Autoradiographic and biochemical assessment of rod outer segment renewal in the vitiligo (C57BL/6-mi...

Exp. Eye Res. (1995) 60, 91-96

Autoradiographic and Biochemical Assessment of Rod Outer Segment Renewal in the Vitil igo (C57BL/6-mivit/mi vit) Mouse

Model of Retinal Degeneration

SYLVIA B. SMITH* AND DENNIS M. DEFOE

Department of Cellular Biology and Anatomy and Department of Ophthalmology, Medical College of Georgia, Augusta, GA 30912, U.S.A.

(Received Houston 9 May 1994 and accepted in revised form 23 September 1994)

Rod outer segment renewal was assessed in vitiligo (C57BL/6-mivit/mi vii) mice using autoradiographic and biochemical methods. This process was examined because the number of phagosomes is reduced in this mouse. Rod outer segment renewal was detectable in the mivi t /mi vit retina. Within 24 hr of intraperitoneal injection of 3H leucine, there was a distinct band of radioactivity present at the junction of the ROS and RIS in mutant mice that was similar to controls. The displacement of the radioactive band progressed normally in the peripheral regions of the vitillgo retina, but did not in the posterior retina. Morphometric analysis of the posterior region of mutant retinas, indicated that the band of radioactivity became less distinct between 1 and 3 days post-inlection. In vitiligo retinas it remained at 2.23 #m, whereas in controls, the band migrated 6.16/zm from the ROS base. When posterior regions of retinas were evaluated 8 days post-injection, there was no band discernible in the vitiligo retinas, but a very dense band at the ROS apex in controls. Assessment of incorporation of radioactivity into rhodopsin using SDS-PAGE indicated a progressive displacement of radio-labeled rhodopsin through the RER, but not as complete a progression through the outer segments. The elongation of the outer segments in the posterior regions of the mutant retina suggests impaired shedding. This, plus the lack of attachment in the posterior retina of photoreceptor cells to RPE in this mouse, seem to be likely causes for the decreased number of phagosomes.

Key words: vitiligo mouse; microphthalmia; photoreceptor cell; retinal degeneration.

The present study describes the renewal of rod outer segments (ROS) in the vitiligo (C57BL/6-miVit/mi vit) mouse. The vitiligo mutation maps to the micro- phthalinia (mi) locus of mouse chromosome 6 (Lamo- reux et al., 1992 ; Tang et al., 1992); mutations at this locus are associated with defects in a basic-helix-loop- helix transcription factor (Hodgkinson et al., 1993; Hughes et al., 1993). The most striking feature of the vitiligo mouse is the depigmentation of skin and pelage. A more subtle feature of the mouse is its slowly progressing retinal degeneration. The vitiligo mouse loses central photoreceptor cell nuclei at a rate of about one row per month beginning at 2 months; ROS lengthen significantly between 4 and 8 weeks, but are severely disrupted by 4 months; macrophage-like cells are present by 6 weeks in the subretinal space; rhodopsin levels decrease gradually (Smith, 1992). Electrophysiologic studies have shown that the electro- retinogram b-wave amplitude and sensitivity of this mutant decrease gradually in a manner that correlates with loss of photoreceptor cells and rhodopsin (Smith and Hamasaki, 1994).

In addition to photoreceptor cell changes, the retinal pigment epithelium (RPE) is unevenly pigmented in the vitiligo mouse (Lerner et al., 1986; Boissy et al.,

* For correspondence at: Department of Cellular Biology and Anatomy, Medical College of Georgia, CB 1619, Augusta, GA 30912-2000, U.S.A.

1987) although the pigmentation of the RPE does not decrease predictably with age as it does in the skin and pelage. There is evidence that two forms of vitamin A, retinyl palmitate and retinol, are accumulating in the RPE of this mutant (Smith et al., 1994b), but the consequence of this accumulation on RPE function is not known. One of the chief functions of the RPE cell, phagocytosis, has been examined in the vitiligo mouse. The number of phagosomes present in the mutant is significantly fewer than in controls, however, there is a measurable, though reduced, peak of phagocytosis that occurs within 2 hr of light onset just as there is in controls (Smith et al., 1994a).

