Stimulating retinal repair

Japanese researchers from RIKEN and Kyoto University have demonstrated retinal regeneration in a mammalian model of retinal degeneration after stimulation of the Wnt signaling pathway. In addition to its better known roles in embryogenesis and development, this pathway also functions as a regulator of some adult stem cell populations.

The team’s work may lead to new therapies for retinal diseases including the degenerative disease called retinitis pigmentosa, in which the rod and cone photoreceptor cells of the retina die off, leading to vision impairment and ultimately blindness.

Previous research by the team, led by Masayo Takahashi at the RIKEN Center for Developmental Biology, Kobe, demonstrated that retinal support cells called Müller glia could de-differentiate, or revert to a cell type from earlier in its developmental pathway, to assume a neuronal fate, but the level of regeneration via this mechanism was very low, occurring in just a few cells.

Now, in an in vitro model of retinal damage, the team has found that retinal cell regeneration can be increased by as much as twenty-fold in the presence of the protein Wnt3a. Their findings are published in the Journal of Neuroscience ¹.

The researchers initially performed experiments in cultured retinas isolated from rats, which can be used as a model for retinal damage. In these so-called explant cultures, the retina is cultured as a unit with no dissociation of retinal cells. After four days in culture, a small proportion of the Müller glial cells re-entered the cell cycle. Immunostaining of the cells further demonstrated that some of these cells were expressing proteins that were characteristic of neural stem cells, suggesting that the Müller glial cells were de-differentiating to neural progenitor cells. When they administered the protein Wnt3a, they found a significant increase in proliferation of these neuronal progenitors from the de-differentiated cells (Fig. 1).

“Newly generated cells constituted almost a layer of cells in the outer nuclear layer,” says Takahashi. “We only observed several cells per field without Wnt treatment. Furthermore, the retinal neurons were regenerated all over the retina.”

This process appears to involve the canonical Wnt signaling pathway, in which Wnt activation protects the β-catenin protein from degradation mediated by a glycogen synthase kinase complex, allowing it to accumulate in the nucleus where it regulates gene transcription (Fig. 2). A number of components of the pathway were shown to be present in the retina, including Wnt receptor and co-receptor proteins, glycogen synthase kinase and β-catenin. Treatment of the retinal explant cultures with Wnt3a caused accumulation of β-catenin in the nucleus and a subsequent increase in the expression of the cell cycle-regulating protein cyclin D1, a known target of the Wnt signaling pathway, compared to the levels of cyclin D1 in untreated cultures.

Takahashi and her colleagues also demonstrated that the Wnt signaling pathway was involved in the retinal regeneration process in vivo. Furthermore, the regeneration process could be stimulated with small molecule inhibitors of glycogen synthase kinase-3β, which normally blocks activation of the pathway, and conversely, is blocked by the application of Wnt3a inhibitor.

Next, the researchers showed that the regenerated cells migrated to the outer nuclear layer of the retina, where, in the presence of retinoic acid (a form of vitamin A), they observed differentiation into rod photoreceptor cells as evidenced by the appearance of cells expressing rhodopsin (Fig. 3).

The team then isolated retinas from a strain of mice with a genetic defect that resembles the human disease retinitis pigmentosa. These mice exhibit a significant loss of photoreceptor cells within one month of birth. Treatment of these retinas with Wnt3a also resulted in the regeneration of retinal cells. This suggests that the Wnt/β-catenin signaling pathway contributes to central nervous system regeneration.

As the researchers had observed in the earlier experiments using retinal explant cultures from rats, a significant increase in cell proliferation was observed after Wnt3a treatment, and the de-differentiated neural progenitors migrated to the outer nuclear layer. But in retinas from mice already exhibiting advanced retinal degeneration, Wnt3a treatment had little or no effect, suggesting that the degree of degeneration prior to treatment affected the ability of the Wnt3a signaling pathway to stimulate repair processes.

Differentiation of the neural progenitor cells into rhodopsin-positive photoreceptor cells was observed following treatment of the Wnt-treated cultures with retinoic acid. A similar result was observed after treatment of the cultures with valproic acid. This compound is an inhibitor of the enzyme histone deacetylase, which regulates expression of another protein involved in photoreceptor development, the transcription factor NeuroD.