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Hereditary cataract of the Nakano mouse
Exp.
Eye Res.
(1990) 50, 671-676
Hereditary
Cataract MAKOTO
Department
of the Nakano
Mouse
TAKEHANA
of Biophysical Chemistry, Faculty of Pharmaceutical Meijo University, Tempaku-ku, Nagoya 468, Japan
Sciences,
The Nakano mouse is a hereditary cataract model whose most characteristic change is a deficiency in lens Na+,K+-ATPase. Consequently, there is a change in lenticuiar sodium and potassium ion levels just before cataract formation. The amounts of calcium ion also change suddenly in the lens, with accumulated levels higher than any other type of cataract. Other biochemical changes coincide with the development of lens opacity, including decreases in the levels of reduced glutathione. ATP, biosynthetic activity of proteoglycans in epitheliai cells, and the permeability of gap junction channels in fiber cells. The decrease in the activity of Na+,K+-ATPase results in changes in a number of key metabolic parameters. resulting in the eventual opacification of the Nakano mouse lens at approximately 30 days of age. Kecr words: Nakano mouse : Na+.K+-ATPase : cation : morphology : lens implantation : glycosaminoglycan : gap junction. .I
1. Introduction Opacification of the Nakano mouse lens was first characterized by Drs Kinoshita and Iwata, who presented the first detailed study of a hereditary cataract in mice (Iwata and Kinoshita, 1971). These and subsequent studies have all suggested that the Nakano mouse cataract may be an excellent model system for the study of possible biochemical changes of human cataractogenesis. The Nakano mouse cataract is inherited as an autosomal recessive trait. It was originally discovered by Dr Kenji Nakano in 1957 (Nakano, 1960). Animals from this strain open their eyes on day 14, the same as normal mice, at which time the lenses of the Nakano mice are still clear as shown in Fig. 1. Homozygous animals develop a pin head opacity in the nucleus of the lens on the 24th postnatal day, and over the next 36 days the cataract becomes mature (Fig. 1) (Iwata and Kinoshita, 1971). This paper will review recent advances in the study of the Nakano mouse system. It will include studies of cation levels. morphology, lens implantation. capsule
glycosaminoglycan synthesis, and predicted gap junctional activity in the lenses from Nakano and normal mice.
2. Experiments Growth
and Results
and Cation Contents
Relative to the lens from control mice, the Nakano mouse lens grow normally until day 14, at which time the eyes open and lens growth rate is decreased. As a result, the adult Nakano mouse lens is only half the size of a normal lens (Hikida and Iwata, 1980a : Iwata, 1980). Figure 2 shows the changes of cation contents in the normal and the Nakano lens with aging. Both potassium and sodium ion levels decrease until the time of opening of the eye at day 14, at which time both ions maintain constant levels in the control ICI? mice. In the Nakano strain, sodium ion levels increase just before lens opacification at 40 days and the cation levels consist of high sodium and low potassium (Iwata and Kinoshita. 1971; Hikida and Iwata,
FIG. 1. Macroscopic appearance of the Nakano mouse eyes at 15 and 60 days old. Oo14-4835/90/06Oh71
+06 $03.00/O
0 1990 Academic Press Limited
672
0
M. TAKEHANA
i= Ln E AT
160
-5
80
+
Y
+ 2
TABLE
Nokono
N
1
Na+,K+-ATPase activities of normal and the Nakano mouselensesi 30 duys 1
120
Strain
40
100
50
ICR Nakano
100
Lensweight Cell number Na+.K--ATPase ( x 10 :‘I (pmolPi cell ’ hr ‘1 (mg) 4.88 3.39
25.8 25.8
2.24 I.15
Age (days) 75.3
ICR
:LNokano
Co’+
Mg2+
50
100 Age
50
100
(days)
FE. 2. Cation levels in the lenses of normal (ICR) and the Nakano mice. 1980a; Iwata, 1980). Taking advantage of these initial observations by Drs Kinoshita and Iwata, many Japanese researchers have measured cation levels in the lens as one of the indicators of cataract progression. Calcium ion content also changes dramatically in the Nakano mouse lens (Fig. 2). This change is more dramatic than in any other types of cataractous lens. Magnesium ion levels remain constant with progression of the cataract (Hikida and Iwata, 1980a). Protein and Gl~tathione It is also well known that soluble protein in the lens aggregates with progression of lens opacity. The percentage of insoluble protein relative to total protein changes little in the normal lens, but in the Nakano, over 60% of the protein in the lens at 60 days old is comprised of insoluble protein (Hikida and Iwata. 1980b). Glutathione levels of the normal lens increase with aging, and the normal adult lens has a value of 10 mM kg-’ H,O. In the Nakano mouse lenses, the GSH level decreases below 50% as compared with normal lens at 60 days old and this level decreases continuously with progression of the cataract (Hikida and Iwata, 1980b).