The decreased number of phagosomes could reflect either a defect in RPE cell phagocytosis or a defect in photoreceptor cell outer segment renewal and shedding since these processes are inextricably linked (Young and Bok, 1969, Anderson and Fisher, 1975; Young, 1977). To determine if the renewal process is altered in vitiligo mice, the present study used autoradiographic techniques on fixed retinal tissue to establish the migration of radioactively-labeled proteins through outer segments. These experiments were based on reports by Young and coworkers (Young, 1967; Young and Droz, 1968 ; Young and Bok, 1969) that injected radioactive amino acids are incorporated into proteins (primarily rhodopsin) of the rough endoplasmic reticulum of the photoreceptor inner segment. Rhodopsin passes through the Golgi complex

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Fie. 1. Light microscope autoradiographs of (A) peripheral and (B) posterior regions of retinas of vitiligo mice injected at 6 weeks with aH leucine and killed at l day (24 hr) post-injection. The arrows point to the band of silver grains. In both retinal areas, the band is dense and is located at the outer segment/inner segment junction. (Magnification x 1210.) Panels (C) and (D) are autoradiographs of retinas of vitiligo mice killed at 8 days post-injection. In the peripheral region (C), the band of silver grains (arrow) is close to the apex of rod outer segments. In the posterior area (D), there is no distinct band. The outer segments are elongated and do not attach to the RPE. [Magnification x 1210 (C) and x 920 (D).]

to the base of the outer segment where it is assembled into proximal membranous disks. In adult mice, the total rod outer segment renewal time is 10 days (LaVail, 1973). In addition to morphometric analysis of the displaced band of radioactivity, the present study also used biochemical techniques to assess the incorporation of 3H leucine into rhodopsin isolated from rod outer segments and rod inner segments.

Details about the lighting and feeding conditions of the animals have been described (Smith, 1992). Mice were injected intraperitoneally at 6 weeks with 3H- labeled leucine in ethanol-water (2:98) solution (specific activity 4 0 - 6 0 Ci mmo1-1, Dupont-NEN) at a dosage of lO/~Ci (g body weight) -1. One group of mi"~t/mi vtt and C57BL/6 control mice were used in histologic experiments and 2-3 mice were killed at 4

ROS RENEWAL IN mivit/mi vit MOUSE 93

TABLE I

The distance from the ROS base to the leading edge of the dense band of radioactivity following injection of 8H leucine into vitiligo and control mice

Area of retina and days after

injection

mF~t/mi v~t C5 7BL/6 mfflt/mi vlt

Distance ~m) Rate of synthesis* Distance (Fm) Rate of synthesis*

Peripheral 1 2'48 _+ 0'68 2"48 1"94 + 0"69 1'94 3 5.01 _+ 0"77 1'67 4"56 + 0"79 1"52 8 11.63+0"63 1-45 9"95+1"55 1.24

Posterior 1 2"77 +_ 0"64 2"77 1"85_ 0"52 1"85 3 6"16 + 0"92 2"05 2"23 + 1"171" 0"74 8 16"25 -t-0"74 2-03 nm:l:

Two or three mice were used at each post-injection time, 20 measurements were made for each retina, ten on each side of optic nerve as mentioned in the text.

* Rate of synthesis = distance divided by number of days post-injection. l Significantly different from controls (three way ANOVA, factors: area, group, time post-injection, /: = 134-92 for interactive effect,

P = 0.00001. post-hoc test (Tukey) was significant (P < 0'05). :~ nm = not measurable.

and 24 hr, 3, 8, 13 and 15 days post-injection. A second group of affected and control mice, used in biochemical studies, were killed at 4 hr, 3, 8 and 13 days post-injection. For biochemical studies, four mice were used per assay, each assay was performed tw!ce.

