Marion Sangster Eckmiller

Defective cone photoreceptor cytoskeleton, alignment, feedback, and energetics can lead to energy depletion in macular degeneration

Macular degeneration (MD) is a puzzling disease characterized by disturbance, and then complete loss, of fine detailed vision in the central macular region of the human retina, as a result of disturbed function and then death of photoreceptor cells. This review describes a possible pathomechanism for MD that involves a causal relationship between mutated genes, altered photoreceptor cytoskeletal proteins, defective cone photoreceptor alignment, and disturbed visual feedback mechanisms that leads to energy depletion and apoptosis of macular cone photoreceptors. MD may be associated with mutations in genes encoding certain photoreceptor proteins (ATP-binding cassette transporter retina, retinitis pigmentosa GTPase regulator, retinal degeneration slow/peripherin) that are components of, or interact with, microtubule-containing cytoskeletal systems at the connecting cilium of rods and cones and at the multiple incisures of rod outer segments (OSs). Vertebrate photoreceptors are directionally sensitive: the longitudinal axis of each cell is actively aligned towards the entrance pupil of the eye, and the cells are most sensitive to light travelling along this axis (as in the Stiles-Crawford effect). The mechanisms responsible for photoreceptor alignment involve movements made by photoreceptors in response to the direction of incident light, using their cytoskeletons. A model is proposed for photoreceptor alignment whereby light absorption in the OS causes fast membrane and slower cytoplasmic changes that spread from the OS to the inner segment myoid, where they activate feedback-controlled motor functions by cytoskeletal elements (microtubules and microfilaments) to produce a differential local bending that adjusts photoreceptor orientation. The fovea at the center of the human macula has specialized features that enable it to provide uniquely high visual acuity, if its cones are accurately aligned. Accordingly, it is proposed that some gene defects in MD cause disturbances at photoreceptor connecting cilia that lead to gradual defects in photoreceptor alignment; misalignment of central macular cones will be initially perceived as blurring, distortions, and decreased acuity of central vision. Although photoreceptors throughout the retina use their cytoskeletons for alignment, the accurate alignment of foveal cones is particularly important because their signals contain fine resolution information that is used in visual feedback systems, e.g., for adjusting accommodation and eye movements. Vision involves multiple feedback loops that are interdependent, e.g., the accuracy of alignment of foveal cones influences how effectively changes in accommodation bring images into focus, and the state of accommodation in turn influences how light entering the eye is projected onto foveal cones. It is proposed that in MD gene defects that disturb the cytoskeleton and alignment of photoreceptors lead to a disturbance in the normal signalling within these feedback systems, causing the mechanisms controlling the alignment of cones in the center of the macula to become progressively more disturbed and the cells to unnecessarily expend energy. These disturbances can lead to local energy depletion within the metabolically fragile central macular cones that trigger them to die, producing central areas of blindness. If this line of reasoning is correct, it may be possible to treat the local energy depletion within macular cones in MD by energy supplementation.

Microtubules in a rod-specific cytoskeleton associated with outer segment incisures.

In many vertebrate retinas the outer segments of rod photoreceptors have multiple incisures, that is, there are numerous indentations in the highly curved membrane forming the edge of their disks and in the plasma membrane enclosing the entire stack of disks. Immunofluorescent localization of tubulin in amphibian photoreceptors yielded a novel series of thin, parallel, fluorescent lines in rod outer segments that extended their full length and coincided with their multiple incisures. Electron-microscopic examination of amphibian retinas revealed the structures responsible for this fluorescence: longitudinally oriented microtubules were associated with incisures at heights throughout rod outer segments. These microtubules were located between the disk rims and the overlying plasma membrane, in the small cytoplasmic compartment at the mouth of incisures; the microtubules and membranes were separated from each other by distances that were uniform, as though interconnected by filaments described in other studies. Thus, in amphibian rod outer segments the incisures mark the site of a cytoskeletal system containing longitudinal microtubules distinct from those of the ciliary axoneme, linked by filaments to the adjacent membranes. This cytoskeleton is expected to be important for the normal structure, function, and renewal of rod outer segments. In amphibian cone outer segments, which do not have incisures, the only anti-tubulin immunofluorescence and the only microtubules were at the axoneme. These findings may help elucidate the diverse properties of rods and cones in many vertebrate retinas and could prove relevant for human retinal degenerations.

