Targeting Necroptosis: A Novel Therapeutic Option for Retinal Degenerative Diseases

Abstract

The discovery of the necroptosis, a form of regulated necrosis that is mediated by receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like pseudokinase (MLKL), represents a major breakthrough that has dramatically altered the conception of necrosis - traditionally thought of as uncontrolled cell death - in various human diseases. Retinal cell death is a leading cause of blindness and has been identified in most retinal diseases, e.g., age-related macular degeneration, glaucoma, retinal detachment, retinitis pigmentosa, etc. Increasing evidence demonstrates that retinal degenerative diseases also share a common mechanism in necroptosis. Exacerbated necroptotic cell death hinders the treatment for retinal degenerative diseases. In this review, we highlight recent advances in identifying retinal necroptosis, summarize the underlying mechanisms of necroptosis in retinal degenerative diseases, and discuss potential anti-necroptosis strategies, such as selective inhibitors and chemical agents, for treating retinal degenerative diseases.

Keywords: RIPK1; RIPK3; Retinal degenerative diseases; age-related macular degeneration; glaucoma; necroptosis.

Figure 1

Molecular mechanisms of necroptosis. (A) Stimulation of TNFR1 by TNFα promotes the formation of a membrane-associated protein complex (complex I), which is composed of TRADD, RIPK1, TRAF2, cIAP1/2, LUBAC, TAK1, and the IKK complex. In complex I, cIAP1/2 and LUBAC promote RIPK1 ubiquitination that recruits TAK1, TAB2/3, and the IKK complex composed of IKK1/2 and NEMO. Complex I can induce expressions of both pro-survival and pro-inflammatory genes via NFκB activation. NFκB activation, in turn, blocks the formation of FADD and caspase-8 complex (also called complex IIa-RIA) through A20 and cFLIP to modulate RIPK1-independent apoptosis. When complex I is unstable or the ubiquitination of RIPK1 is inhibited, stimulation of TNFR1 leads to the dimerization and activation of RIPK1, which interacts with FADD and caspase-8 to form complex IIa-RDA and mediates subsequent apoptosis. When the activity of caspase 8 is blocked, the activated RIPK1 recruits RIPK3 and MLKL, leading to the formation of complex IIb and subsequent phosphorylation of MLKL. (B) In the necrosome, RIPK3 phosphorylates MLKL and leads to its activation and oligomerization. Then the oligomerized MLKLs translocate to the plasma membrane to form a pore-forming complex, causing plasma membrane rupture and release of DAMP molecules, which provoke further inflammation and secondary injury. ESCRT-III, a downstream regulator of MLKL, can counteract a limited amount of plasma membrane perforation mediated by MLKL through shedding of the affected segments. (C) During the process of necroptosis, the necrosome can also phosphorylate PGAM5 and lead to a PGAM5-mediated mitochondrial dysfunction. The activated PGAM5 dephosphorylates DRP1 and causes its translocation from the cytosol to the mitochondrion to stimulate mitochondrial fission and ROS generation. The increasing levels of ROS subsequently induce PARP1 activation, which stimulates the release of tAIF from the mitochondria to the cytosol and, from there, to the nucleus, which provokes DNA degradation and subsequent cell damage.

Figure 2

Detecting the necroptosis in retina. (A) Cell loss occurs widely in different retina injuries/diseases and leads to visual impairment, owing to ischemia/reperfusion, excitotoxicity, and inflammatory reactions. In rat retinas following high intraocular pressure (HIOP) injury (an in vivo animal model of retinal ischemia/reperfusion), hematoxylin and eosin staining indicated significant cell loss in the ganglion cell layer caused by excessive pressure. Cell membrane rupture played a critical role in the cell loss caused by HIOP injury - a notable observation, given that membrane rupture is a typical feature of necroptosis. (B) In vitro observation of R28 cells (a retinal precursor cell line) provides a feasible way to show retinal cell swelling (the other hallmark of necroptosis; upper left) and membrane rupture (upper right) following oxygen-glucose deprivation/recovery injury (an in vitro cell model of retinal ischemia/reperfusion). TEM enables an ultra-high-resolution characterization of retinal cell swelling and membrane rupture of necroptotic cell death (lower right) compared with the normal microscopy (lower left). (C) TNFα/Smac-mimetic/Z-VAD-FMK (TSZ)-induced cell necroptosis is a classical tool to investigate necroptotic signaling. As indicated by flow cytometry with propidium iodide/Annexin V staining, TSZ and TSZ+DMSO treatment showed a large number of necrotic cells (PI positive; upper left and/or right quadrants of each panel) compared with the normal group. Necrostatin-1 (Nec-1), a selective inhibitor of RIPK1 that can reverse necroptotic cell death, is widely used as a positive control to determine the existence of necroptosis by flow cytometry. (D) Upregulation of RIPK1, RIPK3, and MLKL indicated by immunofluorescence or immunohistochemistry are key biomarkers to identify necroptotic cell death. As showed by immunofluorescence assay, the expression of MLKL was significantly enhanced in retinal ganglion cells following HIOP injury, indicating the presence and execution of necroptosis.

Figure 3

Domain organization of necroptotic signaling proteins and their targeted chemical inhibitors. (A) Functional domains of RIPK1, RIPK3, and MLKL. RIPK1 and RIP3 have very similar domain features, both of them containing the N-terminal serine/threonine kinase domain (KD), intermediate domain (ID), and RIP homotypic interaction motif (RHIM) domain. RHIM mediates the assembly of the RIPK1:RIPK3 complex and is crucial for KD activation and induction of necroptosis. RIPK1 also has a C-terminal death domain (DD), which can bind to the death receptors of TNFR1 and Fas to activate apoptosis. In MLKL, the regulatory brace (RB) domain connects the N-terminal execution domain (NED) and C-terminal pseudokinase domain (psKD). The psKD is usually catalytically inactive and contains an unusual pseudoactive site. The NED is sufficient to form channels that can induce membrane depolarization and cell death. (B) Tertiary structures of RIPK1, RIPK3, and MLKL and their inhibitors. Nec-1 can bind to the kinase domain of RIPK1 to stabilize it in an inactive conformation. GSK'872 can be caged in the kinase domain of RIPK3 to inhibit its kinase activity. Necrosulfonamide can bind to the N-terminal execution domain of MLKL to block its activation.