Haohua Qian

Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration

Retinitis pigmentosa, caused predominantly by mutations in photoreceptor genes, currently lacks comprehensive treatment. We discover that retinal microglia contribute non‐cell autonomously to rod photoreceptor degeneration by primary phagocytosis of living rods. Using rd10 mice, we found that the initiation of rod degeneration is accompanied by early infiltration of microglia, upregulation of phagocytic molecules in microglia, and presentation of “eat‐me” signals on mutated rods. On live‐cell imaging, infiltrating microglia interact dynamically with photoreceptors via motile processes and engage in rapid phagocytic engulfment of non‐apoptotic rods. Microglial contribution to rod demise is evidenced by morphological and functional amelioration of photoreceptor degeneration following genetic ablation of retinal microglia. Molecular inhibition of microglial phagocytosis using the vitronectin receptor antagonist cRGD also improved morphological and functional parameters of degeneration. Our findings highlight primary microglial phagocytosis as a contributing mechanism underlying cell death in retinitis pigmentosa and implicate microglia as a potential cellular target for therapy.

Deletion of aryl hydrocarbon receptor AHR in mice leads to subretinal accumulation of microglia and RPE atrophy.

PURPOSE The aryl hydrocarbon receptor (AHR) is a ligand-activated nuclear receptor that regulates cellular response to environmental signals, including UV and blue wavelength light. This study was undertaken to elucidate AHR function in retinal homeostasis. METHODS RNA-seq data sets were examined for Ahr expression in the mouse retina and rod photoreceptors. The Ahr(-/-) mice were evaluated by fundus imaging, optical coherence tomography, histology, immunohistochemistry, and ERG. For light damage experiments, adult mice were exposed to 14,000 to 15,000 lux of diffuse white light for 2 hours. RESULTS In mouse retina, Ahr transcripts were upregulated during development, with continued increase in aging rod photoreceptors. Fundus examination of 3-month-old Ahr(-/-) mice revealed subretinal autofluorescent spots, which increased in number with age and following acute light exposure. Ahr(-/-) retina also showed subretinal microglia accumulation that correlated with autofluorescence changes, RPE abnormalities, and reactivity against immunoglobulin, complement factor H, and glial fibrillary acidic protein. Functionally, Ahr(-/-) mice displayed reduced ERG c-wave amplitudes. CONCLUSIONS The Ahr(-/-) mice exhibited subretinal accumulation of microglia and focal RPE atrophy, phenotypes observed in AMD. Together with a recently published report on another Ahr(-/-) mouse model, our study suggests that AHR has a protective role in the retina as an environmental stress sensor. As such, its altered function may contribute to human AMD progression and provide a target for pharmacological intervention.

Wnt signaling in age-related macular degeneration: human macular tissue and mouse model

BackgroundThe wingless-type MMTV integration site (Wnt) signaling is a group of signal transduction pathways. In canonical Wnt pathway, Wnt ligands bind to low-density lipoprotein receptor-related protein 5 or 6 (LRP5 or LRP6), resulting in phosphorylation and activation of the receptor. We hypothesize that canonical Wnt pathway plays a role in the retinal lesion of age-related macular degeneration (AMD), a leading cause of irreversible central visual loss in elderly.MethodsWe examined LRP6 phosphorylation and Wnt signaling cascade in human retinal sections and plasma kallistatin, an endogenous inhibitor of the Wnt pathway in AMD patients and non-AMD subjects. We also used the Ccl2−/−/Cx3cr1−/−/rd8 and Ccl2−/−/Cx3cr1gfp/gfp mouse models with AMD-like retinal degeneration to further explore the involvement of Wnt signaling activation in the retinal lesions in those models and to preclinically evaluate the role of Wnt signaling suppression as a potential therapeutic option for AMD.ResultsWe found higher levels of LRP6 (a key Wnt signaling receptor) protein phosphorylation and transcripts of the Wnt pathway-targeted genes, as well as higher beta-catenin protein in AMD macula compared to controls. Kallistatin was decreased in the plasma of AMD patients. Retinal non-phosphorylated-β-catenin and phosphorylated-LRP6 were higher in Ccl2−/−/Cx3cr1−/−/rd8 mice than that in wild type. Intravitreal administration of an anti-LRP6 antibody slowed the progression of retinal lesions in Ccl2−/−/Cx3cr1−/−/rd8 and Ccl2−/−/Cx3cr1gfp/gfp mice. Electroretinography of treated eyes exhibited larger amplitudes compared to controls in both mouse models. A2E, a retinoid byproduct associated with AMD was lower in the treated eyes of Ccl2−/−/Cx3cr1−/−/rd8 mice. Anti-LRP6 also suppressed the expression of Tnf-α and Icam-1 in Ccl2−/−/Cx3cr1−/−/rd8 retinas.ConclusionsWnt signaling may be disturbed in AMD patients, which could contribute to the retinal inflammation and increased A2E levels found in AMD. Aberrant activation of canonical Wnt signaling might also contribute to the focal retinal degenerative lesions of mouse models with Ccl2 and Cx3cr1 deficiency, and intravitreal administration of anti-LRP6 antibody could be beneficial by deactivating the canonical Wnt pathway.