Morphogenesis and renewal of cone outer segments

Abstract This chapter describes details of the morphogenesis and the renewal of outer segments (OS) in vertebrate cones that distinguish them from rods. The findings suggest that in cones new membrane is distributed, via the ciliary stalk, to partial growing lamellae that occur both as evaginations at the base and as invaginations throughout distal levels of the OS. During photoreceptor morphogenesis in the developing retina of the amphibian Xenopus , cone outer segments (COS) continually decrease in taper (basal width divided by length) and their distal lamellae contain partial membrane infoldings, termed distal invaginations (DI). Assuming that the growth of a DI splits one pre-existing lamellae into two or more daughter lamellae, the formation of DI at various levels throughout developing COS can decrease their taper. Accordingly, a model has been proposed whereby new membrane entering developing COS is distributed into basal evaginations (BE) at the base and also flows distally along the ciliary membrane to enter lamellae containing growing DI; in developing rod outer segments (ROS) all new membrane enters BE. During OS renewal in the adult Xenopus retina, the shape of mature COS is remodeled in a somewhat different way. During the night COS become short truncated frustums by shedding their narrow tips, during the day COS expand in size and become conical by re-forming narrow tips. Mature COS contain DI, whose formation could participate in remodeling COS shape. Light microscopic observation showed step-like irregularities in the external outline of many COS during day times. Electron microscopic observation showed that these irregularities were on the non-ciliary side of COS, and that the number of DI per COS varied with time but was always greater than the number of BE. These findings suggest that the growth of DI at various levels can remodel the shape of a COS, re-establishing a smooth external outline and generating a new narrow tip. Thus, the model proposed for how membrane is distributed within developing COS can, with slight modification, also explain how the shape of mature COS is remodeled during renewal. New lamellae are likewise added to the COS by the formation of BE and DI, but in mature COS the formation of DI can decrease lamellar width. Because BE formation does not change the local OS width, the taper of vertebrate photoreceptor OS presumably depends upon the relative amount of membrane that is distributed into BE versus DI. The proposed model for membrane distribution within COS is supported by an autoradiographic study showing a shift in the distribution of labeling over COS in the Xenopus retina after short survival times. The model is also indirectly supported by immunocytochemical and ultrastructural observations of the ciliary axoneme microtubules in photoreceptor OS from this retina. In rods the axoneme is not as long as the OS, but in cones the axoneme extends from the base to the distal end of COS at all diurnal times and axonemal microtubules were discarded when the COS tip was shed. During OS renewal the ciliary axoneme is thus replaced in cones, but apparently not replaced in rods. These observations are consistent with the proposed model because elongation of the axoneme can participate in moving membrane along the ciliary stalk into distal regions of the OS where DI are growing; both of these occur in cones but not in rods. These findings provide convincing evidence that in developing and mature cones some new membrane entering the OS is distributed into BE and moves distally along the ciliary membrane to enter pre-existing lamellae in which DI are expanding, but that in ROS all new membrane enters BE. These fundamentally different features of the OS in rods and cones may help to clarify their different photosensitivities, their evolutionary origins, and certain photoreceptor degredations.

RENEWAL OF THE CILIARY AXONEME IN CONE OUTER SEGMENTS OF THE RETINA OF XENOPUS LAEVIS

Abstract.The ciliary axoneme in photoreceptors from the retina of Xenopus laevis was examined by immunofluorescent staining of tubulin throughout the light/dark cycle. The immunofluorescent axoneme extended along only part of the length of rod outer segments but the entire length of cone outer segments. Both the cone axoneme and outer segment elongated during the day and shortened (presumably by shedding) during the night. Fragments of immunofluorescent axonemes were found within packets of outer segment material breaking off from cone tips. These findings show that the ciliary axoneme in the Xenopus retina is replaced during the renewal of outer segments in cones. No evidence has been found for renewal of the axoneme in rods, and thus the stability of the ciliary axoneme may differ in rod and cone photoreceptors.

