Phospholipid scramblase Xkr8 is required for developmental axon pruning via phosphatidylserine exposure.

The mature mammalian brain connectome emerges during development via the extension and pruning of neuronal connections. Glial cells have been identified as key players in the phagocytic elimination of neuronal synapses and projections. Recently, phosphatidylserine has been identified as neuronal "eat-me" signal that guides elimination of unnecessary input sources, but the associated transduction systems involved in such pruning are yet to be described. Here, we identified Xk-related protein 8 (Xkr8), a phospholipid scramblase, as a key factor for the pruning of axons in the developing mammalian brain. We found that mouse Xkr8 is highly expressed immediately after birth and required for phosphatidylserine exposure in the hippocampus. Mice lacking Xkr8 showed excess excitatory nerve terminals, increased density of cortico-cortical and cortico-spinal projections, aberrant electrophysiological profiles of hippocampal neurons, and global brain hyperconnectivity. These data identify phospholipid scrambling by Xkr8 as a central process in the labeling and discrimination of developing neuronal projections for pruning in the mammalian brain.

Genetically encoded phosphatidylserine biosensor for in vitro, ex vivo and in vivo labelling.

BACKGROUND: The dynamics of phosphatidylserine in the plasma membrane is a tightly regulated feature of eukaryotic cells. Phosphatidylserine (PS) is found preferentially in the inner leaflet of the plasma membrane. Disruption of this asymmetry leads to the exposure of phosphatidylserine on the cell surface and is associated with cell death, synaptic pruning, blood clotting and other cellular processes. Due to the role of phosphatidylserine in widespread cellular functions, an efficient phosphatidylserine probe is needed to study them. Currently, a few different phosphatidylserine labelling tools are available; however, these labels have unfavourable signal-to-noise ratios and are difficult to use in tissues due to limited permeability. Their application in living tissue requires injection procedures that damage the tissue and release damage-associated molecular patterns, which in turn stimulates phosphatidylserine exposure. METHODS: For this reason, we developed a novel genetically encoded phosphatidylserine probe based on the C2 domain of the lactadherin (MFG-E8) protein, suitable for labelling exposed phosphatidylserine in various research models. We tested the C2 probe specificity to phosphatidylserine on hybrid bilayer lipid membranes by observing surface plasmon resonance angle shift. Then, we analysed purified fused C2 proteins on different cell culture lines or engineered AAVs encoding C2 probes on tissue cultures after apoptosis induction. For in vivo experiments, neurotropic AAVs were intravenously injected into perinatal mice, and after 2 weeks, brain slices were collected to observe C2-SNAP expression. RESULTS: The biophysical analysis revealed the high specificity of the C2 probe for phosphatidylserine. The fused recombinant C2 proteins were suitable for labelling phosphatidylserine on the surface of apoptotic cells in various cell lines. We engineered AAVs and validated them in organotypic brain tissue cultures for non-invasive delivery of the genetically encoded C2 probe and showed that these probes were expressed in the brain in vivo after intravenous AAV delivery to mice. CONCLUSIONS: We have demonstrated that the developed genetically encoded PS biosensor can be utilised in a variety of assays as a two-component system of C2 and C2m2 fusion proteins. This system allows for precise quantification and PS visualisation at directly specified threshold levels, enabling the evaluation of PS exposure in both physiological and cell death processes.

Errant gardeners: glial-cell-dependent synaptic pruning and neurodevelopmental disorders.

The final stage of brain development is associated with the generation and maturation of neuronal synapses. However, the same period is also associated with a peak in synapse elimination - a process known as synaptic pruning - that has been proposed to be crucial for the maturation of remaining synaptic connections. Recent studies have pointed to a key role for glial cells in synaptic pruning in various parts of the nervous system and have identified a set of critical signalling pathways between glia and neurons. At the same time, brain imaging and post-mortem anatomical studies suggest that insufficient or excessive synaptic pruning may underlie several neurodevelopmental disorders, including autism, schizophrenia and epilepsy. Here, we review current data on the cellular, physiological and molecular mechanisms of glial-cell-dependent synaptic pruning and outline their potential contribution to neurodevelopmental disorders.

Inhibition of UDP/P2Y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo.

