Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

Table 12.1 Annexin antibodies used in this study.

H2O2 in PBS for 20 min at room temperature. Sections were washed in PBS for 10 min and then incubated in 5% BSA and 0.3% triton X-100 in PBS for 2 hours. The sections were then probed with antibodies or anti-serum (below) in 1% BSA in PBS overnight at 4°C. For the control incubations, mouse IgG1 or rabbit normal serum at a concentration corresponding to that of the primary antibody or serum was used. After washing in 0.3% triton X-100 in PBS and incubation in biotin conjugated anti-mouse orrabbit IgG antibody, the sections were washed in PBS and incubated in avidin and biotin in PBS for 30 min, the sections were washed in PBS and then developed with DAB for 2 minutes. After dehydration, the sections were mounted with mounting medium. The sections were examined by light microscopy and the images were digitized using a Hamamatsu CCD camera and processed with Adobe Photoshop 4.0 software on a Power Macintosh computer.

3. DISCUSSION AND RESULTS

Annexin I immunoreactivity was intense in drusen, both on the surface and in the interior. Annexin I antibody also intensely labeled Bruch’s membrane and the choroid. Annexin II immunoreactivity was present on the surface of drusen associated with remaining basal lamina of the RPE that had not been completely removed when the Bruch’s membrane/choroid preparations had been initially isolated. No immunoreactivity was present in the interior of drusen, but punctate labeling was evident in the choriocapillaris. Annexin IV showed no immunoreactivity in drusen or in Bruch’s membrane, but the choroid appeared to be lightly labeled. Annexin VI showed heavy labeling of drusen and Bruch’s membrane. Additionally, this antibody stained the surface of drusen more intensely than any of the other annexin antibodies employed. Examples of this staining pattern are shown in the accompanying figures.

Of the four antibodies used, only annexin I and annexin VI clearly indicate that these two proteins are present in the interior of drusen. Annexin II antibody labeled the basal lamina of the RPE that remained associated with the drusen surface. It was not seen in the interior of drusen, and therefore this protein that was identified in our previous proteomic analysis of drusen must be considered as a part of closely associated cellular contaminants in the drusen preparation utilized (Crabb et al, 2002).

Only annexin IV antibody failed to label any component of the Bruch’s membranechoroid sample. We did observe annexin IV immunoreactivity at the level of the inner limiting membrane of the retina (not shown). Either annexin IV is only occasionally found in drusen and the three samples we have studied did not contain this annexin type, or the antibody employed does not function well with the fixation techniques employed. Since this antibody stains the outer limiting membrane of the retina, this latter interpretation is unlikely.

Annexins are a family of proteins that bind calcium ions and acidic phospholipids. Possible functions include being anti-inflammatory via the inhibition of phospholipase A2

(annexins I-VI), inhibiting blood coagulation (annexins I-VI), mediating exocytosis via membrane to membrane fusion, and regulating cytoskeletal organization via actin binding (annexins 1 and 2). Numerous studies are available on identification of annexins in the vascular and skeletal systems, but few on annexins in the visual system. There are reports of annexins identified in the rat cornea during inflammation (Matsuda et al, 1999) and in galac- tose-induced cataracts in rats (Koshimoto et al, 1992). Zernii et al (2003) recently detected and isolated annexin IV in bovine retinal rod outer segments. Further studies are needed to determine the role of the annexins in the retina and posterior layers of the eye.

4. ACKNOWLEDGEMENTS

Supported by NIH grants EY14240, EY15638 and a Research Center grant from The Foundation Fighting Blindness.

5. REFERENCES

Crabb, J. W., Miyagi, M., Gu, X., Shadrach, K., West, K., Sakaguchi, H., Kamei, M., Hasan, A., Yan, L., Rayborn, M. E., Salomon, R., and Hollyfield, J. G., 2002, Drusen proteome analysis: an approach to the etiology of age-related macular degeneration. Proc Natl Acad Sci. (USA) 99:14682-14687.

Kojima, K., Yamamoto, K., Irimura, T., Osawa, T., Ogawa, H., and Matsumoto. I., 1996, Characterization of car- bohydrate-binding protein p33/41 relation with annexin IV, molecular basis of the doublet forms (p33 and p41), and modulation of the carbohydrate binding activity by phospholipids. J Biol Chem. 271:7679-7685.

