Identification of a Novel Axon Regeneration Role for Noncanonical Wnt Signaling in the Adult Retina after Injury

Canonical and noncanonical Wnt signaling pathways are essential for development and maintenance of the CNS. Whereas the roles of canonical Wnt pathways in neuronal survival and axonal regeneration in adult CNS have been described, the functions of noncanonical Wnt pathways are not well understood. Furthermore, the role of noncanonical Wnt ligands in the adult retina has not been investigated. Noncanonical Wnt signaling shares receptors with canonical Wnt ligands but functions through calcium and c-Jun N-terminal kinase (JNK) signaling pathways. Noncanonical ligands, such as the prototypic ligand Wnt5a, have varying effects in the developing CNS, including inhibiting or promoting axonal growth. To identify a role for noncanonical Wnt signaling in the developed retina after injury, we characterized the effect of Wnt5a on neurite outgrowth in cultured retinal ganglion cell (RGC) neurons and on axonal regeneration in the injured optic nerve in the mouse. Endogenous Wnt5a was upregulated after injury and exogenous Wnt5a significantly enhanced neurite growth of primary RGCs and led to extensive axonal regeneration after optic nerve crush (ONC) injury. Wnt5a also significantly increased RGC survival. Furthermore, Wnt5a induced phosphorylation of CamKII and JNK and induced expression of their downstream pathway components. Therefore, these results demonstrate for the first time that Wnt5a promotes axonal growth and protects RGCs in the adult retina

Lipid profiling dataset of the Wnt3a-induced optic nerve regeneration

We present lipid profiling data from mouse retina and optic nerve after optic nerve crush and during Wnt3a-induced axonal regeneration at 7 and 15 days post-crush. This data is available at the Metabolomics Workbench, http://www.metabolomicsworkbench.org (Project ID: PR000718)

Increased Neuroprotective Microglia and Photoreceptor Survival in the Retina from a Peptide Inhibitor of Myeloid Differentiation Factor 88 (MyD88)

Myeloid differentiation factor 88 (MyD88) is an adaptor protein for the Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) families of innate immunity receptors that mediate inflammatory responses to cellular injury. TLR/IL1R/MyD88 signaling is known to contribute to retinal degeneration, although how MyD88 regulates neuronal survival, and the effect of MyD88 on the inflammatory environment in the retina, is mostly unknown. In this study, we tested the hypothesis that blocking MyD88-mediated signaling early in retinal degeneration promotes transition of microglia towards a neuroprotective anti-inflammatory phenotype, resulting in enhanced photoreceptor survival. We also tested whether systemic delivery of a pharmacologic MyD88 inhibitor has therapeutic potential. The rd10 mouse model of retinal degeneration was injected intraperitoneally with increasing doses of a MyD88 blocking peptide or control peptide early in degeneration, and inflammatory responses and photoreceptor survival were measured at specific time points using flow cytometry, cytokine profiling, and electroretinograms. Our results demonstrated that rd10 mice injected with a low dose of MyD88 inhibitor peptide showed increased rod photoreceptor function and reduced apoptosis compared with control peptide and uninjected mice. MyD88 inhibition also resulted in fewer microglia/macrophage cells in the photoreceptor layer whereas total peripheral and retinal macrophage were not changed. Furthermore, increased number of cells expressing the Arg1 marker of neuroprotective microglia in the photoreceptor layer and higher MCP-1 and anti-inflammatory cytokine IL-27 were associated with photoreceptor survival. Therefore, these data suggest that the MyD88 inhibitor modified the retina environment to become less inflammatory, leading to improved photoreceptor function and survival

Quantitative proteomic analysis after neuroprotective MyD88 inhibition in the retinal degeneration 10 mouse

Progressive photoreceptor death occurs in blinding diseases such as retinitis pigmentosa. Myeloid differentiation primary response protein 88 (MyD88) is a central adaptor protein for innate immune system Toll‐like receptors (TLR) and induces cytokine secretion during retinal disease. We recently demonstrated that inhibiting MyD88 in mouse models of retinal degeneration led to increased photoreceptor survival, which was associated with altered cytokines and increased neuroprotective microglia. However, the identity of additional molecular changes associated with MyD88 inhibitor‐induced neuroprotection is not known. In this study, we used isobaric tags for relative and absolute quantification (iTRAQ) labelling followed by LC‐MS/MS for quantitative proteomic analysis on the rd10 mouse model of retinal degeneration to identify protein pathways changed by MyD88 inhibition. Quantitative proteomics using iTRAQ LC‐MS/MS is a high‐throughput method ideal for providing insight into molecular pathways during disease and experimental treatments. Forty‐two proteins were differentially expressed in retinas from mice treated with MyD88 inhibitor compared with control. Notably, increased expression of multiple crystallins and chaperones that respond to cellular stress and have anti‐apoptotic properties was identified in the MyD88‐inhibited mice. These data suggest that inhibiting MyD88 enhances chaperone‐mediated retinal protection pathways. Therefore, this study provides insight into molecular events contributing to photoreceptor protection from modulating inflammation

The cytokine IL-27 reduces inflammation and protects photoreceptors in a mouse model of retinal degeneration

