Biochemical and Biophysical Research Communications
Lens transcriptome profile during cataract development in Mip-null mice
Introduction
The major intrinsic protein of the mammalian ocular lens is an evolutionarily conserved member of the aquaporin family of water channels also known as aquaporin-0 (AQP0) [1]. In the mouse lens, MIP accounts for over 40% of the lens membrane proteome [2] and, despite relatively modest aquaporin activity, facilitates approximately 80% of water transport across fiber cell membranes [3], [4], [5], [6]. In addition to water transport, MIP has been implicated in the formation of junctional micro-domains between the tightly packed concentric layers of fiber cells that make-up the crystalline mass of the lens and, in particular, MIP is enriched at interlocking protrusions decorating the vertices of fiber cell membranes – suggesting a role in cell-cell junction formation and adhesion [7], [8], [9], [10]. The combined water-transport and cell-adhesion functions of MIP have been proposed to play a synergistic role in establishing the refractive index gradient and biomechanical properties of the lens including its fine-focusing power or accommodation reflex [11], [12], [13].
Mutations in the human MIP gene (MIP) mostly resulting in missense substitutions have been linked with autosomal dominant forms of cataract that present during infancy or childhood exhibiting wide variability in size, shape, and density of lens opacities [14], [15] [http://cat-map.wustl.edu/]. Similarly, mutations in the rodent MIP gene (Mip) have been associated with semi-dominant cataract in the Shrivelled/Fraser (Svl/Fr), lens opacity (Lop), hydropic fibers (Hfi), and Tohoku (Tohm) mouse strains and with recessive cataract in the Kyoto Fancy Rat Stock 4 (kfrs4) strain [16], [17], [18], [19], [20]. In addition to these mutant strains, mice functionally lacking (null) or deficient (hemizygous) in MIP also develop fully penetrant cataract [4].
The pathogenetic mechanisms underlying MIP-related cataract are only partly understood.
Ectopic expression of several human MIP mutants in Xenopus oocytes or cultured cells results in abnormal intracellular accumulation and impaired water transport across the plasma membrane [14], [21], [22], [23]. In lenses of Mip-mutant mice, the mutant forms of MIP have been shown to accumulate within intracellular endoplasmic reticulum (ER)-like membranes rather than plasma membranes of lens fiber cells [16], [17], [18], [19]. Further, in the case of the heterozygous Lop mutant (Lop/+) intracellular accumulation of mutant MIP (p.Ala51Pro) was associated with ER-stress and activation of the unfolded response coupled with oxidative stress and calpain hyper-activation [24]. However, in contrast to mutant forms of MIP that underlie autosomal dominant forms of cataract, less is known about the pathogenic mechanisms associated with ‘recessive’ cataract resulting from MIP loss-of-function. Here, we have undertaken a transcriptome profiling approach to detect potential pathogenic mechanisms associated with cataract development in Mip-null (Mip−/−) lenses.
Section snippets
Mice and lenses
Mice functionally null for MIP (Mip−/−) were generated by gene-trap technology on a hybrid 129/B6J background as described [4] then backcrossed (>6 generations) to C57BL/6J (B6J, Jackson Lab, Bar Harbor ME; stock # 000664) and genotyped as described [24]. Heterozygous (Mip+/-) mice were bred to generate Mendelian ratios of homozygous and wild-type (+/+) littermates for lens transcript profiling on postnatal day 1 (P1) and P7. Following euthanasia, eyes were removed and fixed or lenses were
Mip−/− lens phenotype
Dark-field microscopy revealed that the Mip−/− lens ex vivo (P7) exhibited a translucent grey central opacity with a clear cortex and was smaller in size to that of wild-type (Fig. 1b). Histochemical staining of Mip−/− lens sections (P7) revealed severe degeneration of fiber cells within the central core region flanked by many displaced cell nuclei from the disturbed bow-regions and the equatorial axis appeared misshapen compared with wild-type (Fig. 1d). Immunofluorescent staining with
Discussion
In this study we have monitored transcriptome changes during cataract development in neonatal lenses from mice functionally null for MIP. Overall, relatively few genes (29/1883, ∼1.5% of transcriptome) were differentially regulated >2-fold in the Mip−/− lens at P1 and P7 with more genes down-regulated (∼18) than up-regulated. Pathway analysis suggested that while water transport and/or homeostasis were impaired in the Mip−/− lens at P7 no specific stress pathway was strongly associated with
Acknowledgements
We thank the Genome Technology Access Center (GTAC) at Washington University School of Medicine for help with microarray analysis, Dr. J. Yu for help with statistical analysis, B. McMahan and G. Ling for histology support, and J. King for technical assistance. This work was supported by NIH/NEI grants EY012284 and EY023549 (to A.S.) and P30 EY02687 (Core Grant for Vision Research), and an unrestricted grant to the Department of Ophthalmology and Visual Sciences from Research to Prevent
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