Histological processing of eyes in JB-4 has been described (Smith, 1992; Smith, Cope and McCoy, 1994). Autoradiography was performed by dipping the slides in Kodak NTB-2 nuclear track emulsion diluted, 1 : 1 with water. After slides were exposed for 60 days and developed in Kodak Dektol developer, they were stained with hematoxylin and eosin. The distance from the ROS base to the leading edge of the radioactive band was determined for 20 points along the retina (ten on each side of the optic nerve). The peripheral areas were represented by five points adjacent to the ora serrata and the posterior retina by five points on each side of the optic nerve. The distance between measurement points was approximately 250 Fro.

The morphological assessment of fixed retinal tissue indicated that dense bands of radioactivity were not observed 4 hr after injection in either controls or affected animals. By 24 h, however, a discrete band was measurable in both groups. In control animals, a dense band of radioactivity persisted in the posterior and the peripheral portions of retinas at 3 and 8 days post-injection, but by 13 and 15 days the grains were diffuse and no distinct band could be measured (photomicrographs not shown). In the vitiligo retina, there was a dense band of radioactivity detectable at the junction of the inner and outer segments when examined at 24 hr post-injection. This dense band was observed in the peripheral retina [Fig. I(A)] and the posterior area [Fig. I(B)]. At 3 days post-injection,

there was a discrete band in the peripheral area, but in the posterior area it was less distinct. Within 8 days of injection, the radioactive band had progressed to the rod outer segment apical tips in the peripheral area of vitiligo retinas [Fig. 1(C)]. In contrast, in the posterior area of the vitiligo retina, no clear band of radioactivity was evident, a l though radioactive grains were numerous [Fig. I(D)]. The outer segments were elongated and appeared fragmented toward the apex. They were separated from the RPE. By 13 days post-injection, there existed only background levels of radioactivity in the peripheral vitiligo retina. In the posterior retina, no discrete band was observed al though numerous grains were present.

The differences observed between the peripheral and posterior retina of affected mice prompted measurements of these areas separately. Table I provides this data. With respect to the peripheral areas, there was not a significant difference in band displacement between affected and control animals. For example, at 3 days post-injection, the aH band had migrated 5"01 # m from the ROS base in control mice and 4.56 Fm in affected animals. Thus, the rate of membrane synthesis in the peripheral area was similar between the two groups. With respect to the posterior area, however, the band of radioactivity did not progress in a manner similar to controls. Between 1 and 3 days post-injection the band had not progressed in vitiligo retinas, but had progressed 4 - 5 # m toward the ROS apex in controls. When retinas were measured 8 days post-injection, there was no band discemable in the posterior region of vitiligo retinas.

Since no dense band of radioactivity was observed in the retinas of either control or affected animals at 13 and 15 days post-injection, the number of grains in

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FIG. 2. Representative radioactivity profiles of SDS-polyacrylamide disk gels of rod outer segments gradient purified from control (O) and vitiligo (0 ) mice injected at 6 weeks with 3H leucine and killed: (A) 4 hr post-injection; (B) 8 days post- injection; (C) 13 days post-injection. Protein migration is from left to right. The gels were sliced and disintegrations per min (dpm) per pair of slices were obtained by scintillation counting. The first gel slice represents proteins with a MW 69 kDa. Arrows indicate gel slices containing rhodopsin as indicated by 14C labeled rhodopsin standard. The lower bar graph (D) shows quantitation of radioactivity in rhodopsin purified from ROS in control (solid dark bar) and vitiligo (light gray bar) mice. The ratio of 3H/'4C dpm in rhodopsin was determined from profiles similar to those shown in panels (A)-(C). The ratios in the rhodopsin band at 3 and 8 days in controls were virtually the same, hence this ratio was considered 100%. All other ratios were compared to this value and ratios were calculated as percent of control. (* = Significantly different from controls, ANOVA F = 9"0, P < 0"05).