Background Retinal degenerative diseases are a group of conditions characterized by photoreceptor death and vision loss. Excessive inflammation and microglial activation contribute to the pathology of retinal degenerations and a major focus in the field is identifying more effective anti-inflammatory therapeutic strategies that promote photoreceptor survival. A major challenge to developing anti-inflammatory treatments is to selectively suppress detrimental inflammation while maintaining beneficial inflammatory responses. We recently demonstrated that endogenous levels of the IL-27 cytokine were upregulated in association with an experimental treatment that increased photoreceptor survival. IL-27 is a pleiotropic cytokine that regulates tissue reactions to infection, neuronal disease and tumors by inducing anti-apoptotic and anti-inflammatory genes and suppressing pro-inflammatory genes. IL-27 is neuroprotective in the brain, but its function during retinal degeneration has not been investigated. In this study, we investigated the effect of IL-27 in the rd10 mouse model of inherited photoreceptor degeneration. Methods Male and female rd10 mice were randomly divided into experimental (IL-27) and control (saline) groups and intravitreally injected at age post-natal day (P) 18. Retina function was analyzed by electroretinograms (ERGs), visual acuity by optomotor assay, photoreceptor death by TdT-mediated dUTP nick-end labeling (TUNEL) assay, microglia/macrophage were detected by immunodetection of IBA1 and inflammatory mediators by cytoplex and QPCR analysis. The distribution of IL-27 in the retina was determined by immunohistochemistry on retina cross-sections and primary Muller glia cultures. Results We demonstrate that recombinant IL-27 decreased photoreceptor death, increased retinal function and reduced inflammation in the rd10 mouse model of retinal degeneration. Furthermore, IL-27 injections led to lower levels of the pro-inflammatory proteins Ccl22, IL-18 and IL-12. IL-27 expression was localized to Muller glia and IL-27 receptors to microglia, which are key cell types that regulate photoreceptor survival. Conclusion Our results identify for the first time anti-inflammatory and neuroprotective activities of IL-27 in a genetic model of retinal degeneration. These findings provide new insight into the therapeutic potential of anti-inflammatory cytokines as a treatment for degenerative diseases of the retina

Wnt signaling induces neurite outgrowth in mouse retinal ganglion cells

Wingless-type (Wnt) signaling pathways mediate axonal growth and remodeling in the embryonic optic nerve, brain and spinal cord. Recent studies demonstrated that the canonical Wnt/β-catenin signaling pathway also induces axonal regeneration after injury in the optic nerve of adult animals. However, the molecular mechanisms of Wnt-mediated axonal growth are not well understood. Additionally, because Wnt signaling is stimulated in neurons as well as neighboring non-neuronal cells, the cell type(s) responsible for Wnt-induced axonal regeneration are not known. The objectives of this study were to investigate potential mechanisms and target cells of Wnt3a stimulated neurite growth using primary retinal ganglion cell (RGC) cultures. We demonstrated that Wnt3a ligand induced dose-dependent increases in average neurite length and number of neurites in RGCs. QPCR analysis of candidate mediators showed that Wnt3a-dependent neurite growth was associated with lower expression of Ripk1 and Ripk3 genes. Additionally, inhibiting Ripk1 signaling with Necrostatin-1s led to increased neurite number per cell but not increased neurite length. Therefore, Ripk signaling may be involved in mediating the effects of Wnt3a on neurite number but Ripk activity does not seem to be required for Wnt3a-dependent regulation of neurite length. This study shows that RGCs are direct cellular targets of Wnt3a-induced axonal growth, and we identified a novel association between Wnt signaling and Rip kinases in neurite formation

Context-Dependent Effects of the Ketogenic Diet on Retinal Ganglion Cell Survival and Axonal Regeneration After Optic Nerve Injury

There is increasing interest in nonpharmacologic approaches to protect retinal ganglion cells (RGCs) after injury and enhance the efficacy of therapeutic molecules. Accumulating evidence demonstrates neuroprotection by the high-fat low-carbohydrate ketogenic diet (KD) in humans and animal models of neurologic diseases. However, no studies to date have examined whether the KD protects RGCs and promotes axonal regrowth after traumatic injury to the optic nerve (ON) or whether it increases efficacy of experimental proregenerative molecules. In this study, we investigated whether the KD promoted RGC survival and axonal regeneration after ON injury in the presence and absence of neuroprotective Wnt3a ligand. Adult mice were placed on a KD or control diet before ON crush injury and remained on the diet until the end of the experiment. Nutritional ketosis was confirmed by measuring serum beta-hydroxybutyrate levels. Mice were intravitreally injected with Wnt3a ligand or phosphate-buffered saline (PBS), and RGC survival, function, axonal regeneration, and inflammatory responses were measured. Mice fed the KD showed increased RGC survival and reduced inflammatory cells in PBS-injected mice. Also, mice fed the KD had increased RGC functional responses but not increased RGC numbers in the presence of Wnt3a, indicating that the KD did not enhance the prosurvival effect of Wnt3a. The KD did not promote axonal regeneration in the presence or absence of Wnt3a. The KD has a complex protective effect after ON injury and cotreatment with Wnt3a. This work sets the foundation for studies identifying underlying molecular mechanisms