Monocyte invasion into the retina restricts the regeneration of neurons from Müller glia.

Endogenous reprogramming of glia into neurogenic progenitors holds great promise for neuron restoration therapies. Using lessons from regenerative species, we have developed strategies to stimulate mammalian Müller glia to regenerate neurons in vivo in the adult retina. We have demonstrated that the transcription factor Ascl1 can stimulate Müller glia neurogenesis. However, Ascl1 is only able to reprogram a subset of Müller glia into neurons. We have reported that neuroinflammation from microglia inhibits neurogenesis from Müller glia. Here we find that the peripheral immune response is a barrier to CNS regeneration. We show that monocytes from the peripheral immune system infiltrate the injured retina and negatively influence neurogenesis from Müller glia. Using CCR2-knockout mice of both sexes we find that preventing monocyte infiltration improves the neurogenic and proliferative capacity of Müller glia stimulated by Ascl1. Using scRNA-seq analysis we identified a signaling axis wherein Osteopontin, a cytokine highly expressed by infiltrating immune cells is sufficient to suppress mammalian neurogenesis. This work implicates the response of the peripheral immune system as a barrier to regenerative strategies of the retina.Significance Statement Regeneration of neurons in the central nervous system is extremely limited in mammals. Transgenic overexpression of the proneural transcription factor Ascl1 enables mammalian retinal glia to regenerate some neurons lost to injury. We found that during this regenerative response to injury, monocytes from the periphery invade the neural retina and these inflammatory cells negatively regulate the ability of Müller glia to reprogram into neurogenic progenitors. When monocytes are inhibited from infiltrating the retina, regeneration of neurons from Müller glia is significantly enhanced. This work implicates peripheral immunomodulation as a tool to enhance endogenous neuronal replacement strategies.

Reprogramming Müller glia to regenerate ganglion-like cells in adult mouse retina with developmental transcription factors.

Many neurodegenerative diseases cause degeneration of specific types of neurons. For example, glaucoma leads to death of retinal ganglion cells, leaving other neurons intact. Neurons are not regenerated in the adult mammalian central nervous system. However, in nonmammalian vertebrates, glial cells spontaneously reprogram into neural progenitors and replace neurons after injury. We have recently developed strategies to stimulate regeneration of functional neurons in the adult mouse retina by overexpressing the proneural factor Ascl1 in Müller glia. Here, we test additional transcription factors (TFs) for their ability to direct regeneration to particular types of retinal neurons. We engineered mice to express different combinations of TFs in Müller glia, including Ascl1, Pou4f2, Islet1, and Atoh1. Using immunohistochemistry, single-cell RNA sequencing, single-cell assay for transposase-accessible chromatin sequencing, and electrophysiology, we find that retinal ganglion-like cells can be regenerated in the damaged adult mouse retina in vivo with targeted overexpression of developmental retinal ganglion cell TFs.

Efficient stimulation of retinal regeneration from Müller glia in adult mice using combinations of proneural bHLH transcription factors.

Regenerative neuroscience aims to stimulate endogenous repair in the nervous system to replace neurons lost from degenerative diseases. Recently, we reported that overexpressing the transcription factor Ascl1 in Müller glia (MG) is sufficient to stimulate MG to regenerate functional neurons in the adult mouse retina. However, this process is inefficient, and only a third of the Ascl1-expressing MG generate new neurons. Here, we test whether proneural transcription factors of the Atoh1/7 class can further promote the regenerative capacity of MG. We find that the combination of Ascl1:Atoh1 is remarkably efficient at stimulating neurogenesis, even in the absence of retinal injury. Using electrophysiology and single-cell RNA sequencing (scRNA-seq), we demonstrate that Ascl1:Atoh1 generates a diversity of retinal neuron types, with the majority expressing characteristics of retinal ganglion cells. Our results provide a proof of principle that combinations of developmental transcription factors can substantially improve glial reprogramming to neurons and expand the repertoire of regenerated cell fates.