Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008
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Table 3 Differences observed between published C57BL/6J SNP data (Petkov et al., 2004) when compared with C57BL/6JOlaHsd SNP data
Marker |
|
|
|
|
(Rs #) |
Chr |
C57BL/6J |
C57BL/6JOlaHsd |
Problem |
|
|
|
|
|
3664528 |
1 |
C:C |
T:C |
Heterozygous |
3674936 |
2 |
T:T |
C:T |
Heterozygous |
3707236 |
18 |
A:A |
G:A |
Heterozygous |
|
|
|
|
|
The genotyping results of 112 SNPs across the entire C57BL/6JOlaHsd genome revealed that three SNPs (2.7%) were heterozygous i.e. they were of a different genotype to the published C57BL/6J SNPs (Table 3). This suggests that C57BL/6JOlaHsd mice may be more separate from the C57BL/6J on the genomic level than previously thought. SNP genotyping was not performed in the C57BL/6 substrain.
4 Discussion
An electroretinographic analysis of three C57BL/6 substrains was performed in order to determine primarily whether a deletion on chromosome 6 in the C57BL/6JOlaHsd (Harlan) substrain may influence the ERG. This deletion has been well characterised and is known to contain two genes, those encoding synuclein alpha (Snca) and multimerin 1 (Mmrn1) (Specht and Schoepfer, 2001; Specht and Schoepfer, 2004). The absence of Snca was confirmed in all C57BL/6JOlaHsd mice used in these experiments (data not shown).
An initial comparison of the C57BL/6JOlaHsd (Harlan) and C57BL/6 (B&K) substrains indicated no observable differences between the light adapted ERG and oscillatory potentials. In the dark-adapted series, an increase of 10–16% was observed in the amplitude of both a- and b-waves of the C57BL/6 (B&K) mouse when compared to C57BL/6JOlaHsd (Harlan). This difference was significant at higher, but not at lower flash intensities ( p ≤ 0.05). There were no significant differences observed between these two substrains (Harlan and B&K) when the dark-adapted timing was analysed. It was hypothesised that the absence of Snca, a putative regulator of dopamine transmission, may depress the C57BL/6JOlsHsd dark-adapted ERG, as observed in this comparison.
In order to explore this hypothesis further, ERG recordings were made on a third C57BL/6 substrain, C57BL/6J (obtained from The Jackson Laboratory) known to contain the Snca locus. If this substrain had a similar dark-adapted ERG then it would add weight to the hypothesis that the comparatively low amplitudes in the C57BL/6JOlaHsd mouse were due to the lack of Snca. The dark-adapted ERG of the C57BL/6J mouse was different to both C57BL/6JOlaHsd and C57BL/6 substrains. Unlike the other substrains, C57BL/6J mice exhibited significantly slower a- and b-wave timings. In the a-wave, the timings were 7–10% slower ( p ≤ 0.05) and in the b-wave timings were 15–23% slower ( p ≤ 0.0001) at all flash intensities (Fig. 1; Tables 1a and 1b). When the dark-adapted amplitudes were analysed,
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C57BL/6J mice had similar a-wave amplitudes to C57BL/6JOlaHsd mice, both of which had decreased amplitudes in comparison to C57BL/6 ( p ≤ 0.005 at higher flash intensities comparing C57BL/6 and C57BL/6J; Fig. 2(A); Table 2a). Thus, absence of Snca is unlikely to be responsible for differences observed in a- wave amplitude between C57BL/6JOlaHsd and C57BL/6 substrains. Analysis of b-wave amplitudes showed that all three substrains were different, C57BL/6 having the highest amplitudes, followed by C57BL/6JOlaHsd, with C57BL/6J having the lowest (Fig. 2(B); Table 45.2b). The difference between C57BL/6 and C57BL/6J amplitudes was highly significant ( p ≤ 0.0001) at all flash intensities. Since both C57BL/6 and C57BL/6J substrains contain the Snca locus, it is extremely unlikely that any differences in amplitude are being mediated through this locus.