Long-term rescue of cone photoreceptor degeneration in retinitis pigmentosa 2 (RP2)-knockout mice by gene replacement therapy

Retinal neurodegenerative diseases are especially attractive targets for gene replacement therapy, which appears to be clinically effective for several monogenic diseases. X-linked forms of retinitis pigmentosa (XLRP) are relatively severe blinding disorders, resulting from progressive photoreceptor dysfunction primarily caused by mutations in RPGR or RP2 gene. With a goal to develop gene therapy for the XLRP-RP2 disease, we first performed detailed characterization of the Rp2-knockout (Rp2-KO) mice and observed early-onset cone dysfunction, which was followed by progressive cone degeneration, mimicking cone vision impairment in XLRP patients. The mice also exhibited distinct and significantly delayed falling phase of photopic b-wave of electroretinogram (ERG). Concurrently, we generated a self-complementary adeno-associated viral (AAV) vector carrying human RP2-coding sequence and demonstrated its ability to mediate stable RP2 protein expression in mouse photoreceptors. A long-term efficacy study was then conducted in Rp2-KO mice following AAV-RP2 vector administration. Preservation of cone function was achieved with a wide dose range over 18-month duration, as evidenced by photopic ERG and optomotor tests. The slower b-wave kinetics was also completely restored. Morphologically, the treatment preserved cone viability, corrected mis-trafficking of M-cone opsin and restored cone PDE6 expression. The therapeutic effect was achieved even in mice that received treatment at an advanced disease stage. The highest AAV-RP2 dose group demonstrated retinal toxicity, highlighting the importance of careful vector dosing in designing future human trials. The wide range of effective dose, a broad treatment window and long-lasting therapeutic effects should make the RP2 gene therapy attractive for clinical development.

Deletion of olfactomedin 2 induces changes in the AMPA receptor complex and impairs visual, olfactory, and motor functions in mice

Olfactomedin 2 (Olfm2) is a secretory glycoprotein belonging to the family of olfactomedin domain-containing proteins. A previous study has shown that a mutation in OLFM2 is associated with primary open angle glaucoma in Japanese patients. In the present study, we generated Olfm2 deficient mice by replacing the Olfm2 gene with the LacZ gene. The loss of Olfm2 resulted in no gross abnormalities. However, Olfm2 null mice showed reduced exploration, locomotion, olfactory sensitivity, abnormal motor coordination, and anxiety related behavior. The pattern of the Olfm2 gene expression was studied in the brain and eye using β-galactosidase staining. In the brain, Olfm2 was mainly expressed in the olfactory bulb, cortex, piriform cortex, olfactory trabeculae, and inferior and superior colliculus. In the eye expression was detected mainly in retinal ganglion cells. In Olfm2 null mice, the amplitude of the first negative wave in the visual evoked potential test was significantly reduced as compared with wild-type littermates. Olfm2, similar to Olfm1, interacted with the GluR2 subunit of the AMPAR complexes and Olfm2 co-segregated with the AMPA receptor subunit GluR2 and other synaptic proteins in the synaptosomal membrane fraction upon biochemical fractionation of the adult mice cortex and retina. Immunoprecipitation from the synaptosomal membrane fraction of the Olfm2 null mouse brain cortex using the GluR2 antibody showed reduced levels of several components of the AMPAR complex in the immunoprecipitates including Olfm1, PSD95 and CNIH2. These results suggest that heterodimers of Olfm1 and Olfm2 interact with AMPAR more efficiently than Olfm2 homodimers and that Olfm2 plays a role in the organization of the AMPA receptor complexes.

