups compared to the control group (P < 1 × 10−4), while aggregates in mutant LSS or cholesterol groups showed no difference to the control group (P > 0.1). c, Human lens progenitor cells were co-transfected with wild-type or mutant LSS plus αA-crystallin(Y118D). αA-crystallin(Y118D) co-expressed with pcDNA3.1-N-Flag was used as a control. After transfection for 4 h and incubation in fresh culture medium for another 24 h, the cells were lysed and centrifuged to separate supernatant and insoluble fractions. LSS and crystallin fusion proteins were detected by antibodies against Flag and GFP, respectively. Red arrows indicate higher crystalline content in the soluble fraction versus in the insoluble fraction in cells containing the WT-LSS. Data were quantified from three independent experiments and summarized in Fig. 3d.

  5. Extended Data Figure 5: Lanosterol significantly reduced the intracellular aggregation caused by various cataract-causing mutant crystallin proteins in a concentration-dependent manner when assayed in HLEB-3 or HeLa cells. (486 KB)

    a, Representative confocal images of HLEB-3 cells transfected with various cataract-causing crystallin mutants. b, Representative confocal images of HeLa cells transfected with various cataract-causing crystallin mutants. Cells were transfected with various crystallin constructs for 4 h and cultured for an additional 24 h in fresh culture medium. Then the cells were treated with 10, 20 and 40 μM lanosterol in 1% (HLEB-3 cells) or 2% DMSO (HeLa cells) for 2 h and cultured for another 12 h. Cells treated with 1% (HLEB-3 cells) or 2% DMSO (HeLa cells) were used as the controls. Formation of intracellular aggregates of various crystallin proteins was visualized by fluorescence of GFP (green) and the nuclei were stained with Hoechst 33342 (blue). Typical intracellular aggregates are indicated by arrows. c, Concentration dependence of the aggregation-dissolving effects of lanosterol when assayed in HLEB-3 cells. d, Concentration dependence of the aggregation-dissolving effects of lanosterol when assayed in HeLa cells.

  6. Extended Data Figure 6: Treatment by lanosterol, but not cholesterol, increased cataract-causing mutant crystallins in soluble fractions when compared to a control group or a mutant LSS group. (368 KB)

    a, Human lens progenitor cells were transfected with mutant crystallin genes for 4 h, and then incubated in fresh culture medium for another 24 h. The cells were harvested and lysed. Supernatant and insoluble fractions were separated by centrifugation and analysed by western blot analysis. LSS and crystallin fusion proteins were identified by antibodies against Flag and GFP tags, respectively. The lanosterol-treated group is highlighted by red boxes. Cells treated with 1% DMSO were used as a control. β-Actin was used as an internal protein loading control of total cell lysates (TCL). S, supernatant; P, insoluble fraction. b, Effect of DMSO (n = 4) and cholesterol (n = 7) on the size changes of αA-crystallin(Y118D) aggregates in human lens progenitor cells evaluated by single-particle tracking in live-cell imaging. c, Evaluation of the effect of lanosterol on the dissolution of crystallin aggregates by turbidity. Crystallin aggregates were formed by incubating 5 mg ml−1 protein solution at 60 °C for 2 h (α-crystallins) or 37 °C for 48 h (β- and γ-crystallins) in the presence of 1 M guanidine chloride. The preformed aggregates were re-suspended in PBS at a final protein concentration of 0.2 mg ml−1 and were treated with 500 μM sterols in 500 μM DPPC liposome and incubated at 37 °C for 24 h. Aggregates treated with 500 μM DPPC liposome only were used as the controls. d, Concentration-dependent effect of lanosterol on the re-dissolution of amyloid-like fibrils by αA-crystallin mutants evaluated by ThT fluorescence. Aggregates treated with 500 μM DPPC liposome only were used as the controls.

  7. Extended Data Figure 7: Grading system of cataractous lenses. (484 KB)

    a, Lenses were placed above a grid and photographed. The degree of transparency was scored as 0, a clear lens and absence of opacification (gridlines clearly visible, a′); 1, a blurry lens and a slight degree of opacification (minimal clouding of gridlines, with gridlines still visible, b′); 2, a cloudy lens and presence of diffuse opacification involving almost the entire lens (moderate clouding of gridlines, with main gridlines visible, c′); or 3, an opaque lens and presence of extensive thick opacification involving the entire lens (total clouding of gridlines, with gridlines not seen at all, d′). b, Lanosterol reduced cataract severity and increased clarity in isolated cataractous rabbit lenses. Rabbit lenses (n = 13) were dissected and incubated with lanosterol for 6 days and subsequently assessed for lens clarity and transparency. Pairs of photographs of each cataractous rabbit lens showing before and after treatment with scores underneath are shown. c, Lanosterol reduced cataract severity and increased lens clarity in dogs. Dog eyes with cataracts (n = 7) were treated with lanosterol for 6 weeks and assessed for lens clarity and transparency. A pair of photographs of each study eye before and after treatment is shown with scores underneath. Three control eyes treated with vehicles alone are also presented.

Extended Data Tables

  1. Extended Data Table 1: Exome sequencing and variants (275 KB)
  2. Extended Data Table 2: Treatment effect of lanosterol in cataractous rabbit lenses and dog cataracts. (168 KB)
  3. Extended Data Table 3: Primers used for sequencing of each exon in the human LSS gene and construction of crystallin mutants (386 KB)

Comments

  1. Report this comment #66417

    Anurag Agrawal said:

    The importance of lanosterol in maintaining crystalline structure of the human lens may further be connected to the increased risk of cataract in patients on common drugs like statins and corticosteroids (1,2). Lanosterol is a precursor of cholesterol as well as steroid hormones. Statins inhibit HMG-CoA Reductase, which is upstream of Lanosterol synthesis and use of oral or high dose inhaled steroids can lead to suppression of endogenous steroid synthesis. I do not find any data on lanosterol levels in patients being treated with either steroids or statins and that can be an avenue of exploration.

    1. Wise SJ, Nathoo NA, Etminan M, Mikelberg FS, Mancini GB. Statin use and risk for cataract: a nested case-control study of 2 populations in Canada and the United States. Can J Cardiol. 2014 Dec;30(12):1613-9.

    2. Wang JJ, Rochtchina E, Tan AG, Cumming RG, Leeder SR, Mitchell P. Use of inhaled and oral corticosteroids and the long-term risk of cataract. Ophthalmology. 2009 Apr;116(4):652-7.

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