20
40
60
Age (days)
FIG. 3. ATP levelsin the lensesof normal ([CR) and the Nakano mice. Na+,K+-ATPase activities of the lensesat 30 days after birth. These activities are expressed as enzyme units per cell. The enzyme activity of the Nakano mouselens is approximately half of the normal lens. The ATP level also drops suddenly at the time of eye opening and remains at approximately one-fourth the level of control lens throughout life (Fig. 3) (Takehana and Iwata, 1979). Concerning the deficiency of Na+,K+ATPase, Drs Kinhoshita and Iwata said in their paper (Iwata and Kinoshita, 19 71) : ‘It may be somewhat surprising that this hereditary diseaseaffects only the lens. Except for the cataract there appears to be no other abnormalities.’ In addition, the presence of a Na+,K+-ATPase inhibitor in the Nakano mouse lens has been shown by Dr Kinoshita and coworkers (Kinoshita, 1976 : Fukui, Merola and Kinoshita, 1978). Furthermore, it had been suggested by lens implantation experiments (Yamamoto and Iwata. 1973) that the cause of cataract formation of the Nakano mouse is an endogenousfactor in the lens (see Lens Implantation section).
Na+,K+-ATPase Activity The most distinctive characteristic of the Nakano mouse is the deficiency of Na+,K+-ATPase. This deficiency was initially reported by Drs Kinoshita and Iwata (Iwata and Kinoshita, 1971). Table I shows the
Morphological Changes Figure 4 summarizes the progress of morphological abnormalities in the Nakano mouse lens (Ilga, 19 8 5 j. Swelling of anterior fiber cells appears at 14 days after
HEREDITARY
CATARACT
OF THE NAKANO
FIG. 4. Schematic drawing
showing
673
MOUSE
the cataractogenesis
This is the time of the pin head opacity. At 25 days of age, both anterior and posterior fiber cells are swollen, there is destruction of the posterior suture. resulting in creation of a liquid space, followed by production of a mature cataract. birth.
Lenslmpfantation Figure 5 summarizes the lens implantation studies done with lensesfrom control and Nakano mice. This experiment was carried out by Dr Yamamoto (Yamamoto and Iwata, 1972, 1973). Adult normal and the Nakano mice, approximately 2 months old, were used as the recipients, and fi-9day-old young mice were used as donors. The initial studies were directed towards testing the feasibility of lens implantation. The young normal lens was implanted into the normal eye. This implanted lens increases in size and remains transparent. The second experiment was directed towards testing the influence of the extralenticular environment upon lens transparency. The normal lens was implanted into the Nakano eye. resulting in normal growth and transparency. The third experiment was a confirmation of the genetic defect in the lens. The young Nakano mouse lens was implanted into normal eye. Additionally, the young Nakano mouse lens was implanted into the adult Nakano mouse eye. Both clear lenses became opaque at the 18th day after implantation, demonstrating that development of lens opacity of the Nakano mouse has already been programmed in the lens.
It is also well known that the anterior capsuleof the Nakano mouse is thicker than the normal lens (Iwata. 1974 : Fukui and Yamashina. 1978) in a manner similar to basement membrane thickening during diabetes (Fisher, 1980). Figure 6 is the analysis of glycosaminoglycans
of the Nakano mouse lens with aging.
