Enzymatic vitamin A2 production enables red-shifted optogenetics.

Optogenetics is a technology using light-sensitive proteins to control signaling pathways and physiological processes in cells and organs and has been applied in neuroscience, cardiovascular sciences, and many other research fields. Most commonly used optogenetic actuators are sensitive to blue and green light, but red-light activation would allow better tissue penetration and less phototoxicity. Cyp27c1 is a recently deorphanized cytochrome P450 enzyme that converts vitamin A1 to vitamin A2, thereby red-shifting the spectral sensitivity of visual pigments and enabling near-infrared vision in some aquatic species.Here, we investigated the ability of Cyp27c1-generated vitamin A2 to induce a shift in spectral sensitivity of the light-gated ion channel Channelrhodopsin-2 (ChR2) and its red-shifted homolog ReaChR. We used patch clamp to measure photocurrents at specific wavelengths in HEK 293 cells expressing ChR2 or ReaChR. Vitamin A2 incubation red-shifted the wavelength for half-maximal currents (λ50%) by 6.8 nm for ChR2 and 12.4 nm for ReaChR. Overexpression of Cyp27c1 in HEK 293 cells showed mitochondrial localization, and HPLC analysis showed conversion of vitamin A1 to vitamin A2. Notably, the λ50% of ChR2 photocurrents was red-shifted by 10.5 nm, and normalized photocurrents at 550 nm were about twofold larger with Cyp27c1 expression. Similarly, Cyp27c1 shifted the λ50% of ReaChR photocurrents by 14.3 nm and increased normalized photocurrents at 650 nm almost threefold.Since vitamin A2 incubation is not a realistic option for in vivo applications and expression of Cyp27c1 leads to a greater red-shift in spectral sensitivity, we propose co-expression of this enzyme as a novel strategy for red-shifted optogenetics.

Chromatin access regulates the formation of Müller glia-derived progenitor cells in the retina.

Chromatin access and epigenetic control over gene expression play important roles in regulating developmental processes. However, little is known about how chromatin access and epigenetic gene silencing influence mature glial cells and retinal regeneration. Herein, we investigate the expression and functions of S-adenosylhomocysteine hydrolase (SAHH; AHCY) and histone methyltransferases (HMTs) during the formation of Müller glia (MG)-derived progenitor cells (MGPCs) in the chick and mouse retinas. In chick, AHCY, AHCYL1 and AHCYL2, and many different HMTs are dynamically expressed by MG and MGPCs in damaged retinas. Inhibition of SAHH reduced levels of H3K27me3 and potently blocks the formation of proliferating MGPCs. By using a combination of single cell RNA-seq and single cell ATAC-seq, we find significant changes in gene expression and chromatin access in MG with SAHH inhibition and NMDA-treatment; many of these genes are associated with glial and neuronal differentiation. A strong correlation across gene expression, chromatin access, and transcription factor motif access in MG was observed for transcription factors known to convey glial identity and promote retinal development. By comparison, in the mouse retina, inhibition of SAHH has no influence on the differentiation of neuron-like cells from Ascl1-overexpressing MG. We conclude that in the chick the activity of SAHH and HMTs are required for the reprogramming of MG into MGPCs by regulating chromatin access to transcription factors associated with glial differentiation and retinal development.

Thyroid hormone receptors mediate two distinct mechanisms of long-wavelength vision.