Koshimoto, H. N., Tokuda, M., Matsui, H., Itano, T., Hasegawa, E., and Hatase, O., 1992, Involvement of lipocortin I in development of galactose induced cataracts in rat. Exp Eye Res. 54:817-820.

Matsuda, A., Tagawa, Y., Yamamoto, K., Matsuda, H., and Kusakabe, M., 1999, Identification and immunohistocemical localization of annexin II in rat cornea. Curr Eye Res. 19:368-375.

Mollenhauer, J., 1997, Annexins. Cell Mol Life Sci. 53:506-507.

Zernii, E. Yu., Tikhimirova, P. P., and Senin, I. I., 2003, Detection of annexin IV in bovine retinal rods. Biochemistry (Mosc). 68:129-160.

PART III

ANIMAL MODELS OF RETINAL DEGENERATION

into the protein sequence, do not have an adverse effect on the enzyme activity of IMPDH1 (Aherne et al., 2004). It appears therefore that lack of enzyme activity alone is not the cause of disease symptoms.

A mouse deficient in the Impdh type 1 gene was generated using standard gene targeting techniques by Beverly Mitchell and colleagues, University of North Carolina, USA, and was kindly made available for this study. This group reported that the Impdh1-/- mouse appears to have a normal phenotype and concluded that IMPDHI enzymatic activity is not essential for normal mouse development or fertility (Gu et al., 2003). In the present study we show following electroretinographic (ERG) and histological analysis, that Impdh1-/- knockout mice display symptoms of a mild retinopathy. In addition, both wild-type and mutant IMPDH1 proteins have been expressed in cell culture systems in an effort to understand the molecular mechanisms causing photoreceptor degeneration in this form of RP. Results from these studies point to a probable dominant negative effect of mutant IMPDH1 protein causing the RP10 form of RP, and indicate that protein misfolding and aggregation, rather than reduced IMPDH1 enzyme activity or simply deficiency of IMPDH1, is the likely cause of photoreceptor degeneration causing the severe RP phenotype experienced by human subjects.

2. MATERIALS AND METHODS

2.1. Retinal Histology and Electroretinography

Eyes were perfused overnight with a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde on 0.1 M phosphate buffer, pH7.4, and processed in Epon. Sections 1 mm thick were cut through the optic nerve head, along the vertical meridian of the eye, and were stained with toludine blue for light microscopy. Mouse ERGs were recorded at 6 weeks of age (2 animals), 5 months (6 animals), 8 months (4 animals) 11 months (5 animals) and 13 months (3 animals). Animals were dark-adapted overnight and prepared for electroretinography under dim red light. ERGs were carried out as described fully in Aherne et al. 2004. Rod isolated responses were recorded using a dim white flash (-25 dB maximal intensity) presented in the dark-adapted state. The maximal combined rod/cone response to the maximal intensity flash was then recorded. Following light adaptation for 10 minutes to a background illumination of 30 candelas per m2 presented in Ganzfeld bowl the coneisolated responses were recorded to the maximal intensity flash (3 candelas per m2 per second) presented as a single flash and 10 Hz flickers.

2.2. Expression of IMPDH1 Proteins in Mammalian Cells

The Arg224Pro and Asp226Asn amino acid substitutions were introduced into the WT IMPDH1 cDNA sequence using the Quikchange site-directed mutagenesis kit (Stratagene). The WT and two mutant IMPDH1 cDNA sequences were sub-cloned into the pcDNA3.1/HisC (Invitrogen) vector, which incorporates an N-terminus His-tag in the resulting protein. Human embryonic kidney 293 (HEK 293T) cells were cultured in DMEM with 10% fetal bovine serum at 37°C and an atmosphere of 5% CO2. Cells were seeded at a density of 106 cells per 10 cm dish overnight and were transiently transfected using 20 mg of DNA and the calcium phosphate method. Cells were harvested by trypsinisation after

