While the location of the L-type VDCC in the RPE surface membrane has not been determined, the only conductances that were found to be present on the basolateral membrane surface of the RPE were Cl− and K+ conductances [122, 179].

Why does BMD manifest only in terms of macular rather than diffuse disease? One critical aspect may be that the expression of BEST-1 is not uniform in the retina with higher levels being expressed in the periphery than in the macula [123]. Bestrophin protein levels were 10–390% greater in the periphery vs. macula and BEST-1 mRNA levels showed a peripheral/macular ratios from 2.4 to 2.8. Such a distribution of bestrophin expression may help to explain why BMD manifests in the anatomic macula and not in the retinal periphery. Clearly, the macular region sustains the greatest exposure to light of the entire retina due to its orthogonal positioning along the axis of the pupil aperture. Given the essential role that BEST-1 plays in establishing at least a substantial part of the basolateral Cl− conductance of the RPE, and the role of the RPE membrane in generating the components of the DC ERG in response to light, the additional light-mediated stresses placed on the macula with a limiting level of BEST-expression could, in fact, contribute to the disease process. This example shows that in an autosomal dominant disease such as BMD, the presence of only approximately 50% of WT protein must be taken in the context of regional expression distributions within the retina. Indeed, haploinsufficiency might be spatially distributed across the retinal cell matrix for a given disease gene.

11.2.3.5  RS1

Comparatively, much less is known about retinoschisin compared to the proteins previously discussed. RS1 is a discoidin-domain containing protein that is expressed and secreted by the photoreceptors, bipolar cells, and ganglion cells and acts to form a matrix of extracellular contacts throughout the outer and inner retina [46, 49, 124, 125, 194]. RS1 is first expressed early in retinal development [125]. The strongest expression is in the photoreceptor cells. RS1 localizes throughout both the inner and outer retina with the greatest immunocytochemical staining in the region of outer plasma membrane surfaces of the photoreceptor inner segments and bipolar cells [49]. Retinoschisin forms disulfide-linked octamers within the intracellular compartment prior to secretion [126]. The highly conserved discoidin domain,

which is also found in clotting factors and other proteins, occupies about 75% of the total protein sequence. Most human RS1 mutations occur within this domain [200]. The role of the secreted protein appears to be in establishing and maintaining cell: cell interactions among the photoreceptors, bipolar cells, and Müller glia and inner retinal neurons extending up to the ganglion cells that are essential for retinal tissue architecture and stability, and which are paramount to proper function (Fig. 11.14) [46, 49, 125, 127, 195, 199]. Müller glial cells play a role in distributing RS1 throughout the retina through a process of transcytosis [128]. While RS1 appears to be synthesized by all retinal neurons during development [125], other experiments have shown that retinoschisin, synthesized and secreted into the subretinal space by photoreceptors, is endocytosed by the apical microvilli of Müller cells, which then transport it internally and secrete it throughout the inner and outer retina at its focus sites in the mature retina [49, 125, 127, 128]. This may be a mechanism to supplement retinoschisin expression throughout the retina. RS1 is a peripheral membrane protein that interacts with the anionic phospholipid head charges on the outer surface of the membranes in a divalent ion-dependent fashion [129]. There is evidence that the discoidin domain of RS1 can form interactions with the extracellular collagen matrix, which would be important for anchoring the cells within the extracellular space of the retina. When there is a deficiency of the secreted protein proper cell: cell interactions, and likely cell: matrix interactions cannot occur, and the stage is set for the clinically evident retinal lesions of XLRS. A recent study has shown that RS1 forms protein: protein interactions with Na+/K+ ATPase in the surface membranes of retinal neurons [130]. This suggests that RS1 may also serve a signaling or physiological role in addition to its apparent role in forming cell: cell and cell: substrate interactions.

11.3  Cellular and Tissue Lesions

in Stargardt’s, Best’s, Juvenile

CRD, and JXRS

11.3.1  STGD

The classical clinical lesions of STGD are small yellow to white spots in the macular region that exist at the level of the RPE. These lesions represent clusters of

RPE cells that are engorged with LF, a highly heterogeneous proteolipid mixture which also contains fluorescent lipids. There may be RPE reaction and clumping around the yellow lesions that is also noted clinically. The progressive accumulation of LF in single RPE cells and the local coalescence of LF-engorged RPE cells leads to regional RPE clusters that have functional compromise (see below). Each single RPE cell provides nutrient exchange (with the choriocapillaris), metabolic support (e.g., retinoid metabolism), and daily phagocytosis for some 25–30 overlying photoreceptors in the perifovea and most of these cells in the perifovea

where STGD emerges are rod photoreceptors [58, 59]. This creates a huge metabolic load – a single RPE cell phagocytizes and digests on the order of about five full photoreceptor outer segments per day. Most of this material originates from the rod photoreceptor cells in the perifovea, especially, since the turnover of mammalian cones may be slower than rods [131]. Local compromise and apoptosis of RPE cell clusters and their overlying photoreceptors are responsible for the small macular scotomas that are found in STGD disease, as measured by visual field maps and multifocal electroretinography. These lesions extend outside the

macula in FF indicating a more widespread cellular perturbation in this form of the disease.

