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Scholarly Works (4 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2017)

Recessive Stargardt disease (STGD1) is a blinding juvenile macular degeneration caused by mutations in ATP-binding cassette subfamily A member 4 (ABCA4), a membrane protein thought to be exclusively expressed in photoreceptor outer segment (OS) discs. Its proposed function is an inward-directed flippase for N-retinylidene-phosphatidylethanolamine (N-ret-PE), the conjugate of retinaldehyde and phosphatidylethanolamine, across OS discs. In STGD1 patients and mice with a null mutation in ABCA4 (Abca4-/-), excess free retinaldehyde irreversibly reacts with N-ret-PE to form toxic bisretinoids, which accumulate as lipofuscin pigments in the retinal pigment epithelium (RPE). Chapter 1 of this dissertation provides the first evidence that ABCA4 is expressed in human and murine RPE, with subcellular localization to endo-phagolysosomal membranes, thus suggesting that bisretinoids may be forming directly in the RPE, in addition to being secondarily deposited from phagocytosed OS. Chapter 2 reveals that transgenic mice expressing ABCA4 in the RPE, but not in the retina, had decreased autofluorescence and lipofuscin granules, greater photoreceptor preservation, and significant reduction of bisretinoid and complement factor C3b accumulation, confirming that RPE-specific ABCA4 plays a functional role in RPE internal membranes, as it does in photoreceptor OS discs. Since bisretinoids in the RPE result in susceptibility to complement-mediated injury and failure to support the neurosensory retina, we attempted in Chapter 3 to rescue the phenotype in Abca4-/- mice by increasing expression of a major complement negative-regulatory protein (CRP), complement receptor 1-like protein y (CRRY), in the RPE. Subretinal injection of recombinant adeno-associated virus containing CRRY (AAV-CRRY) in Abca4-/- mice significantly increased RPE CRRY levels, while reducing the accumulation of C3b, total bisretinoids and RPE lipofuscin. Rescue of Abca4-/- mice using AAV-CRRY gene therapy suggests that modulation of the complement system by increasing expression of CRPs in the RPE may be a potential treatment strategy for retinopathies associated with complement dysregulation. Chapter 4 discusses the implications of this dissertation which 1) presents a previously undiscovered role for RPE-expressed ABCA4 to limit excess toxic bisretinoid formation directly in the RPE and thereby maintain RPE health, and 2) demonstrates that the pathophysiology of STGD1 involves cell-autonomous, bisretinoid-induced susceptibility to complement, which can be reduced by complement modulation in the RPE.

    Cover page: Cell-Autonomous Retinal Pigment Epithelium Dysfunction in Stargardt Disease
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2017)
    Recessive Stargardt macular degeneration (STGD1) is caused by mutations in the gene for the ABCA4 transporter in photoreceptor outer segments. STGD1 patients and Abca4-/- (STGD1) mice exhibit buildup of bisretinoid-containing lipofuscin pigments in the retinal pigment epithelium (RPE), increased oxidative stress, augmented complement activation and slow degeneration of photoreceptors. A reduction in complement negative regulatory proteins (CRPs), possibly owing to bisretinoid accumulation, may be responsible for the increased complement activation seen on the RPE of STGD1 mice. CRPs prevent attack on host cells by the complement system, and complement receptor 1-like protein y (CRRY) is an important CRP in mice. Here we attempted to rescue the phenotype in STGD1 mice by increasing expression of CRRY in the RPE using a gene therapy approach. We injected recombinant adeno-associated virus containing the CRRY coding sequence (AAV-CRRY) into the subretinal space of 4-wk-old Abca4-/- mice. This resulted in sustained, several-fold increased expression of CRRY in the RPE, which significantly reduced the complement factors C3/C3b in the RPE. Unexpectedly, AAV-CRRY-treated STGD1 mice also showed reduced accumulation of bisretinoids compared with sham-injected STGD1 control mice. Furthermore, we observed slower photoreceptor degeneration and increased visual chromophore in 1-y-old AAV-CRRY-treated STGD1 mice. Rescue of the STGD1 phenotype by AAV-CRRY gene therapy suggests that complement attack on the RPE is an important etiologic factor in STGD1. Modulation of the complement system by locally increasing CRP expression using targeted gene therapy represents a potential treatment strategy for STGD1 and other retinopathies associated with complement dysregulation.
      Cover page: Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2017)
      Recessive Stargardt macular degeneration (STGD1) is caused by mutations in the gene for the ABCA4 transporter in photoreceptor outer segments. STGD1 patients and Abca4-/- (STGD1) mice exhibit buildup of bisretinoid-containing lipofuscin pigments in the retinal pigment epithelium (RPE), increased oxidative stress, augmented complement activation and slow degeneration of photoreceptors. A reduction in complement negative regulatory proteins (CRPs), possibly owing to bisretinoid accumulation, may be responsible for the increased complement activation seen on the RPE of STGD1 mice. CRPs prevent attack on host cells by the complement system, and complement receptor 1-like protein y (CRRY) is an important CRP in mice. Here we attempted to rescue the phenotype in STGD1 mice by increasing expression of CRRY in the RPE using a gene therapy approach. We injected recombinant adeno-associated virus containing the CRRY coding sequence (AAV-CRRY) into the subretinal space of 4-wk-old Abca4-/- mice. This resulted in sustained, several-fold increased expression of CRRY in the RPE, which significantly reduced the complement factors C3/C3b in the RPE. Unexpectedly, AAV-CRRY-treated STGD1 mice also showed reduced accumulation of bisretinoids compared with sham-injected STGD1 control mice. Furthermore, we observed slower photoreceptor degeneration and increased visual chromophore in 1-y-old AAV-CRRY-treated STGD1 mice. Rescue of the STGD1 phenotype by AAV-CRRY gene therapy suggests that complement attack on the RPE is an important etiologic factor in STGD1. Modulation of the complement system by locally increasing CRP expression using targeted gene therapy represents a potential treatment strategy for STGD1 and other retinopathies associated with complement dysregulation.
        Cover page: Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease.
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2018)
        Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4 -/- mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk -/- but not Abca4 -/- mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4 -/- background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4 -/- mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.
          Cover page: Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration
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