Molecular mechanisms of programmed necrosis in the ischemic retina

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PROJECT SUMMARY Retinal ischemia-reperfusion (IR) injury is a common cause of visual impairment worldwide, affecting both patients' quality of life and functional status. Retinal ganglion cell (RGC) survival is critical for vision. However, these retinal neurons are exquisitely sensitive and many of them undergo necrosis and apoptosis after IR. Since RGC apoptosis is executed by programmed mechanisms and can be regulated, significant attention has been given to this type of cell death. At the same time, RGC necrosis did not receive nearly enough consideration, because it was viewed as an accidental and unregulated cellular event. We now know that necrosis, like apoptosis, can be executed by programmed mechanisms. This form of necrotic cell death is called necroptosis. Our published data and literature indicate that RGC necroptosis contributes to IR-induced retinal injury through direct loss of RGCs and induction of associated inflammatory responses. Therefore, since RGC necroptosis is executed by programmed mechanisms and can be regulated, this field of research is of great importance. However, the signaling cascades, which regulate IR-induced RGC necroptosis, still remain unknown. The long-term objective of this project is to identify the signaling cascades that regulate RGC necroptosis after IR. Based on our published data, preliminary studies and published literature, we proposed a molecular mechanism of IR-induced RGC necroptosis. We suggested that IR-induced Tnf signaling in RGCs facilitates formation of a positive-feedback loop for the sustained production of reactive oxygen species (ROS), which promotes poly (ADP-ribose) polymerase 1 (Parp1) over-activation due to significant oxidative DNA damage. Significant Parp1 over-activation in RGCs mediates ATP depletion, leading to subsequent energy failure, which results in cellular dysfunction and eventually in loss of RGC membrane integrity (necrosis). We will employ a wide range of biochemical, molecular and cell biological techniques as well as animal models to verify the proposed molecular mechanism of IR-induced RGC necroptosis in hypothesis-driven mechanistic experiments outlined in the following specific aims: 1) to test the hypothesis that Tnf signaling promotes IR- induced RGC necroptosis; 2) to test the hypothesis that IR-induced RGC necroptosis is promoted by formation of a positive-feedback loop for sustained ROS production in a Tnf signaling-dependent manner; 3) to test the hypothesis that RGC necroptosis is a result of ATP depletion caused by Parp1 over-activation after IR. Thus, since treatment for IR is limited in part because of a lack of understanding of the molecular events leading to RGC death, a greater understanding of RGC necroptosis after IR will lead to new therapeutic strategies for this important and difficult to treat condition.