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

Grover, S., Fishman, G.A. and Stone, E.M., 2004, A novel IMPDH1 mutation (Arg231Pro) in a family with a severe form of autosomal dominant retinitis pigmentosa. Ophthalmology. 111:1910-1916.

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Kennan, A., Aherne, A., Palfi, A., Humphries, M., McKee, A., Stitt, A., Simpson, D.A., Demtroder, K., Orntoft, T., Ayuso, C., Kenna, P.F., Farrar, G.J., and Humphries, P., 2002, Identification of an IMPDH1 mutation in autosomal dominant retinitis pigmentosa (RP10) revealed following comparative microarray analysis of transcripts derived from retinas of wild-type and Rho (-/-) mice. Hum Mol Genet. 11:547.

Kennan, A., Aherne, A., Bowne, S.J., Daiger, S.P., Farrar, G.J., Kenna, P.F., Humphries, P., 2003, On the role of IMPDH1 in retinal degeneration. Adv Exp Med Biol 533:13-18.

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La Cour, M., 2002, The retinal pigment epithelium. In Kaufman, P.L., Alm, A. (eds), Adler’s Physiology of the Eye. 10th Ed. Mosby, St. Louis, pp. 348-357.

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tions include missense mutations, nonsense mutations, and short deletions.8 AIPL1 mutations linked to LCA are present in either the N-terminal immunophilin like domain (class I) or in the TPR domain (class II). Mutations in the C-terminal proline rich domain (class III) have been linked to dominant cone-rod dystrophy or juvenile retinitis pigmentosa and LCA.9

2. ROLE OF AIPL1 IN RETINAFINDINGS FROM AIPL1 DEFICIENT MICE

To elucidate the role of AIPL1 in the retina and to develop an animal model to study LCA caused by AIPL1 deficiency, we created an AIPL1 knock out mouse. AIPL1-/- mice demonstrate a phenotype consistent with LCA. Specifically, AIPL1-/- mice exhibit no measurable electroretinogram (ERG) response at any age and are completely blind at birth10 (Fig. 14.1). Ultra-structural details of the retina analyzed by electron microscopy show no obvious difference between wild type and knock out mice at post natal day 8 (P8) (Fig. 14.1).10 At P8, AIPL1 deficient mice show a normal complement of rod and cone photoreceptor cells with morphologically normal rod and cone outer segments. This suggests that AIPL1 does not play an essential role in the initial formation of rods and cone photoreceptor cells. At P11, the photoreceptor layer of the retina in the knock out mouse is morphologically indistinguishable form that of wild type by light microscopy, but ultra structural details observed by electron microscopy show disorganized and fragmented outer segments compared to wild type.10 This suggests that AIPL1 plays an essential role in either maintaining the outer segment and/or further photoreceptor cell differentiation after P9 in mice. The photoreceptor nuclear layer is reduced to half at P14 and at P18 the photoreceptor nuclear layer is only 1 cell thick in mice lacking AIPL1.10 By four weeks after birth, the degeneration is complete (Fig. 14.1). Both rod and cone photoreceptor cells degenerate at a similarly rapid rate.10

At the molecular level, differences between wild type and AIPL1-/- retinas appear earlier than the morphological differences. At P8, prior to the onset of retinal degeneration, AIPL1 deficient mice show reduced levels of cGMP phosphodiesterase (PDE) protein.10 The reduction in level of PDE is specific, as the levels of other photoreceptor-specific proteins such as Rhodopsin (Rho), Guanylyl cyclase (GC-E) are normal.10 All three subunits of PDE abg are reduced by 90% despite normal levels of the mRNAs.10 Additionally, no cGMPdependent PDE activity can be detected in the knockout retinas, implying that the PDE that is present in AIPL1-/- is dysfunctional. Consistent with the loss of PDE activity, cGMP levels are high starting at P8.10 Destabilization of rod cGMP PDE as a pathogenic mechanism has precedent in the mouse. The well-characterized rd (retinal dystrophic) mouse results from a truncation mutation and/or a viral insertion in the rod PDEb gene that causes a reduction in PDEb mRNA and loss of rod cGMP PDE.11,12 In rd/rd mice, there is a pattern of rapid photoreceptor cell degeneration similar to the degeneration that occurs in AIPL1-/- mice.12 However, there are significant differences between rd/rd and AIPL1-/- mice.

