Notch-signaling in retinal regeneration and Muller glial plasticity

Eye diseases such as blindness, age-related macular degeneration (AMD), diabetic retinopathy and glaucoma are highly prevalent in the developed world, especially in a rapidly aging population. These sight-threatening diseases all involve the progressive loss of cells from the retina, the light-sensing neural tissue that lines the back of the eye. Thus, developing strategies to replace dying retinal cells or prolonging neuronal survival is essential to preserving sight. In this regard, cell-based therapies hold great potential as a treatment for retinal diseases. One strategy is to stimulate cells within the retina to produce new neurons. This dissertation elucidates the properties of the primary support cell in the chicken retina, known as the Muller glia, which have recently been shown to possess stem-cell like properties, with the potential to form new neurons in damaged retinas. However, the mechanisms that govern this stem-cell like ability are less well understood. In order to better understand these properties, we analyze the role of one of the key developmental processes, i.e., the Notch-Signaling Pathway in regulating proliferative, neuroprotective and regenerative properties of Muller glia and bestow them with this plasticity.The first part of this dissertation is a description of embryonic studies that verify the use of a pharmacological inhibitor of Notch-signaling, DAPT, as well as RNA interference molecules that block downstream effectors of Notch -- Hes1 and Hes5. We find that inhibition of gamma-secretase activity associated with Notch and silencing of the bHLH effectors Hes1 and Hes5 have distinctly different outcomes on cell-fate specification of cultured chicken retinal progenitors. Further, our studies reveal that Notch-signaling plays a limited but important role during retinal regeneration. Components of the Notch-signaling pathways are transiently upregulated in proliferating Muller glia after damage in a chicken retina and blocking Notch after damage enhances some neural regeneration from glial-derived progenitors.In the second part of this dissertation, we analyze the role of the Notch pathway in the postnatal retina in the absence of damage. We find that components of the Notch-signaling pathway are expressed at low levels in most Muller glia in undamaged retina. Further, Notch-signaling influences the phenotype and function of Muller glia in the mature retina low levels of Notch-signaling diminish the neuroprotective capacity of Muller glia, but are required to maintain their ability to become progenitor-like cells. We also find that there is cross-talk between Notch and MAPK pathways -- FGF2, a secreted protein that activates the MAPK pathway, also induces the expression of Notch pathway genes. Further, active Notch-signaling is required for the FGF2-mediated accumulation of p38 MAPK and pCREB in Muller glia.Our data indicate that Notch-signaling is down-stream of and is required for FGF2/MAPK-signaling to drive the proliferation of Muller glia. The third part of this dissertation describes the patterning of the immature zone of cells present at the retinal margin, called the circumferential marginal zone (CMZ). Our data indicates that there is a gradual spatial restriction of this zone of progenitor cells during late stages of embryonic development and that there are regional differences in the maturity of cells within the CMZ. Further, we find that retinal neurons adjacent to the CMZ in far peripheral regions of the temporal retina remain immature and differentiate far more slowly compared to the neurons in central regions of the retina and that the microenvironment at the periphery of the retina that promotes the persistence of a zone of retinal progenitors may also keep some types of neurons immature for extended periods of time.The last part of this dissertation describes the morphological and mechanistic properties of a unique subset of interneurons we discovered in the chicken retina, called the serotonin-accumulating bipolar cells. Even though serotonin is synthesized by amacrine cells, another type of interneuron present in the retina, it transiently accumulates in this distinct type of bipolar neuron. The accumulation of endogenous or exogenous serotonin by bipolar neurons is blocked by selective reuptake inhibitors. Further, inhibition of monoamine oxidase (A) prevents the degradation of serotonin in bipolar neurons, suggesting that MAO(A) is present in these neurons. Our data indicates that serotonin-accumulating bipolar neurons perform glial functions in the retina by actively transporting and degrading serotonin that is synthesized in neighboring amacrine cells.Taken together, the data presented in this dissertation furthers understanding of Muller glial plasticity. This information could be applied to stimulating neural regeneration, harnessing Muller glia as a localized source of stem cells intrinsic to the retina, developing pharmacological therapies targeted to the glia and countering neuronal death in sight-threatening diseases. Additionally, our studies on serotonin-accumulating bipolar cells have implications for understanding the mechanisms of melatonin biosynthesis and retinal circadian rhythms, dysfunctions of which lead to photoreceptor degeneration and loss of vision.