Modulation of the embryonic stem cell state is beneficial for elucidating the innate mechanisms of development and regenerative medicine. Ion flux plays important roles in modulating the transition between stemness and differentiation in mouse embryonic stem cells (mESCs). Optogenetics is a novel tool for manipulating ion flux. To investigate the impact of optical stimulation on embryonic stem cells, optogenetically engineered V6.5 mESCs were used to measure the depolarization mediated by ChR2 on the proliferation, self-renewal, and differentiation of mESCs. Blue light stimulation significantly inhibited ChR2-GFP-V6.5 ESC proliferation and disrupted the cell cycle progression, reducing the proportion of cells in the S phase. Interestingly, optical stimulation could inhibit ChR2-GFP-V6.5 ESC self-renewal and trigger differentiation by activating the extracellular regulated protein kinase (ERK) signaling pathway. Our data suggest that membrane potential changes play pivotal roles in regulating the proliferation, self-renewal and initiation of differentiation of mESCs.

Gamma-amino butyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system, including the retina, and play an important role in both regulating neurogenesis and neural stem cell proliferation. GABAa receptor has been identified in the retina, however, the function of GABAa receptor on retinal progenitor cell (RPC) is unclear. RPCs were cultured to analyze changes in cell proliferation and cell cycle distribution after GABAa receptor activation. The activation of GABAa receptor significantly inhibits RPCs proliferation, cell cycle progress, and self-renewal. Moreover, the activation of GABAa receptor leads to the up-expression of p21 and p27 and down-expression of Nestin, Pax6, Sox2, and Chx10. These results suggest that GABA acts as a negative regulator of RPCs proliferation and self-renewal.

Background It has been found that the extensive use of anticancer drugs containing DNA-alkylating agents not only target cancer cells but also cause retinal inflammation through toxic intermediates. Complement C3 (C3) is a core component of the complement activation pathway, and dysregulation of the complement pathway is involved in several retinal degenerative diseases. However, whether C3 plays a critical role in alkylation-induced retinal degeneration is unclear. Methods Following treatment with the alkylating agent methyl methane sulfonate (MMS), the C3 mRNA and protein level was measured, DNA damage and photoreceptor cell death were assessed in both wild-type (WT) C57BL/6J and C3 knockout (KO) mice. Results We determined that complement pathway is activated following MMS treatment, and C3 knockout (KO) increased the rate of photoreceptor cell survival and preserved visual function. The mRNA levels of nuclear erythroid-related factor 2 (Nrf2) and related genes were higher after MMS application in C3 KO mice. Conclusion In summary, our study found that C3 KO promotes photoreceptor cell survival and activates the Nrf2 signaling pathway in the context of alkylation-induced retinal degeneration.

Retinal degeneration causes vision loss and threatens the health of elderly individuals worldwide. Evidence indicates that the activation of the complement system is associated with retinal degeneration. However, the mechanism of complement signaling in retinal degeneration needs to be further studied. In this study, we show that the expression of C3 and C3a receptor (C3ar1) is positively associated with the inflammatory response and retinal degeneration. Genetic deletion of C3 and pharmacological inhibition of C3ar1 resulted in the alleviation of neuroinflammation, prevention of photoreceptor cell apoptosis and restoration of visual function. RNA sequencing (RNA-seq) identified a C3ar1-dependent network shown to regulate microglial activation and astrocyte gliosis formation. Mechanistically, we found that STAT3 functioned downstream of the C3-C3ar1 pathway and that the C3ar1-STAT3 pathway functionally mediated the immune response and photoreceptor cell degeneration in response to oxidative stress. These findings reveal an important role of C3ar1 in oxidative-induced retinal degeneration and suggest that intervention of the C3ar1 pathway may alleviate retinal degeneration.

Background Age-related macular degeneration (AMD) threatens vision in elderly people globally. Systemic complement C3 has been shown to be tightly associated with AMD. However, the mechanism of how local retinal complement C3 and downstream pathway mediated retinal degeneration is not entirely clear. Methods Wild-type, C3 knockout mice and mice treated with a C3aR inhibitor (SB290157), STAT3 inhibitor (SH-4-54), were exposed to sodium iodine (NaIO3), respectively. C3 localization in degenerated retina was detected by using immunostaining. Retinal function was assessed by electroretinography, followed by histological analyses to assess RPE and photoreceptor cell degeneration. Retinal inflammation and mitochondrion were investigated through Real-time PCR, ELISA and RNA-Seq. Results We reported here that activation of complement was positively associated with retinal degeneration. Strikingly, intracellular C3 was activated in retinal pigment epithelial (RPE) cells, and subsequently promoted the formation of the membrane-attack complex (MAC), which contributed to apoptosis of RPE cells. Intracellular C3 deficiency or pharmalogical inhibition could rescue RPE cells from apoptosis. Moreover, genetic deletion of C3 or inhibition of C3aR/STAT3 results in alleviating immune response and rescue of photoreceptor cells under oxidative stress. Mechanistically, through RNA-Seq, we identified C3/C3aR/STAT3 pathway functionally mediated immune response and photoreceptor cell degeneration. Conclusions These results indicate that inhibition of the C3/C3aR/STAT3 pathway might be a new therapeutic intervention for AMD and other retinal degeneration diseases.