Indoleamine 2,3-dioxygenase overexpression causes kynurenine-modification of proteins, fiber cell apoptosis and cataract formation in the mouse lens

Abstract

Indoleamine 2,3-dioxygenase (IDO) is the first enzyme in the kynurenine pathway. The kynurenines formed in this pathway chemically modify proteins and cause apoptosis in cells. Evidence suggests that kynurenines and their protein modifications are involved in cataract formation, but this has yet to be directly demonstrated. We generated transgenic (Tg) mouse lines that overexpress human IDO in the lens. Homozygous Tg (homTg) lenses had higher IDO immunoreactivity, approximately 4.5 times greater IDO mRNA, and approximately 8 times higher IDO activity compared to lenses from hemizygous Tg (hemTg) animals. The kynurenine content was threefold higher in homTg than in hemTg but was not detected in wild-type (Wt) lenses. Kynurenine modifications were approximately 2.6 times greater in homTg than in hemTg or Wt. HomTg lenses had vacuoles in the epithelium and cortical fiber cells. Kynurenine modifications coincided with apoptosis in the secondary fiber cells of homTg lenses. Caspase-3 and caspase-9 activities were markedly higher in homTg than in hemTg and Wt. The glutathione content was approximately 36% lower in homTg compared to hemTg and Wt lenses. HomTg animals also developed bilateral cataracts within 3 months of birth. Together these data demonstrate that IDO-mediated production of kynurenines results in defects in fiber cell differentiation and their apoptosis and suggest that IDO activity is kept low in the lens to prevent deleterious effects by kynurenines.

FIGURE 1. Transgene

Schematic representation of the hIDO transgenic construct. The chick δ1-crystallin enhancer (δ1) was fused to the mouse αA-crystallin promoter (αA-P) to make the chimeric promoter, δenαA. The hIDO cDNA was inserted between the β-globin intron (β-gloint) and human growth hormone polyA signal (hGH pA, 660 bp).

FIGURE 2. Lens diameter in the transgenic mouse

A. Homozygous transgenic (homTg) mice (right) exhibited considerably smaller lenses. The eye size was normal in age-matched hemizygous transgenic (hemTg) (middle) and wild type (Wt) (left) mice. B. Lens weight was significantly lower in homTg mice compared to Wt, glyoxalase I transgenic (TgGloI), and hemTg mice. C. Lens diameter in homTg mice was significantly shorter than Wt, TgGloI, and hemTg mice. Results are mean ± SD, n = 5 mice (10 lenses). * P < 0.0001.

FIGURE 3. Morphological changes in the lens

Hematoxylin and eosin staining revealed differences in homTg lenses compared to Wt and other transgenic lines. A. In the whole eye, the lens shape and size differed markedly in homTg. B. The epithelial cells of homTg lenses had vacuoles (arrow). C. At the lens equator, the homTg lens had vacuoles (arrow). D. In the lens nucleus, the homTg lens had nucleated fiber cells (arrow). In contrast, all three features were normal in hemTg TgGlo I, and Wt lenses (magnification A, 4X; B, C and D, 10X).

FIGURE 4. Morphological changes in the lens revealed by electron microscopy

The Wt lens had normal fiber cell architecture (panel A) and flat epithelial cells (panel C). The fiber cells in homTg (panel B) were disorganized and had large vacuole-like structures. The epithelial cells in homTg (panel D) were irregularly arranged. Arrowhead indicates space between the epithelial and fiber cells and between fiber cells. Circles indicate gap junctions. Magnification, 10,000X. FC, fiber cell; V, vacuole.

FIGURE 5. Lens opacity in transgenic mice

A. Slit lamp images revealed that lenses from homTg mice were cataractous by 3 months of age. B. Three-month-old homTg lenses had marked opacity. Lenses from age-matched hemTg, TgGlo I, and Wt mice were normal.