In AIPL1-/- mice, both rods and cone photoreceptor cells degenerate at a similar rapid rate, whereas in rd/rd mice, rods degenerate faster than cones.10,12 In AIPL1-/- mice there is no recordable ERG at any age, whereas in rd/rd mice there are some cone responses at postnatal day 12.10,12 This is consistent with the fact that in humans, mutations in AIPL1 cause severe blindness that affects both rods and cones whereas deficiencies in PDE cause retinitis pigmentosa, primarily a rod disease.13

Figure 14.1. Rapid degeneration (a-b) and electrophysiological responses (c-d) in mice lacking AIPL1. Paraffin sections (5 mm) from whole eye stained with hematoxylin and eosin. RPE, retinal pigment epithelium; OS, outer segments; IS, inner segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer a) At P8, there is no difference in the thickness of ONL between wild-type and Aipl1-/- littermates b) By P30, the degeneration of photoreceptor is complete with no ONL. Aipl1-/- section is shown at 100X magnification compared to wild-type, which is shown at 20X magnification. c and d) ERG responses to moderate (300 nJ/cm2, lighter traces) and bright (300 mJ/cm2, darker traces) flashes recorded from P12 wild-type (c) and P12 Aipl1-/- retinas. No ERG responses could be elicited from any of the Aipl1-/- mice examined. (Figure reproduced from Ramamurthy et al., PNAS (2004), 101, 38, 13897-902).

In addition to the knockout mouse, an AIPL1 knockdown mouse was created in which AIPL1 expression was reduced to 20-25% of wild-type levels.14 The knockdown mouse exhibits normal development and normal retinal morphology up to three months of age. At three months, the rod photoreceptor outer segments become disorganized, and by 8 months, more than half of the photoreceptors are lost. Similar to the AIPL1 knock out mouse, PDE protein levels were drastically reduced despite normal mRNA message levels prior to the onset of retinal degeneration.14 The knockdown mice exhibit ERG responses with lower gain and a longer response delay, consistent with a reduced level of rod PDE.14 Surprisingly, cGMP levels were found to be lower in the AIPL1 knockdown rods. This is in contrast to the AIPL1 knockout mice, where reduced levels of PDE result in higher levels of cGMP.14

The phenotype of the AIPL1 knockdown mice further supports the hypothesis that the LCA-related defect in AIPL1 is caused by a defect in the maintenance of rod photoreceptors, as the knockdown mice show normal development and persist with functional photoreceptors for several months prior to onset of the degeneration. The AIPL1 deficient mice suggest that the essential role of AIPL1 in retina is to stabilize the active rod cGMP phosphodiesterase holoenzyme.

3. ROLE OF AIPL1 IN THE STABILITY OF PDE HOLOENZYME

At P8, before the degeneration, AIPL1 deficient mice express 10% or less of all three PDE subunits (a,b and g) despite normal message levels.10 The link between AIPL1 and the stability of PDE holoenzyme is not clear. However, a previous yeast-two hybrid screen together with HEK cell expression studies suggest the involvement of the post-translational modification farnesylation.6

Farnesylation, a type of prenylation, is a post-translational modification that occurs at the C-termini of proteins that contain a C-terminal “CaaX” box signal sequence (Cys- aliphatic-aliphatic-specific amino acid). Farnesylation is a multi-step process. In the first step, farnesyl transferase (FTase) catalyzes the covalent attachment of a farnesyl (C-15) group to the conserved cysteine of the CaaX box. The farnesylated protein is then targeted to the endoplasmic reticulum where the C-terminal three amino acid residues (-aaX) are removed, and the exposed farnesyl cysteine is carboxymethylated.15 Geranylgeranylation is a similar modification that adds a geranylgeranyl (C20) group instead of the farnesyl group to the C-terminus of the protein. PDE-a and PDE-b are known to be farnesylated and geranylgeranylated, respectively.16 The prenylation of PDE-a and b subunits is essential for stability and membrane interactions.17 PDE-a and b subunit mutants, in which conserved CaaX box cysteine is replaced by serine to prevent prenylation, are unstable and degrade rapidly when expressed in insect cells.17 It is not clear whether the methylation or the prenylation modification is essential for the stability of PDE. Inhibition of prenylation in adult rat retina causes the whole retinal cytoarchitecture and photoreceptor structure to fall apart rapidly, suggesting that prenylation is required for photoreceptor structure maintenance.18

