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Lanosterol reverses protein aggregation in cataracts

A Corrigendum to this article was published on 26 August 2015


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.

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Figure 1: Identification of mutations in LSS causing congenital cataracts.
Figure 2: LSS mutations abolished the cyclase enzymatic function.
Figure 3: Lanosterol reduced intracellular aggregation of various crystallin mutant proteins.
Figure 4: Lanosterol re-dissolved pre-formed amyloid-like fibrils of crystallin proteins.
Figure 5: Lanosterol reduced cataract severity and increased clarity.


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We thank the study participants for their support. We thank G. Hannum, M. Kruppa, J. H. Shin, J. Mei, H. Zheng, F. Xu, J. Zhang, M. Kircher, J. Shendure, S. J. Fliesler, J. Gleeson, X.-T. Zuo, Y. Li and Y. Ding for their helpful advice during the course of the experiments and data analysis. This work is supported in part by grants from 973 Project (2015CB94600, 2012CB917304), 863 Program (2014AA021604), NSFC (31327901), State Key Laboratory of Ophthalmology, and State Key Laboratory of Membrane Biology.

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Authors and Affiliations



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.

Corresponding authors

Correspondence to Liangfang Zhang, Yizhi Liu, Yong-Bin Yan or Kang Zhang.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 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.

Extended Data Figure 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).

Extended Data Figure 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.

Extended Data Figure 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.

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.

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.

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.

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.

Extended Data Figure 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.

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

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Zhao, L., Chen, XJ., Zhu, J. et al. Lanosterol reverses protein aggregation in cataracts. Nature 523, 607–611 (2015).

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