Microglia activated through Toll-like receptor (TLR)-2 or -4 can cause neuronal death by phagocytosing otherwise-viable neurons-a form of cell death called "phagoptosis." UDP release from neurons has been shown to provoke microglial phagocytosis of neurons via microglial P2Y6 receptors, but whether inhibition of this process affects neuronal survival is unknown. We tested here whether inhibition of P2Y6 signaling could prevent neuronal death in inflammatory conditions, and whether UDP signaling can induce phagoptosis of stressed but viable neurons. We find that delayed neuronal loss and death in mixed neuronal/glial cultures induced by the TLR ligands lipopolysaccharide (LPS) or lipoteichoic acid was prevented by: apyrase (to degrade nucleotides), Reactive Blue 2 (to inhibit purinergic signaling), or MRS2578 (to specifically block P2Y6 receptors). In each case, inflammatory activation of microglia was not affected, and the rescued neurons remained viable for at least 7 days. Blocking P2Y6 receptors with MRS2578 also prevented phagoptosis of neurons induced by 250 nM amyloid beta 1-42, 5 µM peroxynitrite, or 50 µM 3-morpholinosydnonimine (which releases reactive oxygen and nitrogen species). Furthermore, the P2Y6 receptor agonist UDP by itself was sufficient to stimulate microglial phagocytosis and to induce rapid neuronal loss that was prevented by eliminating microglia or inhibiting phagocytosis. In vivo, injection of LPS into rat striatum induced microglial activation and delayed neuronal loss and blocking P2Y6 receptors with MRS2578 prevented this neuronal loss. Thus, blocking UDP/P2Y6 signaling is sufficient to prevent neuronal loss and death induced by a wide range of stimuli that activate microglial phagocytosis of neurons.

Inflammation induces multinucleation of Microglia via PKC inhibition of cytokinesis, generating highly phagocytic multinucleated giant cells.

Microglia are brain macrophages, which can undergo multinucleation to give rise to multinucleated giant cells that accumulate with ageing and some brain pathologies. However, the origin, regulation and function of multinucleate microglia remain unclear. We found that inflammatory stimuli, including lipopolysaccharide, amyloid ß, α-synuclein, tumour necrosis factor-α and interferon γ, but not interleukin-4, induced multinucleation of cultured microglia: primary rat cortical microglia and the murine microglial cell line BV-2. Inflammation-induced multinucleation was prevented by a protein kinase C (PKC) inhibitor Gö6976 (100 nM) and replicated by a PKC activator phorbol myristate acetate (160 nM). Multinucleation was reversible and not because of cell fusion or phagocytosis, but rather failure of cytokinesis. Time-lapse imaging revealed that some dividing cells failed to abscise, even after formation of long cytoplasmic bridges, followed by retraction of bridge and reversal of cleavage furrow to form multinucleate cells. Multinucleate microglia were larger and 2-4 fold more likely to phagocytose large beads and both dead and live PC12 cells. We conclude that multinucleate microglia are reversibly generated by inflammation via PKC inhibition of cytokinesis, and may have specialized functions/dysfunctions including the phagocytosis of other cells. Inflammation resulted in the accumulation of multiple nuclei per cell in cultured microglia. This multinucleation was reversible and due to a PKC-dependent block of the last step of cell division. Multinucleate microglia were larger and had a greater capacity to phagocytose other cells, suggesting they might remove neurons in the brain.

Integrity assay for messenger RNA in mouse and human brain samples and synaptosomal preparations.

Traditionally, RNA integrity evaluation is based on ribosomal RNAs (rRNAs). Nevertheless, gene expression studies are usually focused on protein-coding messenger RNAs (mRNAs). Here, we present an RT-qPCR-based assay, which estimates mRNA integrity by comparing the abundance of 3' and 5' mRNA fragments. The assay was validated using plasmids with cloned 3'- and 5'-ends of the cDNA reflecting different ratios of 3' and 5' cDNA amplicons in partially degraded RNA samples. The accuracy of integrity value was ensured by including primer efficiency. We used 5':3' assay to quantify RNA degradation in heat- and enzyme-degraded mouse and human brain tissue RNA as well as in clinical human brain RNA samples. In addition, the 5':3' assay was suitable for assessing mRNA integrity in synaptosomal preparations that lack rRNAs. We concluded that the 5':3' assay can be used as a reliable method to evaluate mRNA integrity in tissue and subcellular preparations.