The three TPR domains of AIPL1 suggest that the protein is involved in multi-protein complexes with chaperone, transcriptional, cell-cycle regulation, or protein transport activities.19 TPR domains participate in protein-protein interactions by interacting with the C- termini of proteins.19 In agreement with this, AIPL1 interacts with proteins that have a conserved farnesylation signal at their C-termini.6 Furthermore, in cultured human embryonic kidney cells (HEK-293), AIPL1 enhances the farnesylation of proteins.6 However, the mechanism of this enhancement is not presently known. In AIPL1-/- mice, photoreceptor degeneration seems to be primarily due to loss of PDE subunits. AIPL1 specifically enhances the stability of all three PDE subunits. Surprisingly, the stability of other known farnesylated retinal proteins, such as rhodopsin kinase and transducin gamma subunit are not altered in the absence of AIPL1.10,14 This suggests that the role of AIPL1 is complex and that it may not play a general role in enhancing the farnesylation of retinal proteins. Alternatively, this could reflect the importance of prenylation specifically for stability of PDE.

AIPL1 also interacts with Nedd8 ultimate buster (NUB1), a protein ubiquitously expressed and thought to be involved in proteolysis.20 AIPL1-NUB1 interaction could play a role in the stability of PDE. The presence of an immunophilin like domain in AIPL1 suggests that AIPL1 may play a role as a chaperone of PDE subunits. AIPL1 could either stabilize them or aid in the assembly of the three PDE subunits. Further studies are warranted to understand the requirement of AIPL1 for the stability of rod PDE subunits. At present it is not known if AIPL1 also plays a role in the stability of cone PDE subunits. It seems unlikely, as AIPL1 is not expressed in mature human cones.

It has been suggested that AIPL1 like other immunophilin and TPR containing proteins, could be involved in protein transport. However, our experiments so far do not support a role for AIPL1 in the transport of retinal proteins frorm inner to outer segments.10 Most of

the retinal proteins including the residual PDE are localized normally in the absence of AIPL1.

4. AIPL1 AND RETINAL DEVELOPMENT

The phenotype of AIPL1 deficient mice shows that AIPL1 is necessary for either photoreceptor differentiation and/or maintenance.10 It has been suggested that AIPL1 plays a critical role in the early development of both rod and cone photoreceptors. AIPL1 is expressed in both rod and cone photoreceptors in early human retinal development, and mutations in the AIPL1 gene are associated with severe blinding disease.7,9 NUB1, a protein that interacts with AIPL1, is thought to be involved in cell-cycle progression.20 This has lead to the hypothesis that AIPL1 plays an early role in regulating retinal cell fate decision and or retinal progenitor cell proliferation.20 However, we have not seen much evidence so far to support a developmental role for AIPL1.10 Ultrastructural details of retina analyzed at post-natal day 8 do not show any obvious alterations in mice lacking AIPL1.10 A recent AIPL1 knock out mouse model with a rapid retinal degeneration similar to our AIPL1 knockout, shows no significant defects in number of rods, cones or any second order neuronal cells, such as bipolar, amacrine or ganglion cells.21 However, unlike rd/rd mice, which also have a defective PDE, AIPL1-/- never exhibit any electrical response to light at any age tested, reflecting the rapid cone degeneration seen in AIPL1-/- mice in comparison to rd/rd mice.10,21 It is possible that the rapid cone degeneration seen in AIPL1-/- is an indirect effect due to the early and severe rod photoreceptor cell degeneration. In agreement with this, recent studies show that rod photoreceptor cells produce viability factors (RdCVF) that are essential for survival of cones.22 Alternatively, AIPL1 may play an important role in early cones, which is consistent with its early expression in cones.7 In mature human retina, AIPL1 is expressed exclusively in rod photoreceptor cells suggesting that AIPL1 is necessary for maintenance of rod photoreceptors.7 Whether the shift in the expression from developing cone and rod photoreceptor cells to only mature rods reflects a shift in the function of AIPL1 is presently not known. More studies are needed to address the role of AIPL1 in developing cones, as these will be crucial in the design of suitable therapies for treating patients with AIPL1 associated LCA.

