Mutation of the TRPM3 cation channel underlies progressive cataract development and lens calcification associated with pro‐fibrotic and immune cell responses

Abstract

Transient-receptor-potential cation channel, subfamily M, member 3 (TRPM3) serves as a polymodal calcium sensor in diverse mammalian cell-types. Mutation of the human TRPM3 gene (TRPM3) has been linked with inherited forms of early-onset cataract with or without other eye abnormalities. Here, we have characterized the ocular phenotypes of germline "knock-in" mice that harbor a human cataract-associated isoleucine-to-methionine mutation (p.I65M) in TRPM3 (Trpm3-mutant) compared with germline "knock-out" mice that functionally lack TRPM3 (Trpm3-null). Despite strong expression of Trpm3 in lens epithelial cells, neither heterozygous (Trpm3 +/− ) nor homozygous (Trpm3 −/− ) Trpm3-null mice developed cataract; however, the latter exhibited a mild impairment of lens growth. In contrast, homozygous Trpm3-M/M mutants developed severe, progressive, anterior pyramid-like cataract with microphthalmia, whereas heterozygous Trpm3-I/M and hemizygous Trpm3-M/-mutants developed anterior pyramidal cataract with delayed onset and progression-consistent with a semi-dominant lens phenotype. Histochemical staining revealed abnormal accumulation of calcium phosphate-like deposits and collagen fibrils in Trpm3-mutant lenses and immunoblotting detected increased αII-spectrin cleavage products consistent with calpain hyper-activation. Immunofluorescent confocal microscopy of Trpm3-M/M mutant lenses revealed fiber cell membrane degeneration that was accompanied by accumulation of alpha-smooth muscle actin positive (α-SMA+ve) myofibroblastlike cells and macrosialin positive (CD68+ve) macrophage-like cells. Collectively, our mouse model data support an ocular disease association for TRPM3 in humans and suggest that (1) Trpm3 deficiency impaired lens growth but not lens transparency and (2) Trpm3 dysfunction resulted in progressive lens degeneration and calcification coupled with pro-fibrotic (α-SMA+ve) and immune (CD68+ve) cell responses.

Mutation of the EPHA2 Tyrosine-Kinase Domain Dysregulates Cell Pattern Formation and Cytoskeletal Gene Expression in the Lens

“…000664) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Germline Epha2-mutant mice were generated by clustered regularly interspersed short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) gene editing technology in our Genome Engineering and iPSC Center (GEiC) and Mouse Embryo Stem (ES) Cell Core facility using standard protocols as described [39]. Briefly, guide RNAs (gRNAs) were designed in silico flanking the target site and selected based on minimum off-target sites and distance from target site.…”

“…Epha2.g2, 5 -gttcagtgtacttcagttggngg; Epha2.g3, 5 -ttcagtgtacttcagttggtngg; Epha2.ssODNantisense, 5 -ggc cag gtc ccg gtg cac gta gtt cat gtt ggc cag gta ctt cat gcc gga tgc gat acc ctg cag cat gcc cac tag ctg aag tac act gaa ctc acc atc ctt ctc ctg cag aga tag gcc ctc agt gct gac cgg. Correctly edited and 'off-target' founder mice were identified by PCR-amplification and Sanger sequencing with gene-specific primers (Table S1, Figure S1) as described [39] and subsequently bred onto the B6J genetic background to avoid a deletion mutation in the gene for lens beaded-filament-structural-protein-2 (CP49) carried by some inbred strains [40]. Epha2-null mice (B6J background) were genotyped by PCR-amplification as described [34].…”

“…Mice were humanely killed by CO 2 asphyxiation followed by cervical dislocation or decapitation. Eyes were removed from age-and sex-matched littermates and lenses dissected and imaged as described [35,39,41]. All mouse studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Washington University (Protocol No.…”

“…Enucleated eyes were processed using standard formaldehyde-fixed-paraffin-embedded (FFPE) and frozen section techniques followed by immunolabeling with antigen-specific primary antibodies (Table S2) and species-appropriate Alexa Fluor 488-or 546-conjugated secondary antibodies, (Thermo Fisher Scientific, Waltham, MA, USA), counterstaining of cell nuclei with 4 ,6-diamidino-2-phenylindole (DAPI, MilliporeSigma, Burlington, MA, USA), and visualization by confocal imaging (FV1000 microscope, Olympus) performed as described [35,39,41,42].…”

“…Following lens homogenization (Bullet Blender, Next Advance, Troy, NY, USA), lens post-nuclear lysates were quantified (Non-interfering protein assay, G-Bioscience, St. Louis MO, USA) and subjected to SDS-PAGE separation (Novex 4-12% gradient gels and an Invitrogen XCell electrophoresis/blot system, Thermo Fisher Scientific) and immunoblot analysis (Odyssey Infrared Imaging System, Li-Cor, Lincoln, NE, USA) using appropriate primary antibodies (Table S2) and IRDye labelled secondary antibodies (LiCor) as described [39,42]. Immunoprecipitation was performed using the Pierce Classic IP Kit (#26146, Thermo Fisher Scientific) according to the manufacturer's instructions.…”