1. Feasibility
of lens implantation
Implanted
lens
2. Check of extralenticular
Implanted
3. Confirmation
Implanted
4. Reconfirmation
Implanted
Growth
transparency
Growth
and
transparency
Opacity
at 18th
environment
lens
of genetic
and
defect
lens
of hereditary
lens
FIG. 5. Method of lens implantation and the Nakano mouse (C) lenses.
cataract
Opacity
at 18th
between normal
(N)
synthesis by lens epithelial cells of the normal and the Nakano mice (Nakazawa, Takehana and Iwata. 198 5 1. After extraction of glycosaminoglycan fractions, these fractions are treated with either chondroitinase ABC or nitrous acid and then separated by gel chromatography. The results demonstrate levels of 8 5.3 % heparan sulfate and 13.1% chondroitin sulfate
M
TAKEHANA
,
94 kDa 67 kDa
43 kDa
30 kDo
99
:\ .
Nakano
20 kOa
14 kDa
M
da)
COC
FIG. 7. SDS-polyacrylamide
gel electrophoresis patterns of in normal ~tltf!/I and the
membraneintrinsic polypeptides Nakano (CIK) mouse lenses. M -3c I
40
60 Tube
00
C
N
100
number
6. Gel filtration chromatography (Sephacryl S-200) of the glycosaminoglycan fractions treated with either chondroitinase ABC (0) or nitrous acid (a) after Sepharose CL-4B chromatography.
94 kDa
in the normal lens vs. 45.90/, heparan sulfate and 54.1% chondroitin sulfate in the Nakano lens. This abnormality of glycosaminoglycan synthesis has been reported in aortas of the diabetic rat (Cohen and Foglia, 1970). where a decrease in the levels of proteoglycan synthesis was observed. This change of biosynthesis could be one of the causative factors of the capsule abnormalities.
43 kDo
FIG.
67 kDa
30 kDa
20 kDa
Membrane intrinsic Polypeptides Figure 7 shows the membrane polypeptides of the normal and the Nakano mouselenses.’ ddy ’ Designates membrane from the normal lens as a control. The normal lens membrane is comprised of poiypeptides of 23-26 kDa, but the mature cataractous lens of the Nakano mouse is comprised of only a low molecular weight polypeptide of 21 kDa. This low molecular weight polypeptide is also observed in the nucleus of normal bovine lenses, as shown in Fig. 8. The SDS-polyacrylamide gel electrophoresis pattern also shows the membrane polypeptides of bovine lens cortex and nucleus. In the cortex, the 26-kDa peptide is a major component, while the 24-kDa and 22-kDa polypeptides are the major components in the nucleus (Hibino, Takehana and Iwata. 1989).
14 kDa
FIG. 8. SDS-polyacrylamide
gel electrophoresis
patterns of
membraneintrinsic polypeptidesin cortex (C) and nucleus (N) of bovine lens. The channel forming activities of these membrane polypeptides were measured by reconstitution into liposomes (Gooden et al., 1985). An ascorbic acid solution is added to the suspension of the liposomes which are reconstituted membrane polypeptides and trapped cytochrome c. If membrane polypeptides make channels, ascorbic acid will enter the liposomesand
HEREDITARY
CATARACT
OF
THE
NAKANO
676
MOUSE
of the bovine cortex (Fig. 9) (Takehana, 1989). The permeability of the low molecular weight peptides is lower than the 26-kDa peptide. Based on the similarity of membrane polypeptides in bovine lens nucleus and the Nakano mouse lens, it seems likely that the channel forming activity in the Nakano mouse will be more like the bovine nucleus than the cortex. If we assume that the low molecular weight polypeptides of the Nakano lens originate from the 26kDa polypeptide, there is a question of which side is cleaved, the N- or the C-terminus ? N- or C-terminus specific antibodies were prepared from synthetic peptides (Gooden et al., 1985) to answer the question. Figure 10 demonstrates that the polypeptides of the Nakano lens react only with the C-terminal specific antibody, indicating that the membrane polypeptides of the Nakano mouse lens were cleaved from the Nterminal region. Without polypeptide
Cortex
Nucleus
9. Channel forming activities of membrane proteins in cortex and nucleus of bovine lens. FIG.
FIG. 10. Western blot analysis of membrane proteins of normal (ddy) and the Nakano (cat) mouse lenses using antisera against N-terminus (l-9) and C-terminus (2 54263) of the 26-kDa polypeptide.