Seroprevalence of neuronal antibodies in diseases mimicking autoimmune encephalitis.

Detection of neuronal antibodies for autoimmune encephalitis and paraneoplastic neurological syndromes relies on commercially available cell-based assays and lineblots. However, lineblots may reveal the presence of neuronal antibodies in patients with various non-autoimmune etiologies. Herein we describe patients with non-autoimmune etiologies (cohort B) and detectable neuronal antibodies and compare them to definite cases of autoimmune encephalitis (cohort A) for differences in clinical data. All patients positive for at least one neuronal antibody were retrospectively evaluated for autoimmune encephalitis and/or paraneoplastic neurological syndrome between 2016 and 2022. 39 cases in cohort B and 23 in cohort A were identified. In cohort B, most common diagnoses were neurodegenerative disorders in 9/39 (23.1%), brain tumors in 6/39 (15.4%) while most common detected antibodies were anti-titin (N10), anti-recoverin (N11), anti-Yo (N8) and all were detected in serum only. Differential aspects between cohort A and B were CSF pleocytosis (14/23 (60.8%) vs 11/35 (31.4%), p = 0.042, respectively), MRI features suggestive of encephalitis (6/23 (26.1%) vs 0 (0%), p = 0.002, respectively) and epilepsy restricted to temporal lobes (14/23 (60.9%) vs 2/30 (6.7%), p = 0.0003, respectively). A large proportion of lineblot results were non-specific when only serum was tested and were frequently found in non-autoimmune neurological conditions.

Lactadherin/MFG-E8 is essential for microglia-mediated neuronal loss and phagoptosis induced by amyloid ß.

Nanomolar ß-amyloid peptide (Aß) can induce neuronal loss in culture by activating microglia to phagocytose neurons. We report here that this neuronal loss is mediated by the bridging protein lactadherin/milk-fat globule epidermal growth factor-like factor 8 (MFG-E8), which is released by Aß-activated microglia, binds to co-cultured neurons and opsonizes neurons for phagocytosis by microglia. Aß stimulated microglial phagocytosis, but did not opsonize neurons for phagocytosis. Aß (250 nM) induced delayed neuronal loss in mixed glial-neuronal mouse cultures that required microglia and occurred without increasing neuronal apoptosis or necrosis. This neuronal death/loss was prevented by antibodies to MFG-E8 and was absent in cultures from Mfge8 knockout mice (leaving viable neurons), but was reconstituted by addition of recombinant MFG-E8. Thus, nanomolar Aß caused neuronal death by inducing microglia to phagocytose otherwise viable neurons via MFG-E8. The direct neurotoxicity of micromolar Aß was not affected by MFG-E8. The essential role of MFG-E8 in Aß-induced phagoptosis, suggests this bridging protein as a potential therapeutic target to prevent neuronal loss in Alzheimer's disease.

Inhibition of microglial phagocytosis is sufficient to prevent inflammatory neuronal death.

It is well-known that dead and dying neurons are quickly removed through phagocytosis by the brain's macrophages, the microglia. Therefore, neuronal loss during brain inflammation has always been assumed to be due to phagocytosis of neurons subsequent to their apoptotic or necrotic death. However, we report in this article that under inflammatory conditions in primary rat cultures of neurons and glia, phagocytosis actively induces neuronal death. Specifically, two inflammatory bacterial ligands, lipoteichoic acid or LPS (agonists of glial TLR2 and TLR4, respectively), stimulated microglial proliferation, phagocytic activity, and engulfment of ∼30% of neurons within 3 d. Phagocytosis of neurons was dependent on the microglial release of soluble mediators (and peroxynitrite in particular), which induced neuronal exposure of the eat-me signal phosphatidylserine (PS). Surprisingly, however, eat-me signaling was reversible, so that blocking any step in a phagocytic pathway consisting of PS exposure, the PS-binding protein milk fat globule epidermal growth factor-8, and its microglial vitronectin receptor was sufficient to rescue up to 90% of neurons without reducing inflammation. Hence, our data indicate a novel form of inflammatory neurodegeneration, where inflammation can cause eat-me signal exposure by otherwise viable neurons, leading to their death through phagocytosis. Thus, blocking phagocytosis may prevent some forms of inflammatory neurodegeneration, and therefore might be beneficial during brain infection, trauma, ischemia, neurodegeneration, and aging.

