Phagocytosis executes delayed neuronal death after focal brain ischemia

Delayed neuronal loss and brain atrophy after cerebral ischemia contribute to stroke and dementia pathology, but the mechanisms are poorly understood. Phagocytic removal of neurons is generally assumed to be beneficial and to occur only after neuronal death. However, we report herein that inhibition of phagocytosis can prevent delayed loss and death of functional neurons after transient brain ischemia. Two phagocytic proteins, Mer receptor tyrosine kinase (MerTK) and Milk fat globule EGF-like factor 8 (MFG-E8), were transiently up-regulated by macrophages/microglia after focal brain ischemia in vivo. Strikingly, deficiency in either protein completely prevented long-term functional motor deficits after cerebral ischemia and strongly reduced brain atrophy as a result of inhibiting phagocytosis of neurons. Correspondingly, in vitro glutamate-stressed neurons reversibly exposed the “eat-me” signal phosphatidylserine, leading to their phagocytosis by microglia; this neuronal loss was prevented in the absence of microglia and reduced if microglia were genetically deficient in MerTK or MFG-E8, both of which mediate phosphatidylserine-recognition. Thus, phagocytosis of viable neurons contributes to brain pathology and, surprisingly, blocking this process is strongly beneficial. Therefore, inhibition of specific phagocytic pathways may present therapeutic targets for preventing delayed neuronal loss after transient cerebral ischemia.Cerebral ischemia is one of the most common causes of death and disability worldwide and occurs as a result of interrupted blood supply to the brain, resulting in neurodegeneration during stroke and vascular dementia. In the ischemic area where the reduction of blood flow is most pronounced (the core), neuronal death follows rapidly as a result of energy depletion. However, in areas of partial ischemia (the penumbra) and in areas around the infarct (peri-infarct), neurons stressed by ischemia or its consequences are lost only after some delay, providing an opportunity for therapeutic interference hours or days after stroke (1, 2).In addition to the directly neurotoxic effects of ischemia, damage-associated ligands released from dying cells induce a strong inflammatory response, which can cause further neuronal damage. However, inflammation is a self-limiting process and after ischemia and reperfusion, microglia and recruited macrophages clear cell debris and dying cells through phagocytosis (3, 4).Phagocytosis is generally considered to be a beneficial process that leads to clearance of potentially harmful cellular components and may also contribute to the resolution of inflammation (5). It has been assumed that cells are only phagocytosed after they are committed to die (6); therefore, a potential contribution of phagocytosis to pathology has not been investigated. However, it has recently become clear that viable cells exposed to sublethal stimuli may expose the “eat-me” signal phosphatidylserine (PS), leading to their phagocytosis and thus death (7–11).The recognition of cells exposing PS can be mediated by a variety of proteins and is dependent on the inflammatory state of the macrophage (for review, see ref. 12). For example, two phagocytic proteins that have been shown to be up-regulated by inflammatory activation are: (i) Milk fat globule EGF-like factor-8 (MFG-E8), which binds PS on the phagocytic target cell and the vitronectin receptor on macrophages (13); and (ii) Mer receptor tyrosine kinase (MerTK), which detects PS-exposing target cells with the help of PS-binding bridging proteins, such as Gas6 and Protein S (14, 15). However, MerTK can also initiate engulfment because of phosphorylation by focal adhesion kinase downstream of the MFG-E8 receptor, the vitronectin receptor (16).Interestingly, it has been shown that viable neurons in the ischemic penumbra and surrounding brain are labeled by annexin V, indicating PS exposure peaking 3 d after transient ischemia but reversing thereafter (17), and large numbers of stressed neurons and phagocytic microglia are found together in brain regions during and after ischemia (1, 3). We therefore hypothesized that phagocytosis may contribute to delayed neuronal loss after cerebral ischemia. Our data show that phagocytosis executes neuronal death after transient brain ischemia, because blocking phagocytic signaling prevents both the delayed neuronal loss and long-term functional deficits.