cytochrome c will be reduced. The initial velocity of cytochrome c reduction was measured as channel forming activity. The channel forming activity of low molecular weight peptides in the bovine lens nucleus is approximately 60 “/” compared with the polypeptides 4h
3. Discussion Many of the biochemical studies of the Nakano mouse lens which were mentioned in this paper have been the result of the initiating work of Drs Kinoshita and Iwata. The most pronounced biochemical deficiency in the Nakano cataractous lenses is a decrease in the activity of Na+,K+-ATPase. Many biochemical changes such as GSH, ATP, etc. and the changes of cation levels arise from this enzyme deficiency. Overall, it can be concluded that lens opacification is due to an osmotic effect resulting from the accumulation of sodium ions (Hamai, Fukui and Kuwabara, 1974: Iwata, 1980). Concerning glycosaminoglycan synthesis of epithelial cells, Dr Nakazawa concluded that ‘a deficiency in Na+,K+-ATPase activity may act as a trigger on the development of cataract, and the subsequent changes in cation balance may affect protein synthesis, including proteoglycan synthesis (Shinohara and Piatigorsky. 1977). The decreased synthesis of proteoglycan could be one of the cataractogenic factors’ (Nakazawa, Takehana and Iwata. 1985). Morphological changes start with a delay in denucleation of the cells and the swelling of fiber cells at the posterior suture area. Finally, cells of the posterior suture region liquify completely. Similar changes to the bow area and the posterior suture are observed not only in the lens of the Nakano mouse, but in many other types of cataracts as well (Uga. 1985). The result of the gap junction experiments show that it is difficult to form channels with the 24kDa and 22-kDa peptides as compared with the 26-kDa peptide. Supposedly, it demonstrates that the channel forming activities decrease in proportion to the decrease in the molecular weight of the 26-kDa peptide. As a result, it may be difficult to maintain cellto-cell communication, resulting in metabolic abnormalities of the lens. Furthermore. it is clear that the EER50
676 low molecular weight polypeptides are cleaved from the N-terminal side in the Nakano mouse similar to the human senile cataractous lens (Takemoto and Takehana. 1986a, bj.
Acknowledgments I would like to thank Drs Hikida, Uga, Yamamoto and Nakazawa for sharing their data. I am grateful to Dr Larry Takemoto for a review of this manuscript.
References Cohen, M. P. and Fogiia. V. G. (1970). Aortic mucopolysaccharides in experimental diabetes. Diabetes 19. 639-43. Fisher. R. F. (1980). A comparison of the local and remote effects of basement membrane regeneration in normal and diabetic rats. Br. 1. Patho/. 61, 661-71. Fukui, H. N., Merola. L. 0. and Kinoshita. J. H. (1978). A possible cataractogenic factor in the Nakano mouse lens. Exp. Eye Res. 26, 477-85. Fukui, S. and Yamashina, I. (1978). Biochemical alterations of the lens capsule in mice with hereditary cataract. Exp. Eye Res. 26, 499-506. Gooden, M., Rintoul, D.. Takehana, M. and Takemoto. L. (1985). Major intrinsic polypeptide (MIP26K) from lens membrane : Reconstitution into vesicles and inhibition of channel forming activity by peptide antiserum. Biochem. Biophys. Res. Commun. 128. 993-9. Hamai. Y., Fukd, H. N. and Kuwabara. T. (1974). Morphology of hereditary mouse cataract. Exp. Eye Res. 18. 537-46. Hibino. T., Takehana, M. and Iwata, S. (1989) Differences of gap junction channels between cortex and nucleus. 1. Eye 6. 393-5. Hikida, M. and Iwata, S. (1980a). Differences on opaque appearances and biochemical parameters of cataract lenses in mice carrying the dominant or recessive gene. Proc. ]pn. Chap. ICER. 40-l. Hikida. M. and Iwata. S. (1980b). Relationship between opaque appearances and -SH compounds of cataractous lenses in mice carrying the dominant and recessive genes. Abstract of 19th Congress of Catarnct Research Group, Yonago. Japan,
M TAKEHANA