5. CONCLUSIONS

The AIPL1 knockout mouse replicates the human LCA caused by AIPL1 mutations and is a suitable animal model to test novel therapies. Similar to human patients with LCA, mice lacking AIPL1 do not exhibit any electrical responses. Both rod and cone photoreceptor cells degenerate rapidly. The role of AIPL1 in cones is not clear. However, the rapid cone degeneration in AIPL1-/- mice suggests that AIPL1 is necessary for the survival of the cones. Our study shows that the rapid rod photoreceptor cell degeneration is due to lack of stable PDE trimeric holenzyme. The mechanism by which AIPL1 contributes to the stability of PDE is unknown. Further study of the biochemical function of AIPL1 is required to understand the significance of AIPL1 to photoreceptor differentiation and or maintenance. In addition to the direct application to therapies for LCA patients, such information may enhance our understanding of photoreceptor development and long-term photoreceptor survival.

6.REFERENCES

1.Cremers, F.P., van den Hurk, J.A. & den Hollander, A.I. Molecular genetics of Leber congenital amaurosis. Hum Mol Genet 11:1169-76 (2002).

2.Hanein, S. et al. Leber congenital amaurosis: comprehensive survey of the genetic heterogeneity, refinement of the clinical definition, and genotype-phenotype correlations as a strategy for molecular diagnosis. Hum Mutat 23:306-17 (2004).

3.Sohocki, M.M. et al. Mutations in a new photoreceptor-pineal gene on 17p cause Leber congenital amaurosis. Nat Genet 24:79-83 (2000).

4.Sohocki, M.M., Sullivan, L.S., Tirpak, D.L. & Daiger, S.P. Comparative analysis of aryl-hydrocarbon receptor interacting protein-like 1 (Aipl1), a gene associated with inherited retinal disease in humans. Mamm Genome 12:566-8 (2001).

5.van der Spuy, J. et al. The Leber congenital amaurosis gene product AIPL1 is localized exclusively in rod photoreceptors of the adult human retina. Hum Mol Genet 11:823-31 (2002).

6.Ramamurthy, V. et al. AIPL1, a protein implicated in Leber’s congenital amaurosis, interacts with and aids in processing of farnesylated proteins. Proc Natl Acad Sci U S A 100:12630-5 (2003).

7.van der Spuy, J. et al. The expression of the Leber congenital amaurosis protein AIPL1 coincides with rod and cone photoreceptor development. Invest Ophthalmol Vis Sci 44:5396-403 (2003).

8.Dharmaraj, S. et al. The phenotype of Leber congenital amaurosis in patients with AIPL1 mutations. Arch Ophthalmol 122:1029-37 (2004).

9.Sohocki, M.M. et al. Prevalence of AIPL1 mutations in inherited retinal degenerative disease. Mol Genet Metab 70:142-50 (2000).

10.Ramamurthy, V., Niemi, G.A., Reh, T.A. & Hurley, J.B. Leber congenital amaurosis linked to AIPL1: a mouse model reveals destabilization of cGMP phosphodiesterase. Proc Natl Acad Sci U S A 101:13897-902 (2004).

11.Pittler, S.J. & Baehr, W. Identification of a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase beta-subunit gene of the rd mouse. Proc Natl Acad Sci U S A 88:8322-6 (1991).

12.Farber, D.B., Flannery, J.G. & Bowes-Rickman, C. The rd Mouse Story: Seventy Years of Research on an Animal Model of Inherited Retinal Degeneration. Prog in Retinal and Eyes Res 13:31-65 (1994).