Given that so many differences have been observed during the analysis of dark-adapted retinal function in these three substrains, it possible that unknown genomic differences could account for such differences. To date the only reported sequence variation between C57BL/6 substrains has been the Snca locus deletion (MGI Database, 2007), which may have arisen as far back as the 1970s (Specht and Schoepfer, 2004). In a study presented here, 112 SNPs were genotyped in C57BL/6JOlaHsd mice, of which three were found to be heterozygous (commercial genotyping performed by Kbiosciences, UK). While this comprises only 2.7% of the 112 SNPs, it highlights variation between the published SNPs in the C57BL/6J mouse (Petkov et al., 2004) and substrains. A rapid comparison of the ERG variation (standard deviation; Tables 1a, 1b, 2a and 45.2b) shows more variation in C57BL/6JOlaHsd mice when compared to the other substrains. The C57BL/6J mouse, which has the least variation in the dark-adapted ERG is regularly genotyped for genetic purity (Taft et al., 2006; The Jackson Laboratory, 2006). Other mouse strains, such as the 129 strain, have been shown to be heterozygous (Simpson et al., 1997). Genotypes of inbred strains of mice raised in private colonies at different locations can be influenced by genetic drift, neutral mutation occurring randomly within a population. Variation in the dark-adapted ERG among substrains highlights the need to be vigilant about the use of an exact substrain for all experiments, particularly those involving the ERG.
In summary, characteristics associated with Ganzfeld ERG have been studied in three substrains of C57BL/6 mouse. We have observed that whereas there are no differences in oscillatory potentials or in the light-adapted ERG within these substrains, there are significant differences in the dark-adapted ERG responses. Such differences are unlikely to be mediated by synuclein alpha, since the ERG of two substrains containing the Snca locus was different. Studying variation in retinal function, especially in closely related inbred mouse strains, offers a unique opportunity to search for genes controlling such properties. Knowledge of the functions of such genes will lead to a more comprehensive understanding of retinal function and dysfunction.
Acknowledgments This work was supported by grants from the Higher Education Authority of Ireland, Health Research Board of Ireland and Science Foundation Ireland. We thank Sylvia Mehigan and Caroline Woods for technical assistance.
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A2E, A Pigment of RPE Lipofuscin,
is Generated from the Precursor, A2PE by a Lysosomal Enzyme Activity
Janet R. Sparrow, So Ra Kim, Ana M. Cuervo and Urmi Bandhyopadhyayand
1 Introduction
The lipofuscin that accumulates in retinal pigment epithelial cells with age and that is abundant in some retinal disorders, is sequestered within the interior of membrane bound organelles of the lysosomal compartment of the cell. RPE lipofuscin pigments form in large part due to reactions of vitamin A aldehyde. Evidence to support the contention that RPE lipofuscin derives primarily as a byproduct of the visual cyCle, comes from work demonstrating that when the 11-cis-retinal (11-cisRAL) and all-trans-retinal (atRAL) chromophores are absent, as in Rpe65−/− mice, the lipofuscin-specific autofluorescence eminating from RPE whole-mounts is reduced by more than 90% (Katz and Redmond, 2001). RPE lipofuscin is also lacking in patients with early – onset retinal dystrophy associated with mutations in RPE65, a visual cycle protein shown to be the isomerohydrolase essential to the production of 11-cis-retinal (Lorenz et al., 2004). For some time it was thought that these lipofuscin fluorophores might form within the acidic environment of the lysosome. However, the detection of RPE lipofuscin precursors in photoreceptor outer segments, together with studies in the Royal College of Surgeon (RCS) rat showing that when outer segment phagocytosis fails, RPE lipofuscin is substantially diminished (katz et al., 1986), demonstrates an origin from photoreceptor cells. Nevertheless, questions still remain regarding the extent to which processing of the lipofuscin precursors occurs in lysosomes. For instance, the di-retinal conjugate A2E, a prominent pigment of RPE lipofuscin, is generated in photoreceptor outer segments by a multi-step pathway involving the formation of A2PE, the phosphatidyl-pyridinium bisretinoid that is the immediate precursor of A2E. Our finding that A2E is generated from A2PE by phosphate hydrolysis and that this cleavage can be mediated by phospholipase-D indicates that enzyme-mediated mechanisms may be important in releasing A2E from its precursor; acid hydrolysis of A2PE occurs at a slow rate
J.R. Sparrow
Departments of Ophthalmology; Pathology and Cell Biology,
Columbia University, New York, NY 10032, Tel: 212-305-9944, Fax: 212-305-9638 e-mail: jrs88@columbia.edu
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(Liu et al., 2000; Ben-Shabat et al., 2002). The significance of enzyme-mediated hydrolysis as the mechanism responsible for generating at RAL-derived lipofuscin chromophores from their precursors, is dependent in part on demonstrating an activity in RPE cell lysosomes that can serve this function. Here we probe for such an activity.