Iwata, S. ( 1974). Process of lens opaciiication and membraue function. Ophthalmic Rrs. 6. 1 38-54. Iwata. S. (1980). Phenotypical analysis and biochemical parameters of hereditary mouse cataracts carrying recessive and dominant genes. In Ageing of the Lens (Eds Regnault. F.. Hockwin. 0. and Courtois, Y.). Pp. 17 l-9. Elsevier/North-Holland Biomedical Press : Amsterdam. Iwata. S. and Kinoshita, J. (1971). Mechanism of development of hereditary cataract in mice, Invest. Ophthuhnol. Vis. Sci. 10. 504-I 2. Kinoshita. J. (1976). Biochemical basis on cataract formation. Acto Sot. Ophtlittlmol. lpn 80. 1 362-71. Nakano. K.. Yamamoto. S.. Kutsukake. C.. Ogawa. H.. Nakajima, A. and Takano. E. (1960). Hereditary cataract in mice. fpn. I. Clin. Ophthufmok 14. 1772-6. Nakazawa. K.. Takehana. M. and Iwata. S. (1985). Biosynthesis of proteoglycans by lens epithelial cells of cataractous mouse (Nakano strain). Esp. Q/a RPS. 40. 609-l 8. Shinohara, T. and Piatigorsky, J. (1977). Regulation of protein synthesis, intra-cellular electrolytes and cataract formation in vitro. Nuture 270. 406-l 1. Takehana. M. (1989) The lens as the independent organ: Scavenging and cell-to-cell communication systems. 1. \pn. Sot. Cuturuct Rrs. 1, 1-8. Takehana. M. and Iwata. S. (1979). Changes of ATP. ADP and AMP levels in the lenses of hereditary cataract mouse. Abstract of 6th Congress for Lens Research, Nagoya, Japan. Takemoto, L. and Takehana, M. (1986a). Covalent change of major intrinsic polypeptide (MIP26K) of lens membrane during human senile cataractogenesis. B~UC~~TJI. Biopltys. Rrs. Commun. 135. 965-71. Takemoto, L. and Takehana. M. (1986b). Major intrinsic polypeptide (MIP26K) from human lens membrane: Characterization of low-molecular-weight forms in the aging human lens. Exp. Eye RPS. 43. 661-7. Uga, S. (1985). Mechanisms of cataract formation: Comparison of human cataractous lens with animal models. 1. Eye 2. 239-42. Yamamoto. Y. and Iwata. S. (1972). implantation of the crystalline lens in mice: A preliminary report. fpn. J. Ophthulmol. 16. 300- 3. Yamamoto, Y. and Iwata, S. (1973). Implantation of crystalline lens in mice: As one of the approaches for cataract study. Actu SW. Ophthulmol. Ipn. 77. 888-96.
Eye Res.
(1990) 50, 671-676
Hereditary
Cataract MAKOTO
Department
of the Nakano
Mouse
TAKEHANA
of Biophysical Chemistry, Faculty of Pharmaceutical Meijo University, Tempaku-ku, Nagoya 468, Japan
Sciences,
The Nakano mouse is a hereditary cataract model whose most characteristic change is a deficiency in lens Na+,K+-ATPase. Consequently, there is a change in lenticuiar sodium and potassium ion levels just before cataract formation. The amounts of calcium ion also change suddenly in the lens, with accumulated levels higher than any other type of cataract. Other biochemical changes coincide with the development of lens opacity, including decreases in the levels of reduced glutathione. ATP, biosynthetic activity of proteoglycans in epitheliai cells, and the permeability of gap junction channels in fiber cells. The decrease in the activity of Na+,K+-ATPase results in changes in a number of key metabolic parameters. resulting in the eventual opacification of the Nakano mouse lens at approximately 30 days of age. Kecr words: Nakano mouse : Na+.K+-ATPase : cation : morphology : lens implantation : glycosaminoglycan : gap junction. .I
1. Introduction Opacification of the Nakano mouse lens was first characterized by Drs Kinoshita and Iwata, who presented the first detailed study of a hereditary cataract in mice (Iwata and Kinoshita, 1971). These and subsequent studies have all suggested that the Nakano mouse cataract may be an excellent model system for the study of possible biochemical changes of human cataractogenesis. The Nakano mouse cataract is inherited as an autosomal recessive trait. It was originally discovered by Dr Kenji Nakano in 1957 (Nakano, 1960). Animals from this strain open their eyes on day 14, the same as normal mice, at which time the lenses of the Nakano mice are still clear as shown in Fig. 1. Homozygous animals develop a pin head opacity in the nucleus of the lens on the 24th postnatal day, and over the next 36 days the cataract becomes mature (Fig. 1) (Iwata and Kinoshita, 1971). This paper will review recent advances in the study of the Nakano mouse system. It will include studies of cation levels. morphology, lens implantation. capsule
glycosaminoglycan synthesis, and predicted gap junctional activity in the lenses from Nakano and normal mice.
