Sodium Iodate-Induced Degeneration Results in Local Complement Changes and Inflammatory Processes in Murine Retina

Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO3)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO3 caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components c1s, c3, c4, cfb and cfh. C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory ccl-2, -3, -5, il-1β, il-33 and tgf-β were increased in the retinas of NaIO3 mice, but vegf-a mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO3 retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO3-dependent retinal degeneration. In conclusion, systemically administered NaIO3 promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy

Transcriptome Analysis Did Not Show Endogenous Stem Cell Characteristics in Murine Lgr5+ Retinal Cells.

Lgr5, an intestinal adult stem cell marker, was recently also found in neuronal tissues. We investigated whether retinal Lgr5+ cells express properties of neural stem cells (NSC) and/or of differentiated interneurons during retinal development. RNA was isolated from Lgr5+ and Lgr5- populations from postnatal day 5 (PN5) and adult retinas of Lgr5EGFP-Ires-CreERT2 knock-in mice sorted by fluorescence-activated cell sorting (FACS). Transcriptome analyses were performed on two RNA samples of each developmental stage (PN5 and adult). The online platform PANTHER (Protein ANalysis THrough Evolutionary Relationships) was used to determine overrepresented gene ontology (GO) terms of biological processes within the set of differentially expressed genes. The detailed evaluation included gene expression in regard to stem cell maintenance/proliferation, cell cycle, and Wnt signaling but also markers of differentiated retinal neurons. None of the enriched GO terms of upregulated genes of Lgr5+ cells showed a positive association to NSC. On the contrary, NSC maintenance and proliferation rather prevail in the Lgr5- cell population. Furthermore, results suggesting that Wnt signaling is not active in the Lgr5+ population. Therefore, our transcriptome analysis of Lgr5+ retinal cells suggest that these cells are differentiated neurons, specifically glycinergic amacrine cells

The TGFβ/Notch axis facilitates Müller cell-to-epithelial transition to ultimately form a chronic glial scar.

BACKGROUND Contrasting with zebrafish, retinal regeneration from Müller cells (MCs) is largely limited in mammals, where they undergo reactive gliosis that consist of a hypertrophic response and ultimately results in vision loss. Transforming growth factor β (TGFβ) is essential for wound healing, including both scar formation and regeneration. However, targeting TGFβ may affect other physiological mechanisms, owing its pleiotropic nature. The regulation of various cellular activities by TGFβ relies on its interaction with other pathways including Notch. Here, we explore the interplay of TGFβ with Notch and how this regulates MC response to injury in zebrafish and mice. Furthermore, we aimed to characterize potential similarities between murine and human MCs during chronic reactive gliosis. METHODS Focal damage to photoreceptors was induced with a 532 nm diode laser in TgBAC (gfap:gfap-GFP) zebrafish (ZF) and B6-Tg (Rlbp1-GFP) mice. Transcriptomics, immunofluorescence, and flow cytometry were employed for a comparative analysis of MC response to laser-induced injury between ZF and mouse. The laser-induced injury was paired with pharmacological treatments to inhibit either Notch (DAPT) or TGFβ (Pirfenidone) or TGFβ/Notch interplay (SIS3). To determine if the murine laser-induced injury model translates to the human system, we compared the ensuing MC response to human donors with early retinal degeneration. RESULTS Investigations into injury-induced changes in murine MCs revealed TGFβ/Notch interplay during reactive gliosis. We found that TGFβ1/2 and Notch1/2 interact via Smad3 to reprogram murine MCs towards an epithelial lineage and ultimately to form a glial scar. Similar to what we observed in mice, we confirmed the epithelial phenotype of human Müller cells during gliotic response. CONCLUSION The study indicates a pivotal role for TGFβ/Notch interplay in tuning MC stemness during injury response and provides novel insights into the remodeling mechanism during retinal degenerative diseases

Diverse Signaling by TGFβ Isoforms in Response to Focal Injury is Associated with Either Retinal Regeneration or Reactive Gliosis.

Müller cells may have stem cell-like capability as they regenerate photoreceptor loss upon injury in some vertebrates, but not in mammals. Indeed, mammalian Müller cells undergo major cellular and molecular changes summarized as reactive gliosis. Transforming growth factor beta (TGFβ) isoforms are multifunctional cytokines that play a central role, both in wound healing and in tissue repair. Here, we studied the role of TGFβ isoforms and their signaling pathways in response to injury induction during tissue regeneration in zebrafish and scar formation in mouse. Our transcriptome analysis showed a different activation of canonical and non-canonical signaling pathways and how they shaped the injury response. In particular, TGFβ3 promotes retinal regeneration via Smad-dependent canonical pathway upon regulation of junb gene family and mycb in zebrafish Müller cells. However, in mice, TGFβ1 and TGFβ2 evoke the p38MAPK signaling pathway. The activation of this non-canonical pathway leads to retinal gliosis. Thus, the regenerative versus reparative effect of the TGFβ pathway observed may rely on the activation of different signaling cascades. This provides one explanation of the different injury response in zebrafish and mouse retina

Retinal microglia signaling affects Müller cell behavior in the zebrafish following laser injury induction.

Microglia are the resident tissue macrophages of the central nervous system including the retina. Under pathophysiological conditions, microglia can signal to Müller cells, the major glial component of the retina, affecting their morphological, molecular, and functional responses. Microglia-Müller cell interactions appear to be bidirectional shaping the overall injury response in the retina. Hence, microglia and Müller cell responses to disease and injury have been ascribed both positive and negative outcomes. However, Müller cell reactivity and survival in the absence of immune cells after injury have not been investigated in detail in adult zebrafish. Here, we develop a model of focal retinal injury combined with pharmacological treatments for immune cell depletion in zebrafish. The retinal injury was induced by a diode laser to damage photoreceptors. Two pharmacological treatments were used to deplete either macrophage-microglia (PLX3397) or selectively eliminate peripheral macrophages (clodronate liposomes). We show that PLX3397 treatment hinders retinal regeneration in zebrafish, which is reversed by microglial repopulation. On the other hand, selective macrophage elimination did not affect the kinetics of retinal regeneration. The absence of retinal microglia and macrophages leads to dysregulated Müller cell behavior. In the untreated fish, Müller cells react after injury induction showing glial fibrillary acidic protein (GFAP), Phospho-p44/42 MAPK (Erk1/2), and PCNA upregulation. However, in the immunosuppressed animals, GFAP and phospho-p44/42 MAPK (Erk1/2) expression was not upregulated overtime and the reentry in the cell cycle was not affected. Thus, microglia and Müller cell signaling is pivotal to unlock the regenerative potential of Müller cells in order to repair the damaged retina