In cold-blooded vertebrates such as zebrafish, Müller glial cells (MGs) readily proliferate to replenish lost retinal neurons. In mammals, however, MGs lack regenerative capability as they do not spontaneously re-enter the cell cycle unless the retina is injured. Here, we show that gene transfer of β-catenin in adult mouse retinas activates Wnt signaling and MG proliferation without retinal injury. Upstream of Wnt, deletion of GSK3β stabilizes β-catenin and activates MG proliferation. Downstream of Wnt, β-catenin binds to the Lin28 promoter and activates transcription. Deletion of Lin28 abolishes β-catenin-mediated effects on MG proliferation, and Lin28 gene transfer stimulates MG proliferation. We further demonstrate that let-7 miRNAs are critically involved in Wnt/Lin28-regulated MG proliferation. Intriguingly, a subset of cell-cycle-reactivated MGs express markers for amacrine cells. Together, these results reveal a key role of Wnt-Lin28-let7 miRNA signaling in regulating proliferation and neurogenic potential of MGs in the adult mammalian retina.

Diabetic retinopathy (DR), the most common microvascular complication that occurs in diabetes mellitus (DM), is the leading cause of vision loss in working-age adults. The prevalence of diabetic retinopathy is approximately 30% of the diabetic population and untreated DR can eventually cause blindness. For decades, diabetic retinopathy was considered a microvascular complication and clinically staged by its vascular manifestations. In recent years, emerging evidence has shown that diabetic retinopathy causes early neuronal dysfunction and neurodegeneration that may precede vascular pathology and affect retinal neurons as well as glial cells. This knowledge leads to new therapeutic strategies aiming to prevent dysfunction of retinal neurons at the early stage of DR. Early detection and timely treatment to protect retinal neurons are critical to preventing visual loss in DR. This review provides an overview of DR and the structural and functional changes associated with DR, and discusses neuronal degeneration during diabetic retinopathy, the mechanisms underlying retinal neurodegeneration and microvascular complications, and perspectives on current and future clinic therapies.

Axons fail to regenerate after central nervous system (CNS) injury. Modulation of the PTEN/mTORC1 pathway in retinal ganglion cells (RGCs) promotes axon regeneration after optic nerve injury. Here, we report that AKT activation, downstream of Pten deletion, promotes axon regeneration and RGC survival. We further demonstrate that GSK3β plays an indispensable role in mediating AKT-induced axon regeneration. Deletion or inactivation of GSK3β promotes axon regeneration independently of the mTORC1 pathway, whereas constitutive activation of GSK3β reduces AKT-induced axon regeneration. Importantly, we have identified eIF2Bε as a novel downstream effector of GSK3β in regulating axon regeneration. Inactivation of eIF2Bε reduces both GSK3β and AKT-mediated effects on axon regeneration. Constitutive activation of eIF2Bε is sufficient to promote axon regeneration. Our results reveal a key role of the AKT-GSK3β-eIF2Bε signaling module in regulating axon regeneration in the adult mammalian CNS.

Reprogramming glial cells to convert them into neurons represents a potential therapeutic strategy that could repair damaged neural circuits and restore function. Recent studies show that downregulation of the RNA-binding protein PTBP1 leads to one-step conversion of Müller glia (MG) into retinal ganglion cells (RGCs) with a high efficiency. However, the original study did not perform fate-mapping experiments to confirm MG-to-RGC conversion after Ptbp1 downregulation. To address the fundamental question of whether Ptbp1 downregulation can convert MG into RGCs in the mouse retina, we perform fate-mapping experiments to lineage trace MG independent of the adeno-associated virus (AAV)-mediated labeling system. Here, we report that Ptbp1 downregulation by CRISPR-CasRx or small hairpin RNA is insufficient to convert MG to RGCs. The original conclusion of MG-to-RGC conversion is due to leaky labeling of endogenous RGCs. Our results emphasize the importance of using stringent fate mapping to determine glia-to-neuron conversion in cell reprogramming research.

Reprogramming resident glial cells to convert them into neurons in vivo represents a potential therapeutic strategy that could replenish lost neurons, repair damaged neural circuits, and restore function. AAV (adeno-associated virus)-based expression systems are powerful tools for in vivo gene delivery in glia-to-neuron reprogramming, however, recent studies show that AAV-based gene delivery of Neurod1 into the mouse brain can cause severe leaky expression into endogenous neurons leading to misinterpretation of glia-to-neuron conversion. AAV-based delivery systems were modified for improved in vivo delivery of Neurod1, Math5, Ascl1, and Neurog2 in the adult mouse retina and brain. To examine whether bona fide glia-to-neuron conversion occurs, stringent fate mapping experiments were performed to trace the lineage of glial cells. The neuronal leakage is prevalent after AAV-GFAP-mediated delivery of Neurod1, Math5, Ascl1, and Neurog2. The transgene-dependent leakage cannot be corrected after lowering the AAV doses, using alterative AAV serotypes or injection routes. Importantly, we report the development of two new AAV-based tools that can significantly reduce neuronal leakage. Using the new AAV-based tools, we provide evidence that Neurod1 gene transfer fails to convert lineage traced glial cells into neurons. Stringent fate mapping techniques independently of an AAV-based expression system are the golden standard for tracing the fate of glia cells during neuronal reprogramming. The newly developed AAV-based systems are invaluable tools for glia-to-neuron reprogramming in vivo. The work in Chen lab was supported by National Institutes of Health (NIH) grants R01 EY024986 and R01 EY028921, an unrestricted challenge grant from Research to Prevent Blindness, the New York Eye and Ear Infirmary Foundation, and The Harold W. McGraw, Jr. Family Foundation for Vision Research. The work in Zhang lab was supported by NIH (R01 NS127375 and R01 NS117065) and The Decherd Foundation.