posterior and peripheral retinal areas at these post- injection times was determined by camera lucida tracing. These tracings were made with a Wild microscope outfitted with a drawing tube (Wild- Heerbrugg Instruments, Farmingdale, NY, U.S.A.). There were numerous grains detectable in the outer segment area. The number of grains counted in the posterior area of vitiligo retinas was about 40% greater than in controls (343 /3 cm 2 in miVit/mi T M

versus 2 1 7 / 3 cm 2 in controls). The number of grains counted in the peripheral areas was very similar between the affected animals (153 /3 cm 2) and con- trois 159 /3 cm2).

The lack of a discrete band of radioactivity in the posterior outer segments of the affected animal is striking and may be a reflection of poor ROS-RPE contact. The key difference in the peripheral and posterior retinal areas of the miVit/mi vit mouse is that in the periphery, the contact between the outer segments and RPE is secure, whereas it clearly is not in the posterior area by 7 weeks post-natally [Fig. I(D)]. In order to observe a distinct band of radioactivity in the ROS, outer segments must be well aligned and have sound contact with the retinal pigment epithelium. Upon histological fixation, this lack of contact manifests as fragmented and disrupted

ROS R E N E W A L IN miVit/mi vit M O U S E

ROS. The elongation of the outer segments and apparent lack of contact with the retinal pigment epithelium were noted in an earlier study (Smith, 1992), although the difference between peripheral and posterior regions was not reported.

The unusual migration of the band of radioactivity in the posterior region of outer segments of the affected animals prompted the biochemical assessment of this process. The experiments permitted quantitation of radioactivity in rhodopsin isolated from outer segments and from inner segments. Retinas were removed and rhodopsin was purified from outer segments using sucrose density gradients. The 3H leucine-labeled ROS samples were electrophoresed on polyacrylamide disk gels along with a 14C leucine-labeled rhodopsin standard purified from mouse retinas. These pro- cedures and information about processing of gels by scintillation counting to quantify incorporation of 3H leucine into the band on the gel corresponding to rhodopsin have been described (Smith, St Jules and O'Brien, 1991). The disintegrations per minute (dpm) obtained from counting the radioactivity in the rhodopsin band of gel slices resulted in profiles of incorporation of [3H/~4C] leucine into this protein. Figure 2 shows representative profiles from control and vitiligo mice at three times post-injection. The amount of radioactivity in controls at 4 hr post- injection [Fig. 2(A)] was not as great as at 8 days post-injection [Fig. 2 (B)]. By 13 days post-injection the amount had diminished significantly [Fig. 2(C)]. The pattern in vitiligo mice was similar to controls at 4 hr and 8 days post-injection [Fig. 2(A) and (B)], but was not similar at 13 days post-injection [Fig. 2(C)]. At 13 days post-injection, there was still a significant level of radioactivity detectable in the rhodopsin band purified from the ROS of vitiligo mice.

The ratios [3H/~4C] determined in each gel at four times post-injection in miVit/miv~t mice were compared with C57BL/6 controls and are expressed as percentage of controls in Fig. 2(D). It should be noted that for the purpose of comparing counts at other time points, the amount of radioactivity in the rhodopsin band at 3 and 8 days in controls was considered ' 100%'. This is based on the report of Hall, Bok and Bacharach (1969) that rhodopsin is an integral component of the disk and remains associated with the outer segment until it is lost by shedding at the distal tips. At 4 hr post-injection, the percentage of radioactivity in controls was only 62 % of the level achieved at 3 and 8 days post injection when presumably the radioactivity has advanced into the outer segment completely, but none has been lost. By 13 days post- injection in C57BL/6 controls, the level had dropped to less than 20%. In the miv~t/mi vit outer segments, at 4 h post-injection the percentage of radioactivity was 29% that of controls, it increased by 3 days post- injection and was nearly 100% by 8 days post- injection. Interestingly, the level of radioactivity at 13 days did not drop in vitiligo mice as it had in controls.

