Pathological Effects of Mutant C1QTNF5 (S163R) Expression in Murine Retinal Pigment Epithelium.

The retinal pigment epithelium is a polarized monolayer of postmitotic epithelial cells located between the neural retina and the vascular choroid. On its apical side, it contains numerous microvilli extending into the interphotoreceptor matrix, and surrounding the outer segments of rods and cones. On the basal side is Bruch's membrane, a thin, multilayered, extracellular matrix1 in which pathological changes are integral to AMD.2,3 The retinal pigment epithelium performs functions essential for both the structural integrity and survival of photoreceptor cells, including transport of nutrients, retinoids and waste products, phagocytosis of outer segments, synthesis of the visual chromophore11-cis retinal, and production of growth factors.4,5 The mutation S163R in the globular gC1q domain of complement C1q tumor necrosis factor–related protein-5 (C1QTNF5/CTRP5) causes a rare autosomal dominant disorder known as late-onset retinal degeneration (L-ORD).6 The earliest symptoms appear in midlife, manifested as difficulties in dark adaptation and loss of night vision.7–9 Over time, the disorder progresses to severe loss of both central and peripheral vision, with some patients also developing choroidal neovascularization in late stages of the disease10 that mimic neovascular AMD. In addition, patients also present with abnormally long anterior zonular insertions on the lens capsule which serve as an early disease phenotype.11,12 The hallmark of L-ORD is the presence of abnormal extracellular deposits between the RPE and Bruch's membrane.7,13 These thick deposits, which are histopathologically similar to those found in AMD patients, have a widespread distribution, extending outside the macular region into the peripheral retina.14 Although there is evidence that supplementation with vitamin A may be beneficial early in the disease course when patients exhibit dark-adaptation abnormalities,11 there is no current treatment that prevents the progressive retinal degeneration leading to vision loss. C1QTNF5 belongs to the C1q and tumor necrosis factor-related superfamily (C1q/TNF), characterized by an N-terminal signal peptide, a short variable region, a collagen repeat domain, and a C-terminal globular domain (gC1q) that is homologous to the immune complement component 1q. All members of the C1q/TNF family are secreted multimeric proteins, with specific tissue expression profiles, and many are found circulating in plasma, with levels influenced by genetic background, sex, and metabolic status. Members of the C1q/TNF family are of broad clinical interest since they play numerous roles in immunity and inflammation, glucose and lipid metabolism, and vascular maintenance.15,16 C1QTNF5 is a membrane-associated and secreted protein of unknown function, whose expression is the highest in the RPE and ciliary epithelium.11,17,18 It colocalizes with ZO1, an RPE tight-junction protein, at the hexagonal RPE membrane, and is also secreted into the interphotoreceptor matrix.17,19 C1QTNF5 can also be found in other ocular tissues, including the lens.17 It is secreted by the adipose tissue, and circulates abundantly in human sera, potentially playing an important role in energy homeostasis and obesity-related inflammation.16,20 The crystal structure of the trimeric globular domain of C1QTNF5 was determined at 1.34 A resolution by Tu and Palczewski,21 who also showed that wild-type C1QTNF5 can multimerize into a bouquet-like octadecameric assembly containing six trimers.22 Previous in vitro studies have demonstrated that the S163R mutant is not secreted, has the tendency to aggregate, and is retained primarily within the endoplasmic reticulum.17,23 Interestingly, C1QTNF5 is expressed as a dicistronic transcript with MFRP, encoding the membrane-type frizzled related protein.6,24 The complete open reading frame of C1QTNF5 is located within the 3′ untranslated region of the final exon of MFRP. The two proteins were shown to directly interact; however, the functional significance of this interaction is not currently understood.18,22,23 Mutations in the two genes are associated with distinct disease phenotypes in humans, with the dominant S163R mutation in C1QTNF5 leading to L-ORD, whereas mutations in MFRP cause autosomal recessive retinitis pigmentosa (RP), nanophthalmos, foveoschisis, and optic disc drusen.25–27 We have previously shown that subretinal delivery of a fast-acting AAV8-733YF vector expressing wild-type MFRP led to robust transgene expression at its normal RPE location on the apical membrane and microvilli, prevented retinal degeneration, and rescued photoreceptor function in the rd6 mouse model of RP.28 In this study, we used the recombinant AAV vector technology to examine the pattern of mutant S163R transgene expression and its pathological effects following specific RPE targeting. We show that AAV-expressed wild-type C1QTNF5 is secreted apically toward photoreceptor cells and outer limiting membrane (OLM). In contrast, AAV-expressed S163R mutant forms large spherical intracellular aggregates within the RPE cells of normal mice, and gradually accumulates as thick, widespread extracellular basal deposits, leading to local regions of RPE atrophy and retinal degeneration. These findings have important mechanistic and therapeutic implications for L-ORD and perhaps for AMD as well.