Thyroid hormone (TH) signaling plays an important role in the regulation of long-wavelength vision in vertebrates. In the retina, thyroid hormone receptor ß (thrb) is required for expression of long-wavelength-sensitive opsin (lws) in red cone photoreceptors, while in retinal pigment epithelium (RPE), TH regulates expression of a cytochrome P450 enzyme, cyp27c1, that converts vitamin A1 into vitamin A2 to produce a red-shifted chromophore. To better understand how TH controls these processes, we analyzed the phenotype of zebrafish with mutations in the three known TH nuclear receptor transcription factors (thraa, thrab, and thrb). We found that no single TH nuclear receptor is required for TH-mediated induction of cyp27c1 but that deletion of all three (thraa-/-;thrab-/-;thrb-/- ) completely abrogates its induction and the resulting conversion of A1- to A2-based retinoids. In the retina, loss of thrb resulted in an absence of red cones at both larval and adult stages without disruption of the underlying cone mosaic. RNA-sequencing analysis revealed significant down-regulation of only five genes in adult thrb-/- retina, of which three (lws1, lws2, and miR-726) occur in a single syntenic cluster. In the thrb-/- retina, retinal progenitors destined to become red cones were transfated into ultraviolet (UV) cones and horizontal cells. Taken together, our findings demonstrate cooperative regulation of cyp27c1 by TH receptors and a requirement for thrb in red cone fate determination. Thus, TH signaling coordinately regulates both spectral sensitivity and sensory plasticity.

Reactive microglia and IL1ß/IL-1R1-signaling mediate neuroprotection in excitotoxin-damaged mouse retina.

BACKGROUND: Microglia and inflammation have context-specific impacts upon neuronal survival in different models of central nervous system (CNS) disease. Herein, we investigate how inflammatory mediators, including microglia, interleukin 1 beta (IL1ß), and signaling through interleukin 1 receptor type 1 (IL-1R1), influence the survival of retinal neurons in response to excitotoxic damage. METHODS: Excitotoxic retinal damage was induced via intraocular injections of NMDA. Microglial phenotype and neuronal survival were assessed by immunohistochemistry. Single-cell RNA sequencing was performed to obtain transcriptomic profiles. Microglia were ablated by using clodronate liposome or PLX5622. Retinas were treated with IL1ß prior to NMDA damage and cell death was assessed in wild type, IL-1R1 null mice, and mice expressing IL-1R1 only in astrocytes. RESULTS: NMDA-induced damage included neuronal cell death, microglial reactivity, upregulation of pro-inflammatory cytokines, and genes associated with IL1ß-signaling in different types of retinal neurons and glia. Expression of the IL1ß receptor, IL-1R1, was evident in astrocytes, endothelial cells, some Müller glia, and OFF bipolar cells. Ablation of microglia with clodronate liposomes or Csf1r antagonist (PLX5622) resulted in elevated cell death and diminished neuronal survival in excitotoxin-damaged retinas. Exogenous IL1ß stimulated the proliferation and reactivity of microglia in the absence of damage, reduced numbers of dying cells in damaged retinas, and increased neuronal survival following an insult. IL1ß failed to provide neuroprotection in the IL-1R1-null retina, but IL1ß-mediated neuroprotection was rescued when expression of IL-1R1 was restored in astrocytes. CONCLUSIONS: We conclude that reactive microglia provide protection to retinal neurons, since the absence of microglia is detrimental to survival. We propose that, at least in part, the survival-influencing effects of microglia may be mediated by IL1ß, IL-1R1, and interactions of microglia and other macroglia.

mTor signaling is required for the formation of proliferating Müller glia-derived progenitor cells in the chick retina.

We investigate the roles of mTor signaling in the formation of Müller glia-derived progenitor cells (MGPCs) in the chick retina. During embryonic development, pS6 (a readout of active mTor signaling) is present in early-stage retinal progenitors, differentiating amacrine and ganglion cells, and late-stage progenitors or maturing Müller glia. By contrast, pS6 is present at low levels in a few scattered cell types in mature, healthy retina. Following retinal damage, in which MGPCs are known to form, mTor signaling is rapidly activated in Müller glia. Inhibition of mTor in damaged retinas prevented the accumulation of pS6 in Müller glia and reduced numbers of proliferating MGPCs. Inhibition of mTor had no effect on MAPK signaling or on upregulation of the stem cell factor Klf4, whereas Pax6 upregulation was significantly reduced. Inhibition of mTor potently blocked the MGPC-promoting effects of Hedgehog, Wnt and glucocorticoid signaling in damaged retinas. In the absence of retinal damage, insulin, IGF1 and FGF2 induced pS6 in Müller glia, and this was blocked by mTor inhibitor. In FGF2-treated retinas, in which MGPCs are known to form, inhibition of mTor blocked the accumulation of pS6, the upregulation of Pax6 and the formation of proliferating MGPCs. We conclude that mTor signaling is required, but not sufficient, to stimulate Müller glia to give rise to proliferating progenitors, and the network of signaling pathways that drive the formation of MGPCs requires activation of mTor.

