Deficiency in Retinal TGFß Signaling Aggravates Neurodegeneration by Modulating Pro-Apoptotic and MAP Kinase Pathways.

Transforming growth factor ß (TGFß) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFß receptor type 2 (Tgfbr2) is upregulated in retinal neurons and Müller cells during retinal degeneration. In this study we investigated if this upregulation of TGFß signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGFß signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and Müller cells (Tgfbr2ΔOC) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGFß signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice (Tgfbr2ΔOC; VPP) by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGFß signaling in retinal neurons and Müller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans.

Transcriptional Profiling Identifies Upregulation of Neuroprotective Pathways in Retinitis Pigmentosa.

Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-ß regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-ß, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-ß, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.

Light Intensity-Dependent Dysregulation of Retinal Reference Genes.

The degeneration of photoreceptors is a common hallmark of ocular diseases like retinitis pigmentosa (RP) or age-related macular degeneration (AMD). To experimentally induce photoreceptor degeneration, the light damage paradigm is frequently used. In this study we show that the exposure to high amounts of cool white light (10,000 lux, 1 h) resulted in a more than 11-fold higher apoptotic rate in the retina compared to light exposure with 5000 lux for 30 min. Consequently, exposure to intense light resulted in a significant downregulation of retinal mRNA expression levels of the reference genes Gapdh, Gnb2l, Rpl32, Rps9, Actb, Ubc or Tbp compared to untreated controls. Investigators performing light-induced photoreceptor degeneration should be aware of the fact that higher light intensities will result in a dysregulation of reference genes.

New Insights into Endothelin Signaling and Its Diverse Roles in the Retina.

The vasoactive peptide endothelin is an effective regulator of blood pressure and vascular homeostasis. In addition, the dysregulation of the endothelin signaling pathway is discussed to contribute to ocular diseases like glaucoma or diabetic retinopathy. Furthermore, our workgroup and others showed a protective effect of endothelin 2 for the survival of photoreceptors. In this study, we analyzed mRNA expression levels of the endothelin signaling family in wild-type mice after a puncture of the eye, intravitreal PBS injections, or light-induced photoreceptor degeneration. We observed elevated endothelin receptor a (Eta), endothelin receptor b (Etb), endothelin 1(Et1), and endothelin 2 (Et2) levels, while endothelin 3 (Et3) mRNA levels were not significantly altered. Our findings indicate an important role of the endothelin signaling pathway in response to ocular trauma or disease. These findings make endothelin signaling a promising target to attenuate retinal degeneration.

Complement Components Showed a Time-Dependent Local Expression Pattern in Constant and Acute White Light-Induced Photoreceptor Damage.

Background: Photoreceptor cell death due to extensive light exposure and induced oxidative-stress are associated with retinal degeneration. A correlated dysregulation of the complement system amplifies the damaging effects, but the local and time-dependent progression of this mechanism is not thoroughly understood. Methods: Light-induced photoreceptor damage (LD) was induced in Balb/c mice with white light illumination either for 24 h with 1000 lux (constant model) or 0.5 h with 5000 lux (acute model). Complement protein and mRNA expression levels were compared at 1 and 3 days post-LD for C1s, complement factor B (CFB), mannose binding lectin A, mannose-binding protein-associated serine protease 1 (MASP-1), C3, C4, C9, and complement factor P in retina and RPE/choroid. Histological analyses visualized apoptosis, microglia/macrophage migration, gliosis and deposition of the complement activation marker C3d. Systemic anaphylatoxin serum concentrations were determined using an ELISA. Results: Apoptosis, gliosis and microglia/macrophage migration into the outer nuclear layer showed similar patterns in both models. Local complement factor expression revealed an early upregulation of complement factor mRNA in the acute and constant light regimen at 1 day post-treatment for c1s, cfb, masp-1, c3, c4 and c9 in the RPE/choroid. However, intraretinal complement mRNA expression for c1s, cfb, c3 and c4 was increased at 1 day in the constant and at 3 days in the acute model. A corresponding regulation on protein level in the retina following both LD models was observed for C3, which was upregulated at 1 day and correlated with increased C3d staining in the ganglion cell layer and at the RPE. In the RPE/choroid C1s-complex protein detection was increased at 3 days after LD irrespectively of the light intensities used. Conclusion: LD in mouse eyes is correlated with local complement activity. The time-dependent local progression of complement regulation on mRNA and protein levels were equivalent in the acute and constant LD model, except for the intraretinal, time-dependent mRNA expression. Knowing the relative time courses of local complement expression and cellular activity can help to elucidate novel therapeutic options in retinal degeneration indicating at which time point of disease complement has to be rebalanced.

SMAD7 deficiency stimulates Müller progenitor cell proliferation during the development of the mammalian retina.

The transforming growth factor-ß (TGF-ß) pathway contributes to maintain the quiescence of adult neural stem and progenitor cells in the brain. In the retina, Müller cells are discussed to represent a glial cell population with progenitor-like characteristics. Here, we aimed to investigate if elevated TGF-ß signaling modulates the proliferation of Müller cells during retinal development. We generated mutant mice with a systemic, heterozygous up-regulation of TGF-ß signaling by deleting its inhibitor SMAD7. We investigated apoptosis, proliferation, and differentiation of Müller cells in the developing retina. We show that a heterozygous deletion of SMAD7 results in an increased proliferation of Müller cell progenitors in the central retina at postnatal day 4, the time window when Müller cells differentiate in the mouse retina. This in turn results in a thickened retina and inner nuclear layer and a higher number of differentiated Müller cells in the more developed retina. Müller cells in mutant mice contain higher amounts of nestin than those of control animals which indicates that the increase in TGF-ß signaling activity during retinal development contribute to maintain some progenitor-like characteristics in Müller cells even after their differentiation period. We conclude that TGF-ß signaling influences Müller cell proliferation and differentiation during retinal development.