Distal invaginations and the renewal of cone outer segments in anuran and monkey retinas

SummaryAlthough it is now clear that the outer segments of mature vertebrate cones are regularly renewed, it is not known how a cone outer segment can maintain a tapered shape if its narrower tip is periodically lost by shedding. This problem was addressed by morphological examination of photoreceptors in retinas of anurans (Xenopus laevis) and monkeys (Macaca fascicularis). Light microscopy revealed a marked daily change in the shape of cone outer segments in X. laevis: at light offset they were long and conical, at light onset they had shed their narrow tips, were sharply truncated, and 40% shorter. Electron microscopy revealed previously undescribed fine-structural features in these mature cone outer segments, most notably the presence of many partial membrane infoldings within their distal lamellae. The growth of each of these “distal invaginations” apparently split 1 pre-existing distal lamella into 2 daughter lamellae of reduced width. The formation of distal invaginations at various heights within a cone outer segment would thus make it longer and narrower. Similar ultrastructural features were also found in cone outer segments of monkey retinas. These findings suggest that during outer segment renewal the tapered shape of mature cone outer segments is maintained via a remodelling process that accompanies the formation of distal invaginations.

Outer segment growth and periciliary vesicle turnover in developing photoreceptors of Xenopus laevis

SummaryIt has been proposed that periciliary vesicles in the photoreceptor inner segment represent newly synthesized membrane en route to the outer segment, and that membrane is delivered to the outer segment via fusion of these vesicles with the plasma membrane at the base of the connecting cilium and sclerad flow of the ciliary membrane. The present research was undertaken to test the periciliary vesicle hypothesis and clarify the dynamics of membrane flow in vertebrate photoreceptors. Light- and electron-microscopic measurements on developing photoreceptors in the retina of Xenopus laevis were used to determine the amount of membrane in outer segments and in periciliary vesicles. No significant diurnal variations were found in outer segment growth rate or size of the periciliary vesicle population. In all rods and in cones at the end of the experiment, the area of periciliary vesicle membrane was proportional to the rate at which membrane was added to the outer segment. Thus, the turnover time for the periciliary vesicle population was similar in rods and cones, supporting the periciliary vesicle hypothesis. Quantification of periciliary vesicle membrane in inner segments provides a method for determining the rate at which membrane is added to outer segments, heretofore not possible for cones.

Calmodulin immunolocalization in outer segments of Xenopus laevis photoreceptors

Abstract. Because adaptation of vertebrate photoreceptors to light is mediated by changes in the level of calcium in their outer segments (OS), proteins that bind calcium are important in phototransduction. This study has used immunofluorescence to investigate the distribution of the calcium-binding protein calmodulin within photoreceptor OS dissociated from amphibian (Xenopus laevis) retinas. The OS of rods and cones had a streak of fluorescence to calmodulin at the ciliary axoneme. The OS of rods (but not cones) also displayed regularly spaced puncta of anti-calmodulin fluorescence along longitudinal lines coinciding with their multiple incisures. This location of calmodulin immunofluorescence closely matches the known location of microtubules within the OS of amphibian rods and cones. These findings provide evidence that calmodulin is closely associated with the microtubules of both the axonemal and incisural cytoskeletal systems in OS, and suggest that this association is important for calmodulin function in photoreceptors.

Diverse Localization of Cyclic Nucleotide Gated Channels in the Outer Segments of Rods and Cones

The spatial distribution of cyclic nucleotide gated (CNG) channel molecules in photoreceptor outer segments (OS) dissociated from amphibian retinas was investigated by performing immunofluorescent localization of spectrin and using the known spectrin immunoreactivity of the beta subunit of the channel in rods to infer the location of CNG channels. In the OS of rods and cones, anti-spectrin immunoreactivity occurred as a bright streak of fluorescence at the ciliary axoneme. Rod OS displayed an additional pattern of staining not present in cone OS, namely a series of thin, discrete, longitudinal lines of fluorescence that extended the entire length of the OS and coincided with incisures. Thus, the location of immunoreactivity to spectrin in the OS of both photoreceptor cell types coincided with locations known to contain arrays of longitudinally-oriented microtubules. These findings provide strong evidence that CNG channel molecules are confined within OS membranes to specific restricted locations in the immediate vicinity of microtubules, eg., CNG channel molecules may be tethered to microtubules via the spectrin-like portion of their beta subunits. Because the localization of CNG channels within photoreceptor OS is expected to influence the spatiotemporal dynamics of phototransduction and adaptation, the diverse localization of channels within the OS could contribute to the different functional properties of rods and cones. Because evidence suggests that the OS of human and amphibian photoreceptors have similar microtubule-containing cytoskeletal systems at similar locations, the spatial distribution of CNG channel molecules described here for amphibian photoreceptor OS is also expected to occur in human photoreceptor OS. A disturbance in the localization of CNG channels, or in their associations with other molecules or microtubules, within photoreceptor OS is expected to disturb OS structure and function, which may be relevant for some human retinal degenerations.