Heparin-binding EGF-like growth factor (HB-EGF) stimulates the proliferation of Müller glia-derived progenitor cells in avian and murine retinas.

Müller glia can be stimulated to de-differentiate, proliferate and form Müller glia-derived progenitor cells (MGPCs) that regenerate retinal neurons. In the zebrafish retina, heparin-binding EGF-like growth factor (HB-EGF) may be one of the key factors that stimulate the formation of proliferating MGPCs. Currently nothing is known about the influence of HB-EGF on the proliferative potential of Müller glia in retinas of birds and rodents. In the chick retina, we found that levels of both hb-egf and egf-receptor are rapidly and transiently up-regulated following NMDA-induced damage. Although intraocular injections of HB-EGF failed to stimulate cell-signaling or proliferation of Müller glia in normal retinas, HB-EGF stimulated proliferation of MGPCs in damaged retinas. By comparison, inhibition of the EGF-receptor (EGFR) decreased the proliferation of MGPCs in damaged retinas. HB-EGF failed to act synergistically with FGF2 to stimulate the formation of MGPCs in the undamaged retina and inhibition of EGF-receptor did not suppress FGF2-mediated formation of MGPCs. In the mouse retina, HB-EGF stimulated the proliferation of Müller glia following NMDA-induced damage. Furthermore, HB-EGF not only stimulated MAPK-signaling in Müller glia/MGPCs, but also activated mTor- and Jak/Stat-signaling. We propose that levels of expression of EGFR are rate-limiting to the responses of Müller glia to HB-EGF and the expression of EGFR can be induced by retinal damage, but not by FGF2-treatment. We conclude that HB-EGF is mitogenic to Müller glia in both chick and mouse retinas, and HB-EGF is an important player in the formation of MGPCs in damaged retinas.

The reactivity, distribution and abundance of Non-astrocytic Inner Retinal Glial (NIRG) cells are regulated by microglia, acute damage, and IGF1.

Recent studies have described a novel type of glial cell that is scattered across the inner layers of the avian retina and possibly the retinas of primates. These cells have been termed Non-astrocytic Inner Retinal Glial (NIRG) cells. These cells are stimulated by insulin-like growth factor 1 (IGF1) to proliferate, migrate distally into the retina, and become reactive. These changes in glial activity correlate with increased susceptibility of retinal neurons and Müller glia to excitotoxic damage. The purpose of this study was to further study the NIRG cells in retinas treated with IGF1 or acute damage. In response to IGF1, the reactivity, proliferation and migration of NIRG cells persists through 3 days after treatment. At 7 days after treatment, the numbers and distribution of NIRG cells returns to normal, suggesting that homeostatic mechanisms are in place within the retina to maintain the numbers and distribution of these glial cells. By comparison, IGF1-induced microglial reactivity persists for at least 7 days after treatment. In damaged retinas, we find a transient accumulation of NIRG cells, which parallels the accumulation of reactive microglia, suggesting that the reactivity of NIRG cells and microglia are linked. When the microglia are selectively ablated by the combination of interleukin 6 and clodronate-liposomes, the NIRG cells down-regulate transitin and perish within the following week, suggesting that the survival and phenotype of NIRG cells are somehow linked to the microglia. We conclude that the abundance, reactivity and retinal distribution of NIRG cells can be dynamic, are regulated by homoestatic mechanisms and are tethered to the microglia.