Figure 13.2. Light micrographs of retinal sections from Impdh1-/- mice. Sections shown are (a) 6 month Impdh1-/-, (b) 10 month Impdh1-/-, (c) 10 month wild-type C57/BL6. The outer nuclear layer thickness of Impdh1-/- retina is virtually indistinguishable to that of wild-type up to 10 months of age. (Figures adapted from Aherne et al., Human Molecular Genetics: On the molecular pathology of neurodegeneration in IMPH1-based retinitis pigmentosa, 2004, 13:641–50, by permission of Oxford University Press).

all ERG responses are unrecordable by the end of the second decade. The Impdh1-/- mouse model at 4 months of age, which we estimate to be approximately equivalent to a teenage human subject in terms of relative lifespan, displays no detectable structural or functional degeneration of the retina. In contrast the response in an affected 17-year old male from a large RP10 family to the maximal intensity flash presented in the dark adapted state indicates that there is no recordable photoreceptor activity (Fig. 13.1d) (Jordan et al., 1993; Kennan et al., 2002). Photoreceptor dysfunction in the Impdh1-/- mouse does not appear to be the result of degeneration of photoreceptor cells, since the outer nuclear layer thickness of the retinas in these mice is similar to wild type retinas up to 10 months of age, and even by 13 months of age we have found that there is only marginal loss of nuclei from the outer nuclear layer of the retina (Fig. 13.2). There is evidence of some degree of disorganisation of photoreceptor outer segments in older animals, but the actual number of ONL nuclei does not appear to be affected. Thus, IMPDH1 does not appear to be essential for normal retinal development or early visual function, and photoreceptor cells appear to survive in it’s absence.

Expression studies were carried out on wild-type and mutant IMPDH1 proteins in mammalian cells and involved transfection of constructs expressing IMPDH1 sequences into cells and subsequent extraction of cytosolic and nuclear protein fractions. This analysis indicated that a significant difference in protein solubility was detectable between the wild-type and two mutant forms of the IMPDH1 protein (Fig. 13.3). This suggests that the mutant forms of IMPDH1 protein may be misfolded, causing them to form insoluble aggregates when expressed at high levels in cells.

4. DISCUSSION

Mutations within the IMPDH1 gene are thought to account for 3-5% of autosomal dominant RP (Daiger et al., 2003). Previous studies carried out in this laboratory have shown that two of the confirmed IMPDH1 mutations studied to date (Arg224Pro and Asp226Asn) do not appear to affect the enzymatic activity of IMPDH1 (Aherne et al., 2004). Expres-

Figure 13.3. Wild-type and mutant IMPDH1 proteins expressed in HEK293T cells. (a) SDS-PAGE with Coomassie Blue staining of protein extracts from cells transfected with pcDNA3.1/His wild-type (WT), Arg224Pro and Asp226Asn mutant IMPDH1 sequences, and pcDNA vector alone (negative control). Whole cell extracts (WCE), soluble cytosolic, nuclear and final pelleted protein fractions were extracted, (b) Western blot probed with Anti-His antibody (Figure adapted from Aherne et al., Human Molecular Genetics: On the molecular pathology of neurodegeneration in IMPH1-based retinitis pigmentosa, 2004, 13:641–50, by permission of Oxford University Press).

sion of IMPDH1 proteins in mammalian cells has shown that a substantial difference in solubility exists between the wild-type and mutant forms of the protein, with the mutant proteins being less soluble. This is most likely caused by a detrimental effect on normal protein folding brought about by the presence of pathogenic mutations. This is believed to be the situation in the case of the Arg224Pro mutation and has also been predicted by computer molecular modelling studies on wild-type and mutant IMPDH1 proteins (Aherne et al., 2004). Analysis of retinal function and structure in the Impdh1-/- mouse has shown that this animal model displays symptoms of a mild retinopathy. The retina has the highest metabolic rate of any tissue of the body, with photoreceptors having a particularly high requirement for GTP in visual transduction processes (La Cour, 2002). Therefore, we suggest that depletion of the GTP pool over time is the most likely explanation for the reduction in ERG amplitudes seen in the retinas of older Impdh1-/- mice.