13.Huang, S.H. et al. Autosomal recessive retinitis pigmentosa caused by mutations in the alpha subunit of rod cGMP phosphodiesterase. Nat Genet 11:468-71 (1995).

14.Liu, X. et al. AIPL1, the protein that is defective in Leber congenital amaurosis, is essential for the biosynthesis of retinal rod cGMP phosphodiesterase. Proc Natl Acad Sci U S A 101:13903-8 (2004).

15.Choy, E. et al. Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell 98:69-80 (1999).

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17.Qin, N. & Baehr, W. Expression and mutagenesis of mouse rod photoreceptor cGMP phosphodiesterase. J Biol Chem 269:3265-71 (1994).

18.Pittler, S.J., Fliesler, S.J., Fisher, P.L., Keller, P.K. & Rapp, L.M. In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure. J Cell Biol 130:431-9 (1995).

19.Blatch, G.L. & Lassle, M. The tetratricopeptide repeat: a structural motif mediating protein-protein interactions. Bioessays 21:932-9 (1999).

20.Akey, D.T. et al. The inherited blindness associated protein AIPL1 interacts with the cell cycle regulator protein NUB1. Hum Mol Genet 11:2723-33 (2002).

21.Dyer, M.A. et al. Retinal degeneration in Aipl1-deficient mice: a new genetic model of Leber congenital amaurosis. Brain Res Mol Brain Res 132:208-20 (2004).

22.Leveillard, T. et al. Identification and characterization of rod-derived cone viability factor. Nat Genet 36:55-9 (2004).

Figure 15.2. Light micrographs of the toluidine blue stained retinal sections show abnormal RPE cells and a loss of photoreceptor cells in the homozygous r15 mice but not in the heterozygous mice. (A) and (C) show relative normal retina of 6.5-month-old r15 heterozygous mouse; (B) and (D) show abnormal RPE cell layer and displaced RPE cells in the subretinal space of 6.5-month-old r15 homozygous mutant retina; (E) is a section of 17.5-month- old r15 heterozygous control; (F) is a section of 17.5-month-old r15 homozygous mutant. Note the reduced photoreceptor layers in the homozygous sections. Arrows (in B, D and F) indicate the abnormal pigmented structures. Scale bars: 20 mm in (A), (B), (E), and (F); 10 mm in (C) and (D).

of photoreceptor nuclear at the age of 17.5-months (Fig. 15.2F). Therefore, the r15 mutation develops a slow degeneration of photoreceptor cells.

Transmission electron microscopic analysis verifies that the RPE cells of homozygous r15 mutant lack apical microvilli and form membranous whorls in the subretinal space (Fig. 15.3B). It suggests that mutant RPE cells may fail to shed the outer segments of photoreceptor cells, and the unphagocytosed outer segments could form membranous whorls at the subretinal space. Mutant RPE cells contain intracellular vacuoles (Fig. 15.3D) and degenerative RPE layer gives rise to the aberrantly displaced structures in the subretinal space (data not shown). Therefore, the r15 mutation recapitulates some of the defects observed in mouse Mertk mutation, this suggests that the causative gene for r15 mutation is essential for the phagocytosis of RPE cells. Currently, it is not clear whether r15 mutation is mechanistically related to the Mertk mutation. We continue our efforts to map the chromosome location of the r15 mutation and to identify the causative gene. We believe that the r15 mutant line provides us an alternative model to study the molecular basis for the regulation and function of the RPE phagocytosis.

2.1. BEMR18 is Another ENU-Induced Mouse Mutation with RPE Abnormality

BEMr18 is also an ENU-mutagenized recessive mutation that shows retinal vessel attenuation. Histology analysis reveals that RPE cell hypertrophy can be observed in the r18 homozygous mice at as early as 4-weeks old (data not shown). RPE cell hypertrophy and RPE layer disorganization become more obvious as the mice grow old (Fig. 15.4D and 4E). Shortening of outer segments and loss of 3-4 layers of photoreceptor cells have been observed in the 1-year-old r18 homozygous mutant, but not in the heterozygous control littermate (Fig. 15.4).