Minzhong Yu

Norrin, Frizzled-4, and Lrp5 Signaling in Endothelial Cells Controls a Genetic Program for Retinal Vascularization

Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled-4 (Fz4) receptor, Lrp5 coreceptor, or Norrin ligand cause retinal hypovascularization, but the mechanisms by which Norrin/Fz4/Lrp signaling controls vascular development have not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, whereas excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is upregulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.

Age-related changes in visual function in cystathionine-beta-synthase mutant mice, a model of hyperhomocysteinemia

Homocysteine is an amino acid required for the metabolism of methionine. Excess homocysteine is implicated in cardiovascular and neurological disease and new data suggest a role in various retinopathies. Mice lacking cystathionine-beta-synthase (cbs(-/-)) have an excess of retinal homocysteine and develop anatomical abnormalities in multiple retinal layers, including photoreceptors and ganglion cells; heterozygous (cbs(+/-)) mice demonstrate ganglion cell loss and mitochondrial abnormalities in the optic nerve. The purpose of the present study was to determine whether elevated homocysteine, due to absent or diminished cbs, alters visual function. We examined cbs(-/-) (3 weeks) and cbs(+/-) mice (5, 10, 15, 30 weeks) and results were compared to those obtained from wild type (WT) littermates. Conventional dark- and light-adapted ERGs were recorded, along with dc-ERG to assess retinal pigment epithelial (RPE) function. The visual evoked potential (VEP) was used to assess transmission to the visual cortex. The amplitudes of the major ERG components were reduced in cbs(-/-) mice at age 3 weeks and VEPs were delayed markedly. These findings are consistent with the early retinal disruption observed anatomically in these mice. In comparison, at 3 weeks of age, responses of cbs(+/-) mice did not differ significantly from those of WT mice. Functional abnormalities were not observed in cbs(+/-) mice until 15 weeks of age, at which time amplitude reductions were noted for the ERG a- and b-wave and the light peak component, but not for other components generated by the RPE. VEP implicit times were delayed in cbs(+/-) mice at 15 and 30 weeks, while VEP amplitudes were unaffected. The later onset of functional defects in cbs(+/-) mice is consistent with a slow loss of ganglion cells reported previously in the heterozygous mutant. Light peak abnormalities indicate that RPE function is also compromised in older cbs(+/-) mice. The data suggest that severe elevations of homocysteine are associated with marked alterations of retinal function while modest homocysteine elevation is reflected in milder and delayed alterations of retinal function. The work lays the foundation to explore the role of homocysteine in retinal diseases such as glaucoma and optic neuropathy.

A Novel Role of Complement in Retinal Degeneration

PURPOSE The association of single nucleotide polymorphisms of components of the complement alternative pathway with the risk of age-related macular degeneration (AMD) indicates that complement signaling plays an important role in retinal physiology. How genetic variation leads to retinal degeneration is unknown. It has been assumed that complement activation augments immune responses, which in turn initiate AMD pathogenesis. To better understand the relationship between complement and the outer retina, we examined mice lacking the main complement component C3 and the receptors for complement activation fragments C3a (C3aR) and/or C5a (C5aR). METHODS Complement mutant mice were studied along with wild-type (WT) littermates from 6 weeks to 14 months of age. Strobe flash electroretinography (ERG) was used to examine outer retinal function and a dc-ERG technique was used to measure ERG components generated by the retinal pigment epithelium. Retinas were examined by histology, immunohistochemistry, and biochemistry. RESULTS Mice lacking C3aR and/or C5aR developed early onset and progressive retinal degeneration, accompanied by cleaved caspase-3 upregulation. Genetic deletion of C3aR and/or C5aR led to cell-specific defects that matched the cellular localization of these receptors in the WT retina. Compared to WT, C3aR(-/-) and C3aR(-/-)C5aR(-/-) mice showed increased retinal dysfunction upon light exposure. C3aR(-/-)C5aR(-/-) mice immunized with 4-hydroxynonenal-adducted protein developed severe retinal impairment unrelated to immune response. CONCLUSIONS C3aR- and C5aR-mediated signaling was necessary to maintain normal retinal function and structure. These receptors may be important biomarkers for predicting retinal degeneration including AMD.

