Neuronal cell death occurs extensively during development and pathology, where it is especially important because of the limited capacity of adult neurons to proliferate or be replaced. The concept of cell death used to be simple as there were just two or three types, so we just had to work out which type was involved in our particular pathology and then block it. However, we now know that there are at least a dozen ways for neurons to die, that blocking a particular mechanism of cell death may not prevent the cell from dying, and that non-neuronal cells also contribute to neuronal death. We review here the mechanisms of neuronal death by intrinsic and extrinsic apoptosis, oncosis, necroptosis, parthanatos, ferroptosis, sarmoptosis, autophagic cell death, autosis, autolysis, paraptosis, pyroptosis, phagoptosis, and mitochondrial permeability transition. We next explore the mechanisms of neuronal death during development, and those induced by axotomy, aberrant cell-cycle reentry, glutamate (excitoxicity and oxytosis), loss of connected neurons, aggregated proteins and the unfolded protein response, oxidants, inflammation, and microglia. We then reassess which forms of cell death occur in stroke and Alzheimer's disease, two of the most important pathologies involving neuronal cell death. We also discuss why it has been so difficult to pinpoint the type of neuronal death involved, if and why the mechanism of neuronal death matters, the molecular overlap and interplay between death subroutines, and the therapeutic implications of these multiple overlapping forms of neuronal death.

Significance Brain ischemia is a major cause of death and disability worldwide, but the cellular mechanisms of delayed neuronal loss and brain atrophy after cerebral ischemia are poorly understood and thus currently untreatable. Surprisingly, we find that after cerebral ischemia, brain macrophages phagocytose viable and functional neurons, causing brain atrophy and motor dysfunction. Our data show that delayed neuronal death and functional impairment after cerebral ischemia can be prevented by blocking specific phagocytic pathways, and therefore highlight new therapeutic targets for stroke and dementia.

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Milk-fat globule EGF factor-8 (MFG-E8, SED1, lactadherin) is known to mediate the phagocytic removal of apoptotic cells by bridging phosphatidylserine (PS)-exposing cells and the vitronectin receptor (VR) on phagocytes. However, we show here that MFG-E8 can mediate phagocytosis of viable neurons during neuroinflammation induced by lipopolysaccharide (LPS), thereby causing neuronal death. In vitro, inflammatory neuronal loss is independent of apoptotic pathways, and is inhibited by blocking the PS/MFG-E8/VR pathway (by adding PS blocking antibodies, annexin V, mutant MFG-E8 unable to bind VR, or VR antagonist). Neuronal loss is absent in Mfge8 knockout cultures, but restored by adding recombinant MFG-E8, without affecting inflammation. In vivo, LPS-induced neuronal loss is reduced in the striatum of Mfge8 knockout mice or by co-injection of an MFG-E8 receptor (VR) inhibitor into the rat striatum. Our data show that blocking MFG-E8-dependent phagocytosis preserves live neurons, implying that phagocytosis actively contributes to neuronal death during brain inflammation.

Airway and/or lung remodeling, involving exaggerated extracellular matrix (ECM) protein deposition, is a critical feature common to pulmonary diseases including chronic obstructive pulmonary disease (COPD), asthma, and idiopathic pulmonary fibrosis (IPF). Fibulin-1 (Fbln1), an important ECM protein involved in matrix organization, may be involved in the pathogenesis of these diseases. We found that Fbln1 was increased in COPD patients and in cigarette smoke–induced (CS-induced) experimental COPD in mice. Genetic or therapeutic inhibition of Fbln1c protected against CS-induced airway fibrosis and emphysema-like alveolar enlargement. In experimental COPD, this occurred through disrupted collagen organization and interactions with fibronectin, periostin, and tenascin-c. Genetic inhibition of Fbln1c also reduced levels of pulmonary inflammatory cells and proinflammatory cytokines/chemokines (TNF-α, IL-33, and CXCL1) in experimental COPD. Fbln1c−/− mice also had reduced airway remodeling in experimental chronic asthma and pulmonary fibrosis. Our data show that Fbln1c may be a therapeutic target in chronic respiratory diseases.