Nanotubes from bacteriophage tail sheath proteins: internalisation by cancer cells and macrophages.

Bionanoparticles comprised of naturally occurring monomers are gaining interest in the development of novel drug transportation systems. Here we report on the stabilisation, cellular uptake, and macrophage clearance of nanotubes formed from the self-assembling gp053 tail sheath protein of the vB_EcoM_FV3 bacteriophage. To evaluate the potential of the bacteriophage protein-based nanotubes as therapeutic nanocarriers, we investigated their internalisation into colorectal cancer cell lines and professional macrophages that may hinder therapeutic applications by clearing nanotube carriers. We fused the bacteriophage protein with a SNAP-tag self-labelling enzyme and demonstrated that its activity is retained in assembled nanotubes, indicating that such carriers can be applied to deliver therapeutic biomolecules. Under physiological conditions, the stabilisation of the nanotubes by PEGylation was required to prevent aggregation and yield a stable solution with uniform nano-sized structures. Colorectal carcinoma cells from primary and metastatic tumours internalized SNAP-tag-carrying nanotubes with different efficiencies. The nanotubes entered HCT116 cells via dynamin-dependent and SW480 cells - via dynamin- and clathrin-dependent pathways and were accumulated in lysosomes. Meanwhile, peritoneal macrophages phagocytosed the nanotubes in a highly efficient manner through actin-dependent mechanisms. Macrophage clearance of nanotubes was enhanced by inflammatory activation but was dampened in macrophages isolated from aged animals. Altogether, our results demonstrate that gp053 nanotubes retained the cargo's enzymatic activity post-assembly and had the capacity to enter cancer cells. Furthermore, we emphasise the importance of evaluating the nanocarrier clearance by immune cells under conditions mimicking a cancerous environment.

Sialic acid metabolism orchestrates transcellular connectivity and signaling in glioblastoma.

BACKGROUND: In glioblastoma (GBM), the effects of altered glycocalyx are largely unexplored. The terminal moiety of cell coating glycans, sialic acid, is of paramount importance for cell-cell contacts. However, sialic acid turnover in gliomas and its impact on tumor networks remain unknown. METHODS: We streamlined an experimental setup using organotypic human brain slice cultures as a framework for exploring brain glycobiology, including metabolic labeling of sialic acid moieties and quantification of glycocalyx changes. By live, 2-photon and high-resolution microscopy we have examined morphological and functional effects of altered sialic acid metabolism in GBM. By calcium imaging we investigated the effects of the altered glycocalyx on a functional level of GBM networks. RESULTS: The visualization and quantitative analysis of newly synthesized sialic acids revealed a high rate of de novo sialylation in GBM cells. Sialyltrasferases and sialidases were highly expressed in GBM, indicating that significant turnover of sialic acids is involved in GBM pathology. Inhibition of either sialic acid biosynthesis or desialylation affected the pattern of tumor growth and lead to the alterations in the connectivity of glioblastoma cells network. CONCLUSIONS: Our results indicate that sialic acid is essential for the establishment of GBM tumor and its cellular network. They highlight the importance of sialic acid for glioblastoma pathology and suggest that dynamics of sialylation have the potential to be targeted therapeutically.

Neuronal death induced by nanomolar amyloid ß is mediated by primary phagocytosis of neurons by microglia.

Alzheimer disease is characterized by neuronal loss and brain plaques of extracellular amyloid ß (Aß), but the means by which Aß may induce neuronal loss is not entirely clear. Although high concentrations of Aß (µM) can induce direct toxicity to neurons, we find that low concentration (nM) induce neuronal loss through a microglia-mediated mechanism. In mixed neuronal-glial cultures from rat cerebellum, 250 nM Aß1-42 (added as monomers, oligomers or fibers) induced about 30% loss of neurons between 2 and 3 days. This neuronal loss occurred without any increase in neuronal apoptosis or necrosis, and no neuronal loss occurred with Aß42-1. Aß greatly increased the phagocytic capacity of microglia and induced phosphatidylserine exposure (an "eat-me" signal) on neuronal processes. Blocking exposed phosphatidylserine by adding annexin V or an antibody to phosphatidylserine or inhibiting microglial phagocytosis by adding either cytochalasin D (to block actin polymerization) or cyclo(RGDfV) (to block vitronectin receptors) significantly prevented neuronal loss. Loss of neuronal synapses occurred in parallel with loss of cell bodies and was also prevented by blocking phagocytosis. Inhibition of phagocytosis prevented neuronal loss with no increase in neuronal death, even after 7 days, suggesting that microglial phagocytosis was the primary cause of neuronal death induced by nanomolar Aß.

