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Autosomal dominant optic atrophy (adOA) is a juvenile onset, progressive ocular disorder characterized by bilateral loss of vision, central visual field defects, colour vision disturbances, and optic disc pallor. adOA is most frequently associated with mutations in OPA1 encoding a dynamin-related large GTPase that localizes to mitochondria. Histopathological studies in adOA patients have shown a degeneration of retinal ganglion cells (RGCs) and a loss of axons in the optic nerve. However little is known about the molecular mechanism and pathophysiology of adOA due to the lack of appropriate in vivo models. Here we report a first mouse model carrying a splice site mutation (c.1065 + 5G → A) in the Opa1 gene. The mutation induces a skipping of exon 10 during transcript processing and leads to an in-frame deletion of 27 amino acid residues in the GTPase domain. Western blot analysis showed no evidence of a shortened mutant protein but a ∼50% reduced OPA1 protein level supporting haploinsufficiency as a major disease mechanism in adOA. Homozygous mutant mice die in utero during embryogenesis with first notable developmental delay at E8.5 as detected by magnetic resonance imaging (MRI). Heterozygous mutants are viable and of normal habitus but exhibit an age-dependent loss of RGCs that eventually progresses to a severe degeneration of the ganglion cell and nerve fibre layer. In addition optic nerves of mutant mice showed a reduced number of axons, and a swelling and abnormal shape of the remaining axons. Mitochondria in these axons showed disorganized cristae structures. All these defects recapitulate crucial features of adOA in humans and therefore document the validity and importance of this model for future research. [ABSTRACT FROM AUTHOR]

Loss of Norrin signalling due to mutations in the Norrie disease pseudoglioma gene causes severe vascular defects in the retina, leading to visual impairment and ultimately blindness. While the emphasis of experimental work so far was on the developmental period, we focus here on disease mechanisms that induce progression into severe adult disease. The goal of this study was the comprehensive analysis of the long-term effects of the absence of Norrin on vascular homeostasis and retinal function. In a mouse model of Norrie disease retinal vascular morphology and integrity were studied by means of in vivo angiography; the vascular constituents were assessed in detailed histological analyses using quantitative retinal morphometry. Finally, electroretinographic analyses were performed to assess the retinal function in adult Norrin deficient animals. We could show that the primary developmental defects not only persisted but developed into further vascular abnormalities and microangiopathies. In particular, the overall vessel homeostasis, the vascular integrity, and also the cellular constituents of the vascular wall were affected in the adult Norrin deficient retina. Moreover, functional analyses indicated to persistent hypoxia in the neural retina which was suggested as one of the major driving forces of disease progression. In summary, our data provide evidence that the key to adult Norrie disease are ongoing vascular modifications, driven by the persistent hypoxic conditions, which are ineffective to compensate for the primary Norrin-dependent defects.

Serum Response Factor (SRF) fulfills essential roles in post-natal retinal angiogenesis and adult neovascularization. These functions have been attributed to the recruitment by SRF of the cofactors Myocardin-Related Transcription Factors MRTF-A and -B, but not the Ternary Complex Factors (TCFs) Elk1 and Elk4. The role of the third TCF, Elk3, remained unknown. We generated a new Elk3 knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In Elk3(-/-) mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in Elk3(-/-) mice from the age of four weeks, and persisted into late adulthood. Tortuous vessels have been observed in human pathologies, e.g. in ROP and FEVR. These human disorders were linked to altered activities of vascular endothelial growth factor (VEGF) in the affected eyes. However, in Elk3(-/-) mice, we did not observe any changes in VEGF or several other potential confounding factors, including mural cell coverage and blood pressure. Instead, concurrent with the post-natal transient delay of radial outgrowth and the formation of adult tortuous arteries, Elk3-dependent effects on the expression of Angiopoietin/Tie-signalling components were observed. Moreover, in vitro microvessel sprouting and microtube formation from P10 and adult aortic ring explants were reduced. Collectively, these results indicate that Elk3 has distinct roles in maintaining retinal artery integrity. The Elk3 knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.

To monitor viability of implanted genetically engineered and microencapsulated human stem cells (MicroBeads) in the mouse eye, and to study the impact of the beads and/or xenogenic cells on retinal integrity.MicroBeads were implanted into the subretinal space of SV126 wild type mice using an ab externo approach. Viability of microencapsulated cells was monitored by noninvasive retinal imaging (Spectralis™ HRA+OCT). Retinal integrity was also assessed with retinal imaging and upon the end of the study by light and electron microscopy. The implanted GFP-marked cells encapsulated in subretinal MicroBeads remained viable over a period of up to 4 months. Retinal integrity and viability appeared unaltered apart from the focal damage due to the surgical implantation, GFAP upregulation, and opsin mistargeting in the immediate surrounding tissue.The accessibility for routine surgery and its immune privileged state make the eye an ideal target for release system implants for therapeutic substances, including neurotrophic and anti-angiogenic compounds or protein based biosimilars. Microencapsulated human stem cells (MicroBeads) promise to overcome limitations inherent with single factor release systems, as they are able to produce physiologic combinations of bioactive compounds.

Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of Crb1 and Crb2 genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.

BackgroundOptical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration.Methodology/principal findingsWe achieved to adapt a commercial 3(rd) generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Leber's congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified.Conclusions/significanceWe found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeutic strategies.