2. Experiments Growth
and Results
and Cation Contents
Relative to the lens from control mice, the Nakano mouse lens grow normally until day 14, at which time the eyes open and lens growth rate is decreased. As a result, the adult Nakano mouse lens is only half the size of a normal lens (Hikida and Iwata, 1980a : Iwata, 1980). Figure 2 shows the changes of cation contents in the normal and the Nakano lens with aging. Both potassium and sodium ion levels decrease until the time of opening of the eye at day 14, at which time both ions maintain constant levels in the control ICI? mice. In the Nakano strain, sodium ion levels increase just before lens opacification at 40 days and the cation levels consist of high sodium and low potassium (Iwata and Kinoshita. 1971; Hikida and Iwata,
FIG. 1. Macroscopic appearance of the Nakano mouse eyes at 15 and 60 days old. Oo14-4835/90/06Oh71
+06 $03.00/O
0 1990 Academic Press Limited
672
0
M. TAKEHANA
i= Ln E AT
160
-5
80
+
Y
+ 2
TABLE
Nokono
N
1
Na+,K+-ATPase activities of normal and the Nakano mouselensesi 30 duys 1
120
Strain
40
100
50
ICR Nakano
100
Lensweight Cell number Na+.K--ATPase ( x 10 :‘I (pmolPi cell ’ hr ‘1 (mg) 4.88 3.39
25.8 25.8
2.24 I.15
Age (days) 75.3
ICR
:LNokano
Co’+
Mg2+
50
100 Age
50
100
(days)
FE. 2. Cation levels in the lenses of normal (ICR) and the Nakano mice. 1980a; Iwata, 1980). Taking advantage of these initial observations by Drs Kinoshita and Iwata, many Japanese researchers have measured cation levels in the lens as one of the indicators of cataract progression. Calcium ion content also changes dramatically in the Nakano mouse lens (Fig. 2). This change is more dramatic than in any other types of cataractous lens. Magnesium ion levels remain constant with progression of the cataract (Hikida and Iwata, 1980a). Protein and Gl~tathione It is also well known that soluble protein in the lens aggregates with progression of lens opacity. The percentage of insoluble protein relative to total protein changes little in the normal lens, but in the Nakano, over 60% of the protein in the lens at 60 days old is comprised of insoluble protein (Hikida and Iwata. 1980b). Glutathione levels of the normal lens increase with aging, and the normal adult lens has a value of 10 mM kg-’ H,O. In the Nakano mouse lenses, the GSH level decreases below 50% as compared with normal lens at 60 days old and this level decreases continuously with progression of the cataract (Hikida and Iwata, 1980b).
20
40
60
Age (days)
FIG. 3. ATP levelsin the lensesof normal ([CR) and the Nakano mice. Na+,K+-ATPase activities of the lensesat 30 days after birth. These activities are expressed as enzyme units per cell. The enzyme activity of the Nakano mouselens is approximately half of the normal lens. The ATP level also drops suddenly at the time of eye opening and remains at approximately one-fourth the level of control lens throughout life (Fig. 3) (Takehana and Iwata, 1979). Concerning the deficiency of Na+,K+ATPase, Drs Kinhoshita and Iwata said in their paper (Iwata and Kinoshita, 19 71) : ‘It may be somewhat surprising that this hereditary diseaseaffects only the lens. Except for the cataract there appears to be no other abnormalities.’ In addition, the presence of a Na+,K+-ATPase inhibitor in the Nakano mouse lens has been shown by Dr Kinoshita and coworkers (Kinoshita, 1976 : Fukui, Merola and Kinoshita, 1978). Furthermore, it had been suggested by lens implantation experiments (Yamamoto and Iwata. 1973) that the cause of cataract formation of the Nakano mouse is an endogenousfactor in the lens (see Lens Implantation section).