The impact of maternal high-fat diet on offspring neurodevelopment.

A maternal high-fat diet affects offspring neurodevelopment with long-term consequences on their brain health and behavior. During the past three decades, obesity has rapidly increased in the whole human population worldwide, including women of reproductive age. It is known that maternal obesity caused by a high-fat diet may lead to neurodevelopmental disorders in their offspring, such as autism spectrum disorder, attention deficit hyperactivity disorder, anxiety, depression, and schizophrenia. A maternal high-fat diet can affect offspring neurodevelopment due to inflammatory activation of the maternal gut, adipose tissue, and placenta, mirrored by increased levels of pro-inflammatory cytokines in both maternal and fetal circulation. Furthermore, a maternal high fat diet causes gut microbial dysbiosis further contributing to increased inflammatory milieu during pregnancy and lactation, thus disturbing both prenatal and postnatal neurodevelopment of the offspring. In addition, global molecular and cellular changes in the offspring's brain may occur due to epigenetic modifications including the downregulation of brain-derived neurotrophic factor (BDNF) expression and the activation of the endocannabinoid system. These neurodevelopmental aberrations are reflected in behavioral deficits observed in animals, corresponding to behavioral phenotypes of certain neurodevelopmental disorders in humans. Here we reviewed recent findings from rodent models and from human studies to reveal potential mechanisms by which a maternal high-fat diet interferes with the neurodevelopment of the offspring.

Lysosome-targeted photodynamic treatment induces primary keratinocyte differentiation.

Photodynamic therapy is an attractive technique for various skin tumors and non-cancerous skin lesions. However, while the aim of photodynamic therapy is to target and damage only the malignant cells, it unavoidably affects some of the healthy cells surrounding the tumor as well. However, data on the effects of PDT to normal cells are scarce, and the characterization of the pathways activated after the photodamage of normal cells may help to improve clinical photodynamic therapy. In our study, primary human epidermal keratinocytes were used to evaluate photodynamic treatment effects of photosensitizers with different subcellular localization. We compared the response of keratinocytes to lysosomal photodamage induced by phthalocyanines, aluminum phthalocyanine disulfonate (AlPcS2a) or aluminum phthalocyanine tetrasulfonate (AlPcS4), and cellular membrane photodamage by m-tetra(3-hydroxyphenyl)-chlorin (mTHPC). Our data showed that mTHPC-PDT promoted autophagic flux, whereas lysosomal photodamage induced by aluminum phthalocyanines evoked differentiation and apoptosis. Photodamage by AlPcS2a, which is targeted to lysosomal membranes, induced keratinocyte differentiation and apoptosis more efficiently than AlPcS4, which is targeted to lysosomal lumen. Computational analysis of the interplay between these molecular pathways revealed that keratin 10 is the coordinating molecular hub of primary keratinocyte differentiation, apoptosis and autophagy.

Sexual dimorphism of microglia and synapses during mouse postnatal development.

Microglia participate in synapse remodeling in the cortex and hippocampus during mouse postnatal development. Although sex differences in microglia activity during embryonic development have been reported in these regions, it remains unexplored whether microglia show sexually dimorphic features during the early postnatal period, a critical window for synapse formation and maturation. Here, we investigated morphological and functional features of microglia across early postnatal development as well as morphological features of both pre- and postsynaptic neuronal compartments in the mouse hippocampus. We found a sex-dependent shift in microglia volume and phagocytic capacity across the first four postnatal weeks. Measurements of synaptic features revealed sex differences in the density of synaptic spines and boutons during the second postnatal week. These data are consistent with a precocious development of both microglia and synapses in the female brain. We further hypothesize that this bias may contribute to sex-specific brain wiring. © 2017 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 78: 618-626, 2018.

Microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction.