FIGURE 6. Indoleamine 2,3-dioxygenase (IDO) activity in the lens

IDO activity was estimated by measuring its product, kynurenine, by reverse-phase HPLC. A. The elution profile of kynurenine showed that the peak between 12–14 min was kynurenine. Top panel, kynurenine standard and bottom panel, kynurenine in homTg lens extract. B. homTg lenses had 8-fold higher IDO activity than hemTg lenses. Methyl-tryptophan (MT)-treated homTg lens extract (homTg-MT) had 40% less activity compared to the untreated sample, whereas the activity in Wt lens extract and heat-treated homTg lens extract (homTg-heat) was negligible. Results shown are mean ± SD of 5 lenses from 5 mice. * P < 0.0001. C. Immunohistochemical localization of IDO in the lens. Prominent DAB staining (dark brown) was observed throughout the homTg lens. In hemTg lens, staining occurred only in the anterior epithelium and to some extent in the bow and outer posterior regions. Wt lens did not show appreciable DAB staining (magnification 4X).The negative control (labeled-control), where the primary was omitted did not show DAB staining. The inset shows Western blotting for IDO (45 kDa) in the water-soluble proteins using the IDO monoclonal antibody (1:5,000 diluted). Lane 1, Wt; lane 2, hemTg; lane 3, homTg. GAPDH loading control (36 kDa) is also shown. D. Quantitative real-time PCR analysis for hIDO mRNA-showed that hIDO mRNA was 4.5-fold higher in homTg lens than in hemTg lens. In the Wt lens no hIDO mRNA was present. Results shown are mean ± SD of 5 lenses from 5 mice. * P < 0.0001.

FIGURE 7. Lens kynurenine content and protein modification

A. Kynurenine content was estimated by reverse-phase HPLC. Kynurenine content was 3-fold greater in homTg than hemTg but was not detected in Wt lens. B. ELISA for kynurenine-induced modifications of lens proteins. Modifications were ~2.6 times greater in homTg lens than hemTg or Wt lens. Results shown are mean ± SD of 5 independent experiments of 5 lenses from 5 mice. * P < 0.0001; ** P= 0.002. C. Immunostaining for kynurenine modification. Marked kynurenine modifications (red) were found in homTg lens (panel C3) especially in the nucleus but not in Wt (panel C1) or hemTg (panel C2) lenses. Panel C4 presents regions at higher magnification to show kynurenine modification of nuclei (top) and fiber cells (bottom). The immune reactivity in homTg lens was markedly reduced in the absence of anti-kynurenine mAb (panel C5) and when the antibody was pre-incubated with kynurenine-modified RNase A (C6). Nuclei were stained with DAPI (blue) (magnification 40X). Figures are representative of images obtained from 3 independent experiments.

FIGURE 8. Kynurenine and GSH in the lens

A. Incubation of lens proteins with Ltryptophan revealed a ~7-fold increase in kynurenine formation in homTg lenses relative to hemTg lenses. The Wt lens proteins did not produce kynurenine. B. Lens organ culture with (black bar) and without (gray bar) L-tryptophan. Kynurenine content increased in transgenic lenses but not in Wt lens. C. Lens GSH content decreased by ~36% in homTg lenses relative to Wt and hemTg lenses. Results shown are mean ± SD of 5 lenses from 5 mice. * P < 0.0001.

FIGURE 9. Apoptosis and kynurenine modification in the lens

A. TUNEL-positive cells (green) were detected in homTg lens but not in Wt and hemTg lenses or in homTg lens incubated without terminal transferase. Nuclei were stained with DAPI (blue) (magnification 40X). B. Cells that were positive for TUNEL (green) were also positive for kynurenine modifications (red) (magnification 40X). Images in the lower panels are sections magnified from the panels above. Figures are representative of images obtained from 3 independent experiments.

FIGURE 10. Caspase activity in the lens

A. Caspase-3 activity in the homTg lens was significantly higher than in Wt and hemTg lenses. B. Caspase-9 activity was significantly higher in homTg lens compared with Wt and hemTg lenses. Results shown are mean ± SD of 5 independent experiments of 5 lenses from 5 mice. *P < 0.0001.

Figure 11. Identification of KYN-modified proteins

Water-soluble lens proteins were incubated with KYN-antibody, Protein A/G Sepharose, gel was pelleted and subjected to SDSPAGE followed by Western blotting using KYN-antibody. The major protein band (thick arrow) was analyzed by mass spectrometry and the results are presented in Table. Lane 1, Wt lens extract; Lane 2, homTg lens extract; Lane 3, Gel + homTg lens extract, Lane 4, Gel + Ab. The heavy and light chains of the antibody are indicated by thin arrows.