Na+,K+-ATPase Activity The most distinctive characteristic of the Nakano mouse is the deficiency of Na+,K+-ATPase. This deficiency was initially reported by Drs Kinoshita and Iwata (Iwata and Kinoshita, 1971). Table I shows the
Morphological Changes Figure 4 summarizes the progress of morphological abnormalities in the Nakano mouse lens (Ilga, 19 8 5 j. Swelling of anterior fiber cells appears at 14 days after
HEREDITARY
CATARACT
OF THE NAKANO
FIG. 4. Schematic drawing
showing
673
MOUSE
the cataractogenesis
This is the time of the pin head opacity. At 25 days of age, both anterior and posterior fiber cells are swollen, there is destruction of the posterior suture. resulting in creation of a liquid space, followed by production of a mature cataract. birth.
Lenslmpfantation Figure 5 summarizes the lens implantation studies done with lensesfrom control and Nakano mice. This experiment was carried out by Dr Yamamoto (Yamamoto and Iwata, 1972, 1973). Adult normal and the Nakano mice, approximately 2 months old, were used as the recipients, and fi-9day-old young mice were used as donors. The initial studies were directed towards testing the feasibility of lens implantation. The young normal lens was implanted into the normal eye. This implanted lens increases in size and remains transparent. The second experiment was directed towards testing the influence of the extralenticular environment upon lens transparency. The normal lens was implanted into the Nakano eye. resulting in normal growth and transparency. The third experiment was a confirmation of the genetic defect in the lens. The young Nakano mouse lens was implanted into normal eye. Additionally, the young Nakano mouse lens was implanted into the adult Nakano mouse eye. Both clear lenses became opaque at the 18th day after implantation, demonstrating that development of lens opacity of the Nakano mouse has already been programmed in the lens.
It is also well known that the anterior capsuleof the Nakano mouse is thicker than the normal lens (Iwata. 1974 : Fukui and Yamashina. 1978) in a manner similar to basement membrane thickening during diabetes (Fisher, 1980). Figure 6 is the analysis of glycosaminoglycans
of the Nakano mouse lens with aging.
1. Feasibility
of lens implantation
Implanted
lens
2. Check of extralenticular
Implanted
3. Confirmation
Implanted
4. Reconfirmation
Implanted
Growth
transparency
Growth
and
transparency
Opacity
at 18th
environment
lens
of genetic
and
defect
lens
of hereditary
lens
FIG. 5. Method of lens implantation and the Nakano mouse (C) lenses.
cataract
Opacity
at 18th
between normal
(N)
synthesis by lens epithelial cells of the normal and the Nakano mice (Nakazawa, Takehana and Iwata. 198 5 1. After extraction of glycosaminoglycan fractions, these fractions are treated with either chondroitinase ABC or nitrous acid and then separated by gel chromatography. The results demonstrate levels of 8 5.3 % heparan sulfate and 13.1% chondroitin sulfate
M
TAKEHANA
,
94 kDa 67 kDa
43 kDa
30 kDo
99
:\ .
Nakano
20 kOa
14 kDa
M
da)
COC
FIG. 7. SDS-polyacrylamide
gel electrophoresis patterns of in normal ~tltf!/I and the
membraneintrinsic polypeptides Nakano (CIK) mouse lenses. M -3c I
40
60 Tube
00
C
N
100
number
6. Gel filtration chromatography (Sephacryl S-200) of the glycosaminoglycan fractions treated with either chondroitinase ABC (0) or nitrous acid (a) after Sepharose CL-4B chromatography.
94 kDa
in the normal lens vs. 45.90/, heparan sulfate and 54.1% chondroitin sulfate in the Nakano lens. This abnormality of glycosaminoglycan synthesis has been reported in aortas of the diabetic rat (Cohen and Foglia, 1970). where a decrease in the levels of proteoglycan synthesis was observed. This change of biosynthesis could be one of the causative factors of the capsule abnormalities.
43 kDo
FIG.
67 kDa
30 kDa
20 kDa
Membrane intrinsic Polypeptides Figure 7 shows the membrane polypeptides of the normal and the Nakano mouselenses.’ ddy ’ Designates membrane from the normal lens as a control. The normal lens membrane is comprised of poiypeptides of 23-26 kDa, but the mature cataractous lens of the Nakano mouse is comprised of only a low molecular weight polypeptide of 21 kDa. This low molecular weight polypeptide is also observed in the nucleus of normal bovine lenses, as shown in Fig. 8. The SDS-polyacrylamide gel electrophoresis pattern also shows the membrane polypeptides of bovine lens cortex and nucleus. In the cortex, the 26-kDa peptide is a major component, while the 24-kDa and 22-kDa polypeptides are the major components in the nucleus (Hibino, Takehana and Iwata. 1989).