Microglia are highly motile glial cells that are proposed to mediate synaptic pruning during neuronal circuit formation. Disruption of signaling between microglia and neurons leads to an excess of immature synaptic connections, thought to be the result of impaired phagocytosis of synapses by microglia. However, until now the direct phagocytosis of synapses by microglia has not been reported and fundamental questions remain about the precise synaptic structures and phagocytic mechanisms involved. Here we used light sheet fluorescence microscopy to follow microglia-synapse interactions in developing organotypic hippocampal cultures, complemented by a 3D ultrastructural characterization using correlative light and electron microscopy (CLEM). Our findings define a set of dynamic microglia-synapse interactions, including the selective partial phagocytosis, or trogocytosis (trogo-: nibble), of presynaptic structures and the induction of postsynaptic spine head filopodia by microglia. These findings allow us to propose a mechanism for the facilitatory role of microglia in synaptic circuit remodeling and maturation.

Amyloid ß induces microglia to phagocytose neurons via activation of protein kinase Cs and NADPH oxidase.

Alzheimer's disease is characterized by brain plaques of amyloid beta and by neuronal loss, but it is unclear how amyloid beta causes neuronal loss and how to prevent this loss. We have previously shown that amyloid beta causes neuronal loss by inducing microglia to phagocytose neurons, and here we investigated whether protein kinase Cs and NADPH oxidase were involved in this. The loss of neurons induced by amyloid beta in co-cultures of primary glia and neurons was completely prevented by inhibiting protein kinase Cs with Gö6976 or Gö6983. Directly activating protein kinase Cs with phorbol myristate acetate stimulated microglial phagocytosis, and induced neuronal loss mediated by MFG-E8/vitronectin receptor pathway of microglial phagocytosis. Blocking phagocytosis by MFG-E8 knockout or receptor inhibition left live neurons, indicating microglial phagocytosis was the cause of neuronal death. Phorbol myristate acetate stimulated the microglial NADPH oxidase, and inhibiting the oxidase prevented neuronal loss. A physiological activator of NADPH oxidase, fMLP, also induced neuronal loss dependent on microglia. Amyloid beta-induced neuronal loss was blocked by NADPH oxidase inhibitors, superoxide dismutase or Toll-like receptor function-blocking antibodies. The results indicate that amyloid beta induces microglial phagocytosis of neurons via activating protein kinase Cs and NADPH oxidase, and that activating the kinases or oxidase is sufficient to induce neuronal loss by microglial phagocytosis. Thus inhibiting protein kinase Cs or NADPH oxidase might be beneficial in Alzheimer's disease or other brain pathologies involving inflammatory neuronal loss mediated by microglia.

Tumour necrosis factor alpha-induced neuronal loss is mediated by microglial phagocytosis.

Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine, expressed in many brain pathologies and associated with neuronal loss. We show here that addition of TNF-α to neuronal-glial co-cultures increases microglial proliferation and phagocytosis, and results in neuronal loss that is prevented by eliminating microglia. Blocking microglial phagocytosis by inhibiting phagocytic vitronectin and P2Y6 receptors, or genetically removing opsonin MFG-E8, prevented TNF-α induced loss of live neurons. Thus TNF-α appears to induce neuronal loss via microglial activation and phagocytosis of neurons, causing neuronal death by phagoptosis.

Analysis of microglial production of reactive oxygen and nitrogen species.

Reactive oxygen and nitrogen species are both regulators and effectors of microglial activation, and assays of these oxidants can be used as a measure of acute and chronic activation of microglial cells. Here we describe quick methods to assess the production of superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite by microglia.

Primary phagocytosis of neurons by inflamed microglia: potential roles in neurodegeneration.

Microglial phagocytosis of dead or dying neurons can be beneficial by preventing the release of damaging and/or pro-inflammatory intracellular components. However, there is now evidence that under certain conditions, such as inflammation, microglia can also phagocytose viable neurons, thus executing their death. Such phagocytic cell death may result from exposure of phosphatidylserine (PS) or other eat-me signals on otherwise viable neurons as a result of physiological activation or sub-toxic insult, and neuronal phagocytosis by activated microglia. In this review, we discuss the mechanisms of phagocytic cell death and its potential roles in Alzheimer's Disease, Parkinson's Disease, and Frontotemporal Dementia.