Carnosic acid slows photoreceptor degeneration in the Pde6b(rd10) mouse model of retinitis pigmentosa.

The photoreceptor cell death associated with the various genetic forms of retinitis pigmentosa (RP) is currently untreatable and leads to partial or complete vision loss. Carnosic acid (CA) upregulates endogenous antioxidant enzymes and has proven neuroprotective in studies of neurodegenerative models affecting the brain. In this study, we examined the potential effect of CA on photoreceptor death in the Pde6brd10 mouse model of RP. Our data shows that CA provided morphological and functional preservation of photoreceptors. CA appears to exert its neuroprotective effects through inhibition of oxidative stress and endoplasmic reticulum stress.

Distinct and Atypical Intrinsic and Extrinsic Cell Death Pathways between Photoreceptor Cell Types upon Specific Ablation of Ranbp2 in Cone Photoreceptors

Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2), a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1+-apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct retinal spatial signatures as well as with other etiologically distinct neurodegenerative disorders.

Disruption of murine Adamtsl4 results in zonular fiber detachment from the lens and in retinal pigment epithelium dedifferentiation

Human gene mutations have revealed that a significant number of ADAMTS (a disintegrin-like and metalloproteinase (reprolysin type) with thrombospondin type 1 motifs) proteins are necessary for normal ocular development and eye function. Mutations in human ADAMTSL4, encoding an ADAMTS-like protein which has been implicated in fibrillin microfibril biogenesis, cause ectopia lentis (EL) and EL et pupillae. Here, we report the first ADAMTSL4 mouse model, tvrm267, bearing a nonsense mutation in Adamtsl4. Homozygous Adamtsl4(tvrm267) mice recapitulate the EL phenotype observed in humans, and our analysis strongly suggests that ADAMTSL4 is required for stable anchorage of zonule fibers to the lens capsule. Unexpectedly, homozygous Adamtsl4(tvrm267) mice exhibit focal retinal pigment epithelium (RPE) defects primarily in the inferior eye. RPE dedifferentiation was indicated by reduced pigmentation, altered cellular morphology and a reduction in RPE-specific transcripts. Finally, as with a subset of patients with ADAMTSL4 mutations, increased axial length, relative to age-matched controls, was observed and was associated with the severity of the RPE phenotype. In summary, the Adamtsl4(tvrm267) model provides a valuable tool to further elucidate the molecular basis of zonule formation, the pathophysiology of EL and ADAMTSL4 function in the maintenance of the RPE.

Complement anaphylatoxin receptors C3aR and C5aR are required in the pathogenesis of experimental autoimmune uveitis

Recent studies have suggested that reagents inhibiting complement activation could be effective in treating T cell mediated autoimmune diseases such as autoimmune uveitis. However, the precise role of the complement anaphylatoxin receptors (C3a and C5a receptors) in the pathogenesis of autoimmune uveitis remains elusive and controversial. We induced experimental autoimmune uveitis in mice deficient or sufficient in both C3a and C5a receptors and rigorously compared their retinal phenotype using various imaging techniques, including indirect ophthalmoscopy, confocal scanning laser ophthalmoscopy, spectral domain optical coherence tomography, topical endoscopic fundus imaging, and histopathological analysis. We also assessed retinal function using electroretinography. Moreover, we performed Ag‐specific T cell recall assays and T cell adoptive transfer experiments to compare pathogenic T cell activity between wild‐type and knockout mice with experimental autoimmune uveitis. These experiments showed that C3a receptor/C5a receptor‐deficient mice developed much less severe uveitis than did control mice using all retinal examination methods and that these mice had reduced pathogenic T cell responses. Our data demonstrate that both complement anaphylatoxin receptors are important for the development of experimental autoimmune uveitis, suggesting that targeting these receptors could be a valid approach for treating patients with autoimmune uveitis.