2 Methods
2.1 Synthesis of A2E and A2PE
A2E was synthesized from at RAL and ethanolamine as described previously (Parish et al., 1998). A2PE was synthesized using atRAL and phosphatidylethanolamine from egg yolk or dipalmitoyl-phosphatidylethanolamine (DP-A2PE) as described previously (Liu et al., 2000).
2.2 Preparation of a Lysosomal Fraction of ARPE-19 Cells
Lysosomes from ARPE-19 cells were isolated by modification of a previously described method (Storrie and Madden, 1990). Briefly, ARPE-19 cells, grown to confluence (11 flasks) and serum-deprived for 24 hours, were scraped from flasks and centrifuged at 500 g for 5 min at 4◦C. Pellets were resuspended in 1–2 ml of 0.25 M sucrose and the cells were disrupted in a nitrogen cavitation chamber at 35 psi pressure, on ice for 7 min. The resulting suspension of fragmented cells was centrifuged at 2500 x g for 15 min 4◦C to sediment nuclei, unbroken cells and heavy mitochondria and the post-nuclear supernatant ( 3 ml) was fractionated on a discontinuous metrizamide/percoll density gradient (35% metrizamide, 17% metrizamide and 6 % Percoll in 0.25% sucrose) with centrifugation (20,000 rpm, 35 min, 4◦C; SW40.1 rotor). The fraction containing lysosomes and light mitochondria (band 2) was collected, adjusted to 40% metrizamide and separated in a second discontinuous metrizamide density gradient (17% metrizamide, 5% metrizamide and 0% metrizamide in 0.25% sucrose) by centrifugation (20,000 rpm, 35 min, 4◦C). In addition, from male Wistar rats (200–250 g) fasted for 24 hours, liver lysosomes were isolated from a light mitochondrial-lysosomal fraction in a discontinuous metrizamide density gradient as described (Cuervo et al., 1997). Lysosomal matrices and membranes were isolated after hypotonic shock and centrifugation (200,000 g, 20 min, 4◦C) (Ohsumi et al., 1983).
2.3 Incubation with Phospholipase-D and the Lysosomal Fraction
Fifteen microliters of 2 mM DP-A2PE in DMSO was added to 285 l of 40 mM morpholinepropanesulfonic acid buffer (pH 6.5) containing 300 units/ml phospholipase-D from S. chromofuscus (Sigma-Aldrich, St. Louis, MO) and 15 mM
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CaCl2 or 100 g protein of the lysosomal fraction of ARPE-19 cells or rat liver. The mixtures were incubated for 3 h at 37◦C and were then extractedwith 2:1 v/v chloroform/methanol containing 0.1 % TFA, dried under argon and re-dissolved in 300 l of 50% methanolic chloroform. In some experiments, calphostin C (5 M; Sigma-Aldrich, St. Louis, MO), and 100 L of a protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO) were added to inhibit the activity of phospholipase- D (Ben-Shabat et al., 2002). A2PE and A2E were detected by HPLC using a reverse phase C4 column, monitoring at 430 nm and injection of authentic standards of A2PE or A2E.
2.4 HPLC Analysis
For quantification of A2E and A2PE an Alliance system (Waters, Corp, Milford, MA) equipped with 2695 Separation Module, and 2996 Photodiode Array Detector and operating with Empower software was used with a C4 column
Fig. 1 An activity present in lysosomes isolated from ARPE-19 cells cleaves A2PE to generate A2E. A2PE was incubated in the absence (A) and presence (B) of the lysosomal fraction or with phospholipase-D (C). Samples were analyzed by reverse phase HPLC with 430 nm detection. A peak with UV-visible absorbance and retention time indicative of A2E appears in B and C
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(4 × 250 mm, 5 m). A2PE and A2E were eluted with the following gradients ofacetonitrile in water (containing 0.1% trifluoroacetic acid): 75% (5 min; flow rate, 1 ml/min), 75–100% (5 min; flow rate, 1.5 ml/min), and 100% (10 min; flow rate, 1.5 ml/min), and were monitored at 430 nm. Injection volumes were 10 L; each sample was injected three times for reliability.