Synaptic activity promotes resistance to diverse apoptotic insults, the mechanism behind which is incompletely understood. We show here that a coordinated downregulation of core components of the intrinsic apoptosis pathway by neuronal activity forms a key part of the underlying mechanism. Activity-dependent protection against apoptotic insults is associated with inhibition of cytochrome c release in most but not all neurons, indicative of anti-apoptotic signaling both upstream and downstream of this step. We find that enhanced firing activity suppresses expression of the proapoptotic BH3-only member gene Puma in a NMDA receptor-dependent, p53-independent manner. Puma expression is sufficient to induce cytochrome c loss and neuronal apoptosis. Puma deficiency protects neurons against apoptosis and also occludes the protective effect of synaptic activity, while blockade of physiological NMDA receptor activity in the developing mouse brain induces neuronal apoptosis that is preceded by upregulation of Puma. However, enhanced activity can also confer resistance to Puma-induced apoptosis, acting downstream of cytochrome c release. This mechanism is mediated by transcriptional suppression of apoptosome components Apaf-1 and procaspase-9, and limiting caspase-9 activity, since overexpression of procaspase-9 accelerates the rate of apoptosis in active neurons back to control levels. Synaptic activity does not exert further significant anti-apoptotic effects downstream of caspase-9 activation, since an inducible form of caspase-9 overrides the protective effect of synaptic activity, despite activity-induced transcriptional suppression of caspase-3. Thus, suppression of apoptotic gene expression may synergize with other activity-dependent events such as enhancement of antioxidant defenses to promote neuronal survival.

BackgroundMicroglia are resident brain macrophages that can phagocytose dead, dying or viable neurons, which may be beneficial or detrimental in inflammatory, ischaemic and neurodegenerative brain pathologies. Cell death caused by phagocytosis of an otherwise viable cell is called ‘primary phagocytosis’ or ‘phagoptosis’. Calreticulin (CRT) exposure on the surface of cancer cells can promote their phagocytosis via LRP (low-density lipoprotein receptor-related protein) on macrophages, but it is not known whether this occurs with neurons and microglia.MethodsWe used primary cultures of cerebellar neurons, astrocytes and microglia to investigate the potential role of CRT/LRP phagocytic signalling in the phagocytosis of viable neurons by microglia stimulated with lipopolysaccharide (LPS) or nanomolar concentrations of amyloid-β peptide1-42 (Aβ). Exposure of CRT on the neuronal surface was investigated using surface biotinylation and western blotting. A phagocytosis assay was also developed using BV2 and PC12 cell lines to investigate CRT/LRP signalling in microglial phagocytosis of apoptotic cells.ResultsWe found that BV2 microglia readily phagocytosed apoptotic PC12 cells, but this was inhibited by a CRT-blocking antibody or LRP-blocking protein (receptor-associated protein: RAP). Activation of primary rat microglia with LPS or Aβ resulted in loss of co-cultured cerebellar granule neurons, and this was blocked by RAP or antibodies against CRT or against LRP, preventing all neuronal loss and death. CRT was present on the surface of viable neurons, and this exposure did not change in inflammatory conditions. CRT antibodies prevented microglia-induced neuronal loss when added to neurons, while LRP antibodies prevented neuronal loss when added to the microglia. Pre-binding of CRT to neurons promoted neuronal loss if activated microglia were added, but pre-binding of CRT to microglia or both cell types prevented microglia-induced neuronal loss.ConclusionsCRT exposure on the surface of viable or apoptotic neurons appears to be required for their phagocytosis via LRP receptors on activated microglia, but free CRT can block microglial phagocytosis of neurons by acting on microglia. Phagocytosis of CRT-exposing neurons by microglia can be a direct cause of neuronal death during inflammation, and might therefore contribute to neurodegeneration and be prevented by blocking the CRT/LRP pathway.

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