11.3.2  BMD

The classical clinical lesion of Bests disease is a larger egg yolk-shaped elevated yellow singular lesion at the level of the RPE and in the foveal region. This lesion emerges over time and eventually dissipates to form a geographic region of foveal RPE disturbance. A central scotoma may be left in this wake and generally central vision is compromised. This focal clinical lesion poorly represents the underlying pathophysiology recognized in the pathognomonic suppression of the EOG, which represents an electrophysiological response of the RPE of the entire retina. The underlying biochemical lesion is at the level of the surface membrane Cl− ionic channels, and perhaps intracellular Cl− channels which play roles in membrane transport by the RPE in its role to maintain deturgescence of the subretinal space, and possibly in lysosomal function of the degrading outer

segment debris. Prior studies by histology and recent studies by OCT have demonstrated that the initial lesion of Best’s (vitelliform) is due to the accumulation of material between the RPE apical surface and the photoreceptor outer segments (i.e., the subretinal space) [132, 133]. The yellowish character of this lesion suggests retinoid content and, in fact, recent studies have also shown that the accumulation of A2E plays a dominant role in the pathogenesis of Best’s disease [134]. The lesion itself points to a failure of both the transepithelial RPE fluid transport from the subretinal space to the choriocapillaris, and an impairment in phagocytosis of the shed rod and cone outer segment fragments. The lesion of BMD undergoes a characteristic temporal progression. Initially, the lesion is formed into an egg yolk-like sac and suggests a nonfluid accumulation. Later, the material appears to enter into a less viscous or more fluid state and is able to settle with respect to gravity, or the soluble materials settle out leaving only a clear fluid behind. It is at this point that the underlying RPE, as is still commonly intact, can be viewed through the relatively transparent retina above the

yellow pseudohypopyon. Later, as the material resolves with RPE reaction, there is often the emergence of RPE atrophy and central geographic atrophy not unlike that which occurs in dAMD.

animal models of juvenile macular diseases have emerged. Some of these models are also useful for investigative efforts to understand and treat adult macular degenerative diseases (e.g., AMD) as well.

The classical lesions of JXRS are a pinwheel-shaped arrangement of the inner retinal cystic cavities surrounding the fovea, and larger schisis cavities in the midperipheral and usually inferior retina [46]. Defi­ ciency of RS1 causes a generalized disruption of the retinal laminar architecture with loss of integrity of the outer plexiform and inner nuclear layers and profound loss of photoreceptors. Structural delamination of the inner retina is a characteristic of JXRS, and “vitreous veils” of the inner retina separated from the bulk retina appear in the clinical retinal anatomic exam. While the disease is often stationary and benign, marked loss of central vision can result from foveal lesions, and severe complications can arise from larger retinal schisis cavities including retinal detachment, vitreous hemorrhage, and neovascular glaucoma. Macular cystic cavities found in youth often disappear to leave only an altered foveal reflex in adults.

Also retinoschisin has been localized within the outer plexiform layer at the synapse between the photoreceptors and bipolar cells [125]. This localization of RS1 to the synapse coincides in development with the appearance of the b-wave of the ERG, indicating the importance of RS1 to synaptic architecture and maintenance. Breakdown of the photoreceptor: bipolar synapse is a characteristic feature of JXRS, which is indicated by a loss of b-wave in the ERG of affected individuals.

11.4  Animal Models Available

for Research of Disease

Mechanism and Therapeutics

Animal models are critical both to understand the mechanisms of retinal and macular degenerative diseases and to test candidate therapeutic strategies. A number of both small and large-scale mammalian

There are murine animal models for STGD disease that include a knockout of ABCR [135], a transgenic model for the 5 bp human deletion in ELOVL4 [94, 95], and a knock-in at the mouse ELOVL4 locus of a genomic expression construct of the human 5 bp deletion of ELOVL4 [74, 75, 136]. The ABCR knockout accumulates LF and A2E and has slowed dark adaptation but no early onset retinal degeneration as would be seen in STGD. Nevertheless, this is a useful model for understanding the mechanism of A2E formation, accumulation, and toxicity at the level of the RPE. The ELOVL4 transgenic mouse model has an altered formation of VLCFAs, accumulates LF and A2E, and sustains an early onset degeneration biased mostly to the center of retina which manifests as ERG a-wave and b-wave changes and histological evidence for photoreceptor and RPE degeneration. The time of onset and the rate of degeneration were proportional to the level of expression of the mutant human transgene in different lines of mice. This is a realistic model for the pathophysiology of STGD, and also dAMD, and a suitable model to test the therapeutics for rescue and toxicity. The ELOVL4 mutant knock-in resulted in a heterozygous mouse with one copy of human mutant ELOVL4 and another copy of WT mouse ELOVL4, with each gene controlled by its intrinsic promoter. This model accurately simulates the dose load for autosomal dominant STGD, and provides appropriate regulation of the gene. This model demonstrates LF accumulation in the RPE as well as ERG and histological evidence for cone–rod degeneration (CRD) which simulates STGD. Evidence for reductions in long-chain fatty acid synthesis was also demonstrated, including EPA and DHA. The cone degeneration appears to affect only short wave-sensitive cones and not middle wavelength-sensitive cones. This mouse model appears to be an excellent resource to understand both the mechanism of autosomal dominant STGD3 disease, and to test the safety and efficacy of therapeutics.