95

The level was about three times more than control values suggesting an accumulation of labeled rhodopsin in the outer segment.

To determine if there was an accumulation of radioactively-labeled rhodopsin in the inner segments, rhodopsin from this portion of the photoreceptor cell was purified by sucrose density gradients and con- canavalin A chromatography followed by SDS gel electrophoresis (Smith and O'Brien, 1991). The ratios of 3H leucine in the rhodopsin band of the sample to the 14C leucine band in the standard was similar between controls and vitifigo mice, regardless of the time post-injection. In controls at 4 hr, 8 days and 13 days post-injection the ratios were 9.85, 6"9 and 6.2, respectively. In miVit/mi vit at the same times post- injection, the ratios were 9"98, 7"02 and 6.5. It seems unlikely that the explanation for the accumulation of 3H counts in the ROS is due to a lag of protein in the RER. The gel electrophoretic analysis of the incorporation of aH leucine into rhodopsin purified from inner and outer segments of mFit/mF" mice over the time course indicated that the rhodopsin was being transported through the inner segment and into the outer segment.

At 13 days post-injection, when the radioactivity in rhodopsin purified from ROS in controls had diminished to low levels, there was still detectable radioactivity in rhodopsin purified from the ROS of vitiligo retinas. The levels were about 50% of the levels detected at 8 days post-injection. This would suggest that some of the radioactively labeled rhodopsin is being progressively transported to the ROS apical tips and phagocytosed by the RPE and some is not. This partial diminution in radioactively labeled rhodopsin presumably reflects the differences observed in the peripheral versus posterior retina. It is also possible that some labeled ROS material may be shed into the subretinal space and may be phagocytosed by wan- dering macrophages.

The finding of altered outer segment renewal is of particular importance because diminished phagocytosis has been observed in the miV'/mi v~t mouse (Smith et al., 1994a). Between the ages of 4 and 8 weeks, the number of phagosomes was reduced markedly to a value that was only about 16 % that of controls. There were many more phagosomes in the peripheral vitiligo retina than in the posterior retina. The decreased number of phagosomes may reflect the lack of contact between RPE/ROS which is pro- nounced in the posterior area. This is also the area where the outer segments are considerably elongated. The cause of the functional differences between posterior and peripheral retinal photoreceptors in this mutant remains to be determined.

Acknowledgements

This work was supported by Public Health Service research grants EY09682 (SBS) and EY07036 (DMD) from

96 S.B. SMITH AND D.M. DEFOE

the National Eye Institute. The authors thank Dr M. Wetzel for helpful suggestions about this experiment, and Drs Michael Mulroy and Paul Sohal for use of microscopic equipment. The C 57BL/6- miv*t/mi v~ mice used in this study were the offspring from our colony of breeding pairs which were provided by Dr R. L. Sidman, New England Regional Primate Research Center, Southboro, MA, U.S.A. (funded by NEI grant EY06859). The technical assistance of J. McCoy, D. McCool, B. Cope and B. Headrick is gratefully ack- nowledged. We thank Mr Ellis Cobb and D. McCool for excellent animal care.

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Smith, S. B., Duncan, T., Kutty, G., Kutty, R. K. and Wiggert, B. (1994b). Elevation of retinyl palmitate in eyes and livers and of IRBP in eyes of vitiligo mutant mice. Biochemical Journal 300, 63-8.

Tang, M., Neumann, P.E., Kosaras, B., Taylor, B. A. and Sidman, R. L. (1992). Vitiligo maps to mouse chromosome 6 within or close to the mi locus. Mouse Genome 90, 441-3.

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Young, R.W. (1977). The daily rhythm of shedding and degradation of cone outer segment membranes in the lizard retina. ]. Ultrastruct. Res. 61, 172-85.

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