Deficiency of CC chemokine ligand 2 and decay-accelerating factor causes retinal degeneration in mice

CC chemokine ligand 2 (CCL2) recruits macrophages to reduce inflammatory responses. Decay-accelerating factor (DAF) is a membrane regulator of the classical and alternative pathways of complement activation. In view of the link between complement genes and retinal diseases, we evaluated the retinal phenotype of C57BL/6J mice and mice lacking Ccl2 and/or Daf1 at 12 months of age, using scanning laser ophthalmoscopic imaging, electroretinography (ERG), histology, immunohistochemistry, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis. In comparison to C57BL/6J mice, mutant mice had an increased number of autofluorescent foci, with the greatest number in the Ccl2(-/-)/Daf1(-/-) retina. ERG amplitudes in Ccl2(-/-)/Daf1(-/-), Ccl2(-/-) and Daf1(-/-) mice were reduced, with the greatest reduction in Ccl2(-/-)/Daf1(-/-) mice. TUNEL-positive cells were not seen in C57BL/6J retina, but were prevalent in the outer and inner nuclear layers of Ccl2(-/-)Daf1(-/-) mice and were present at reduced density in Ccl2(-/-) or Daf1(-/-) mice. Cell loss was most pronounced in the outer and inner nuclear layers of Ccl2(-/-)/Daf1(-/-) mice. The levels of the endoplasmic reticulum chaperone GPR78 and transcription factor ATF4 were significantly increased in the Ccl2(-/-)/Daf1(-/-) retina. In comparison to the C57BL/6J retina, the phosphorylation of NF-κB p65, p38, ERK and JNK was significantly upregulated while SIRT1 was significantly downregulated in the Ccl2(-/-)/Daf1(-/-) retina. Our results suggest that loss of Ccl2 and Daf1 causes retinal neuronal death and degeneration which is related to increased endoplasmic reticulum stress, oxidative stress and inflammation.

Visual abnormalities associated with enhanced optic nerve myelination

Expression of the constitutively active serine/threonine kinase Akt in oligodendrocytes results in enhanced myelination in the CNS. Here, we have examined the effects of this Akt overexpression on optic nerve structure and on optic nerve function, assessed using the visual evoked potential (VEP). Transgenic mice have been generated with the Plp promoter driving expression of a modified form of Akt, in which aspartic acids are substituted for Thr308 and Ser473. These Plp-Akt-DD (Akt-DD) mice, and littermate controls, were studied at different ages. Optic nerves were examined anatomically at 2 and 6 months of age. At 2 months of age, optic nerves were substantially thicker in Akt-DD mice, reflecting an increase in myelination of optic nerve axons. By electron microscopy, myelin thickness was increased in Akt-DD optic nerve, with extended paranodal domains having excess paranodal loops, and the density of nodes of Ranvier was reduced, relative to control mice. We recorded VEPs in response to strobe flash ganzfeld stimuli presented after overnight dark- and light-adapted conditions at ages ranging from 1 to 10 months. It was possible to record a clear VEP from Akt-DD mice at all ages examined. At 1 month of age, VEP implicit times were somewhat shorter in Akt-DD transgenic mice than in control animals. Beyond 6months of age, VEP latencies were consistently delayed in Akt-DD transgenic mice. These abnormalities did not reflect an alteration in retinal function as there were no significant differences between ERGs obtained from control or Akt-DD transgenic mice. In young mice, the somewhat faster responses may reflect improved transmission due to increased myelination of optic nerve axons. In older mice, where the Akt-DD optic nerve is markedly thicker than control, it is remarkable that optic nerves continue to function.

Mutations in CTNNA1 cause butterfly-shaped pigment dystrophy and perturbed retinal pigment epithelium integrity

Butterfly-shaped pigment dystrophy is an eye disease characterized by lesions in the macula that can resemble the wings of a butterfly. Here we report the identification of heterozygous missense mutations in the CTNNA1 gene (encoding α-catenin 1) in three families with butterfly-shaped pigment dystrophy. In addition, we identified a Ctnna1 missense mutation in a chemically induced mouse mutant, tvrm5. Parallel clinical phenotypes were observed in the retinal pigment epithelium (RPE) of individuals with butterfly-shaped pigment dystrophy and in tvrm5 mice, including pigmentary abnormalities, focal thickening and elevated lesions, and decreased light-activated responses. Morphological studies in tvrm5 mice demonstrated increased cell shedding and the presence of large multinucleated RPE cells, suggesting defects in intercellular adhesion and cytokinesis. This study identifies CTNNA1 gene variants as a cause of macular dystrophy, indicates that CTNNA1 is involved in maintaining RPE integrity and suggests that other components that participate in intercellular adhesion may be implicated in macular disease.