Abstract
Degenerative retinopathies are the leading causes of irreversible visual impairment in the elderly, affecting hundreds of millions of patients. Müller glia cells (MGC), the main type of glia found in the vertebrate retina, can resume proliferation in the rodent adult injured retina but contribute weakly to tissue repair when compared to zebrafish retina. However, postnatal and adult mouse MGC can be genetically reprogrammed through the expression of the transcription factor (TF) Achaete-scute homolog 1 (ASCL1) into induced neurons (iNs), displaying key hallmarks of photoreceptors, bipolar and amacrine cells, which may contribute to regenerate the damaged retina. Here, we show that the TF neurogenin 2 (NEUROG2) is also sufficient to lineage-reprogram postnatal mouse MGC into iNs. The efficiency of MGC lineage conversion by NEUROG2 is similar to that observed after expression of ASCL1 and both TFs induce the generation of functionally active iNs. Treatment of MGC cultures with EGF and FGF2 prior to Neurog2 or Ascl1 expression enhances reprogramming efficiencies, what can be at least partially explained by an increase in the frequency of MGCs expressing sex determining region Y (SRY)-box 2 (SOX2). Transduction of either Neurog2 or Ascl1 led to the upregulation of key retina neuronal genes in MGC-derived iNs, but only NEUROG2 induced a consistent increase in the expression of putative retinal ganglion cell (RGC) genes. Moreover, in vivo electroporation of Neurog2 in late progenitors from the neonatal rat retina, which are transcriptionally similar to MGCs, also induced a shift in the generation of retinal cell subtypes, favoring neuronal differentiation at the expense of MGCs and resuming the generation of RGCs. Altogether, our data indicate that NEUROG2 induces lineage conversion of postnatal rodent MGCs into RGC-like iNs in vitro and resumes the generation of this neuronal type from late progenitors of the retina in vivo.
Highlights
The retina is a unique tissue with highly organized architecture, known to be one of the most energetically demanding systems in the nervous system (Wong-Riley, 2010)
We show that cells isolated in culture and expressing several hallmarks of Müller cells, the main type of glia found in the vertebrate retina, can be reprogrammed into neurons when transduced with plasmids encoding either of the basic Helix loop Helix transcription factors Neurog2 or Ascl1
Cell–lineage reprogramming is affected by treatment with epidermal growth factor (EGF) and FGF2 during Müller glia cells (MGC) enrichment, and led to the expression of typical neuronal markers, MAP-2 and TUBB3
Summary
The retina is a unique tissue with highly organized architecture, known to be one of the most energetically demanding systems in the nervous system (Wong-Riley, 2010). Trauma, or genetic mutations, gradual and irreversible cell death affects specific neuronal types in the retina (Athanasiou et al, 2013). Retinal ganglion cells (RGCs) and their axons degenerate in glaucoma, a neurodegenerative disease associated with increased intraocular pressure, eventually leading to blindness (Kimura et al, 2017). In the last 5 years almost 65 million people worldwide were diagnosed with glaucoma (Gill et al, 2016; Liang et al, 2017), which is the leading cause of visual impairment in developed countries (WHO). Recent progress in cell-based therapy may, provide novel means to restore vision in glaucoma patients (Abu-Hassan et al, 2015; Chamling et al, 2016)
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