3 Results
To assay for an enzyme activity that can release A2E from its precursor A2PE, lysosomes were isolated from ARPE-19 cells and rat liver by differential centrifugation followed by centrifugation through a discontinuous metrizamide gradient. Incubation of an HPLC purified sample of DP-A2PE (Fig. 1A) with the RPE lysosomal fraction followed by HPLC analysis revealed the appearance of a chromatographic peak that on the basis of UV-visible absorbance and retention time could be identified as A2E. The peak attributable to A2PE was concomitantly decreased in height (Fig. 1B). Incubation of A2PE in the presence of phospholipase-D also resulted in the appearance of A2E in the chromatographic profile (Fig. 1C). A hydrolytic activity that could mediate phosphate cleavage of A2PE was also present in the liver lysosomal fraction (Fig. 2A, B). Additionally, the activity in liver lysosomes that released A2E from A2PE was efficiently suppressed by the phospholipase-D inhibitor calphostin C (Fig. 2C) and by a protease inhibitor cocktail (Fig. 2D).
Fig. 2 An activity in liver lysosomes generates A2E from A2PE and is suppressed by inhibitors of phospholipase-D. A2PE was incubated in the absence (A) and presence (B, C, D) of a liver lysosomal fraction. The phospholipase-D inhibitor calphostin C (C) and a protease inhibitor mixture with phospholipase-D inhibitory activity (D) were added to some reaction mixtures. Reverse phase HPLC chromatograms with 430 nm detection
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4 Conclusions
Understanding the steps involved in the biosynthesis of A2E is vital to the development of therapies that would reduce the deposition of A2E in RPE cells. In the current work we have shown that an enzyme activity is present in RPE cell lysosomes that can generate A2E by phosphate hydrolysis of the precursor A2PE. The ability of phospholipase-D inhibitors to suppress the cleavage activity in the lysosomal fractions, indicates that phospholipase-D may be the enzyme in RPE cell lysosomes. The detection of small amounts of A2E in extracts of photoreceptor cell outer segments (Ben-Shabat et al., 2002), together with reports of a phospholipase-D activity in outer segments isolated from bovine eyes suggests that some A2PE may also undergo hydrolysis before internalization by RPE cells (Salvador and Giusto, 1998).
A2PE, the precursor of the lipofuscin pigment A2E, forms in outer segments (Liu et al., 2000; Ben-Shabat et al., 2002). Because shedding of outer segment membrane with deposition in RPE cells leads to the complete replacement of the outer segment every 10–14 days (Young, 1971), A2PE is not continually amassed in outer segments; rather it is transferred to RPE cells along with the phagocytosed outer segment material. Nonetheless, A2PE is not detectable in RPE cells, because as we have shown here, A2PE is efficiently processed to generate A2E. However, although other macromolecules phagocytosed by the RPE are degraded by lysosomal enzymes to small molecules that can diffuse out of the lysosome (Feeney-Burns and Eldred, 1983), after phosphate cleavage of A2PE no further degradation of the molecule occurs. This is likely because the structure of A2E is unprecedented and as such is not recognized by the lysosomal enzymes of the RPE cell. Being undigestible it accumulates. The presence of an enzyme activity in RPE lysosomes that can release di-retinal RPE lipofuscin pigments and phosphatidic acid from precursors is also significant to more recently characterized lipofuscin pigments. Specifically, we have shown that the RPE pigment atRAL dimer-E can be generated by phosphate cleavage of atRAL dimer-PE, a compound that is generated when two molecules of atRAL condense to form an aldehyde-bearing dimer that then reacts with PE to form a protonated Schiff base. The pigments atRAL dimer-PE and atRAL dimer-E have absorbances in the visible spectrum at 510 nm and structures and biosynthetic pathways that are distinct from A2E/isoA2E. We have identified these compounds in RPE isolated from human eyes and in the lipofuscin-filled RPE of mice homozygous for a null mutation in the Abca4/Abcr gene (Fishkin et al., 2004).
Acknowledgments The work was supported by National Institutes of Health Grant EY12951 and Research to Prevent Blindness. JRS is the recipient of an Alcon Research Institute Award.
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