The Impdh1-/- mouse model displays relatively normal ERG amplitudes and histological features up to the age of 5 months, which when compared to other mouse models of RP that have been generated, would not be considered a severe degenerative retinopathy. Therefore, it appears unlikely that the autosomal dominant segregation pattern which is characteristic of the RP10 form of retinitis pigmentosa, is caused as a result of ‘haploinsufficiency’ for the normal IMPDH1 gene product. Rather, disease pathology is probably caused by a gain-of-function or dominant-negative phenotypic effect exerted by mutant protein. Protein misfolding now appears to be a common theme in discussions of the molecular mechanisms of degeneration in adRP. A number of groups have reported that misfolded RP-associated proteins can initiate the ‘unfolded protein response’ (UPR) in cells, which is a stress

response activated by the appearance of misfolded and/or aggregated proteins in the endoplasmic reticulum (ER) of the cell. There is evidence that rhodopsin and the recently identified carbonic anhydrase IV mutant proteins can upregulate the production of a number of proteins associated with the UPR (Rebello et al., 2004; Frederick et al., 2001). Differences in solubility between wild-type and mutant proteins, similar to those seen here with IMPDH1, have previously been noted with the RP-associated splicing factor protein, PRPF31 (Deery et al., 2002). In addition, protein folding defects associated with rhodopsin mutations have been demonstrated to cause the formation of insoluble aggresomes in cells (Illing et al., 2002; Saliba et al., 2002).

Interestingly, the results shown here may present a new option for therapy in this form of RP. It is possible that the suppression of both wild-type and mutant IMPDH1 alleles may, in principle, be sufficient to remove the major dominant negative pathological effect of mutant IMPDH1 protein. This may be particularly beneficial given that using current gene suppression technologies such as RNA interference, it has proven difficult to target single base pair mutations (Miller et al., 2003). Targeting both wild-type and mutant IMPDH1 alleles may result in a situation in which photoreceptor neurons continue to survive and remain sufficiently functional such as to provide useful vision, possibly well into adult life. In addition, supplementation with XMP or GTP molecules may be sufficient to ameliorate disease symptoms, without the need for gene replacement. The current focus of our research is on the generation of dominant animal models of RP caused by mutations within the IMPDH1 gene and on the assessment of novel therapies for this form of RP.

5. ACKNOWLEDGEMENTS

The Ocular Genetics Unit of TCD is supported by grants from the from the Wellcome Trust, the Health Research Board of Ireland, Science Foundation Ireland, the British RP Society, the Foundation Fighting Blindness (USA), Fighting Blindness Ireland, and the European Union 5th Framework Programme. The Ocular Genetics Unit is a member of the HEA-Ireland-sponsored Biopharmaceutical Science Network.

6. REFERENCES

Aherne, A., Kennan, A., Kenna P.F., McNally N., Lloyd D.G., Alberts I.L., Kiang A.S., Humphries M.M., Ayuso C., Engel P.C., Gu J.J., Mitchell B.S., Farrar G.J., Humphries P., 2004, On the molecular pathology of neurodegeneration in IMPDH1-based retinitis pigmentosa. Hum Mol Genet. 13:641-650.

Bowne, S.J., Sullivan, L.S., Blanton, S.H., Cepko, C.L., Blackshaw, S., Birch, D.G., Hughbanks-Wheaton, D., Heckenlively, J.R., and Daiger, S.P., 2002, Mutations in the inosine monophosphate dehydrogenase 1 gene (IMPDH1) cause the RP10 form of autosomal dominant retinitis pigmentosa. Hum Mol Genet. 11:559.

Daiger, S.P., Sullivan, L.S., Bowne, S.J., Kennan, A., Humphries, P., Birch, D.G., Heckenlively, J.R.; RP1 Consortium, 2003, Identification of the RP1 and RP10 (IMPDH1) genes causing autosomal dominant RP. Adv Exp Med Biol. 533:1-11.

Deery, E.C., Vithana, E.N., Newbold, R.J., Gallon, V.A., Bhattacharya, S.S., Warren, M.J., Hunt, D.M. and Wilkie, S.E., 2002, Disease mechanism for retinitis pigmentosa (RP11) caused by mutations in the splicing factor gene PRPF31. Hum Mol Genet. 11:3209-3219.

Frederick, J.M., Krasnoperova, N.V., Hoffmann, K., Church-Kopish, J., Ruther, K., Howes, K., Lem,, J. and Baehr, W., 2001, Mutant rhodopsin transgene expression on a null background. Invest Ophthalmol Vis Sci. 42:826833.