14 kDa
FIG. 8. SDS-polyacrylamide
gel electrophoresis
patterns of
membraneintrinsic polypeptidesin cortex (C) and nucleus (N) of bovine lens. The channel forming activities of these membrane polypeptides were measured by reconstitution into liposomes (Gooden et al., 1985). An ascorbic acid solution is added to the suspension of the liposomes which are reconstituted membrane polypeptides and trapped cytochrome c. If membrane polypeptides make channels, ascorbic acid will enter the liposomesand
HEREDITARY
CATARACT
OF
THE
NAKANO
676
MOUSE
of the bovine cortex (Fig. 9) (Takehana, 1989). The permeability of the low molecular weight peptides is lower than the 26-kDa peptide. Based on the similarity of membrane polypeptides in bovine lens nucleus and the Nakano mouse lens, it seems likely that the channel forming activity in the Nakano mouse will be more like the bovine nucleus than the cortex. If we assume that the low molecular weight polypeptides of the Nakano lens originate from the 26kDa polypeptide, there is a question of which side is cleaved, the N- or the C-terminus ? N- or C-terminus specific antibodies were prepared from synthetic peptides (Gooden et al., 1985) to answer the question. Figure 10 demonstrates that the polypeptides of the Nakano lens react only with the C-terminal specific antibody, indicating that the membrane polypeptides of the Nakano mouse lens were cleaved from the Nterminal region. Without polypeptide
Cortex
Nucleus
9. Channel forming activities of membrane proteins in cortex and nucleus of bovine lens. FIG.
FIG. 10. Western blot analysis of membrane proteins of normal (ddy) and the Nakano (cat) mouse lenses using antisera against N-terminus (l-9) and C-terminus (2 54263) of the 26-kDa polypeptide.
cytochrome c will be reduced. The initial velocity of cytochrome c reduction was measured as channel forming activity. The channel forming activity of low molecular weight peptides in the bovine lens nucleus is approximately 60 “/” compared with the polypeptides 4h
3. Discussion Many of the biochemical studies of the Nakano mouse lens which were mentioned in this paper have been the result of the initiating work of Drs Kinoshita and Iwata. The most pronounced biochemical deficiency in the Nakano cataractous lenses is a decrease in the activity of Na+,K+-ATPase. Many biochemical changes such as GSH, ATP, etc. and the changes of cation levels arise from this enzyme deficiency. Overall, it can be concluded that lens opacification is due to an osmotic effect resulting from the accumulation of sodium ions (Hamai, Fukui and Kuwabara, 1974: Iwata, 1980). Concerning glycosaminoglycan synthesis of epithelial cells, Dr Nakazawa concluded that ‘a deficiency in Na+,K+-ATPase activity may act as a trigger on the development of cataract, and the subsequent changes in cation balance may affect protein synthesis, including proteoglycan synthesis (Shinohara and Piatigorsky. 1977). The decreased synthesis of proteoglycan could be one of the cataractogenic factors’ (Nakazawa, Takehana and Iwata. 1985). Morphological changes start with a delay in denucleation of the cells and the swelling of fiber cells at the posterior suture area. Finally, cells of the posterior suture region liquify completely. Similar changes to the bow area and the posterior suture are observed not only in the lens of the Nakano mouse, but in many other types of cataracts as well (Uga. 1985). The result of the gap junction experiments show that it is difficult to form channels with the 24kDa and 22-kDa peptides as compared with the 26-kDa peptide. Supposedly, it demonstrates that the channel forming activities decrease in proportion to the decrease in the molecular weight of the 26-kDa peptide. As a result, it may be difficult to maintain cellto-cell communication, resulting in metabolic abnormalities of the lens. Furthermore. it is clear that the EER50
676 low molecular weight polypeptides are cleaved from the N-terminal side in the Nakano mouse similar to the human senile cataractous lens (Takemoto and Takehana. 1986a, bj.
Acknowledgments I would like to thank Drs Hikida, Uga, Yamamoto and Nakazawa for sharing their data. I am grateful to Dr Larry Takemoto for a review of this manuscript.
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