Distinctive patterns of alterations in proton efflux from goldfish retinal horizontal cells monitored with self-referencing H + -selective electrodes

The H+ hypothesis of lateral feedback inhibition in the outer retina predicts that depolarizing agents should increase H+ release from horizontal cells. To test this hypothesis, self‐referencing H+‐selective microelectrodes were used to measure extracellular H+ fluxes from isolated goldfish horizontal cells. We found a more complex pattern of cellular responses than previously observed from horizontal cells of other species examined using this technique. One class of cells had an initial standing signal indicative of high extracellular H+ adjacent to the cell membrane; challenge with glutamate, kainate or high extracellular potassium induced an extracellular alkalinization. This alkalinization was reduced by the calcium channel blockers nifedipine and cobalt. A second class of cells displayed spontaneous oscillations in extracellular H+ that were abolished by cobalt, nifedipine and low extracellular calcium. A strong correlation between changes in intracellular calcium and extracellular proton flux was detected in experiments simultaneously monitoring intracellular calcium and extracellular H+. A third set of cells was characterized by a standing extracellular alkalinization which was turned into an acidic signal by cobalt. In this last set of cells, addition of glutamate or high extracellular potassium did not significantly alter the proton signal. Taken together, the response characteristics of all three sets of neurons are most parsimoniously explained by activation of a plasma membrane Ca2+ ATPase pump, with an extracellular alkalinization resulting from exchange of intracellular calcium for extracellular H+. These findings argue strongly against the hypothesis that H+ release from horizontal cells mediates lateral inhibition in the outer retina.

Maturation arrest in early postnatal sensory receptors by deletion of the miR-183/96/182 cluster in mouse

Significance MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally. The evolutionarily conserved miR-183/96/182 cluster, consisting of three related miRNAs, is highly expressed in maturing sensory receptor cells. However, its role in the functional maturation of sensory receptors has not been adequately addressed due to the lack of appropriate in vivo models. We show that deletion of miR-183/96/182 in mice leads to severe deficits in vision, hearing, balance, and smell. These deficits arise from defects in the timing and completion of terminal differentiation in sensory receptor cells associated with dysregulation of networks of genes involved in key processes, such as chromatin remolding and ciliogenesis. Thus, the miR-183/96/182 cluster has an essential role for the maturation of sensory receptors. The polycistronic miR-183/96/182 cluster is preferentially and abundantly expressed in terminally differentiating sensory epithelia. To clarify its roles in the terminal differentiation of sensory receptors in vivo, we deleted the entire gene cluster in mouse germline through homologous recombination. The miR-183/96/182 null mice display impairment of the visual, auditory, vestibular, and olfactory systems, attributable to profound defects in sensory receptor terminal differentiation. Maturation of sensory receptor precursors is delayed, and they never attain a fully differentiated state. In the retina, delay in up-regulation of key photoreceptor genes underlies delayed outer segment elongation and possibly mispositioning of cone nuclei in the retina. Incomplete maturation of photoreceptors is followed shortly afterward by early-onset degeneration. Cell biologic and transcriptome analyses implicate dysregulation of ciliogenesis, nuclear translocation, and an epigenetic mechanism that may control timing of terminal differentiation in developing photoreceptors. In both the organ of Corti and the vestibular organ, impaired terminal differentiation manifests as immature stereocilia and kinocilia on the apical surface of hair cells. Our study thus establishes a dedicated role of the miR-183/96/182 cluster in driving the terminal differentiation of multiple sensory receptor cells.

Repopulating retinal microglia restore endogenous organization and function under CX3CL1-CX3CR1 regulation

Chemokine signaling via CX3CL1-CX3CR1 helps regulate homeostasis in the population of microglia in the adult mouse retina. Microglia have been discovered to undergo repopulation following ablation. However, the functionality of repopulated microglia and the mechanisms regulating microglia repopulation are unknown. We examined microglial homeostasis in the adult mouse retina, a specialized neural compartment containing regular arrays of microglia in discrete synaptic laminae that can be directly visualized. Using in vivo imaging and cell-fate mapping techniques, we discovered that repopulation originated from residual microglia proliferating in the central inner retina that subsequently spread by centrifugal migration to fully recapitulate pre-existing microglial distributions and morphologies. Repopulating cells fully restored microglial functions including constitutive “surveying” process movements, behavioral and physiological responses to retinal injury, and maintenance of synaptic structure and function. Microglial repopulation was regulated by CX3CL1-CX3CR1 signaling, slowing in CX3CR1 deficiency and accelerating with exogenous CX3CL1 administration. Microglial homeostasis following perturbation can fully recover microglial organization and function under the regulation of chemokine signaling between neurons and microglia.