Alzheimer's disease-associated R47H TREM2 increases, but wild-type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss.

Triggering receptor on myeloid cells 2 (TREM2) is an innate immune receptor, upregulated on the surface of microglia associated with amyloid plaques in Alzheimer's disease (AD). Individuals heterozygous for the R47H variant of TREM2 have greatly increased risk of developing AD. We examined the effects of wild-type (WT), R47H and knock-out (KO) of human TREM2 expression in three microglial cell systems. Addition of mouse BV-2 microglia expressing R47H TREM2 to primary mouse neuronal cultures caused neuronal loss, not observed with WT TREM2. Neuronal loss was prevented by using annexin V to block exposed phosphatidylserine, an eat-me signal and ligand of TREM2, suggesting loss was mediated by microglial phagocytosis of neurons exposing phosphatidylserine. Addition of human CHME-3 microglia expressing R47H TREM2 to LUHMES neuronal-like cells also caused loss compared to WT TREM2. Expression of R47H TREM2 in BV-2 and CHME-3 microglia increased their uptake of phosphatidylserine-beads and synaptosomes versus WT TREM2. Human iPSC-derived microglia with heterozygous R47H TREM2 had increased phagocytosis of synaptosomes vs common-variant TREM2. Additionally, phosphatidylserine liposomes increased activation of human iPSC-derived microglia expressing homozygous R47H TREM2 versus common-variant TREM2. Finally, overexpression of TREM2 in CHME-3 microglia caused increased expression of cystatin F, a cysteine protease inhibitor, and knock-down of cystatin F increased CHME-3 uptake of phosphatidylserine-beads. Together, these data suggest that R47H TREM2 may increase AD risk by increasing phagocytosis of synapses and neurons via greater activation by phosphatidylserine and that WT TREM2 may decrease microglial phagocytosis of synapses and neurons via cystatin F.

Neu1 Is Released From Activated Microglia, Stimulating Microglial Phagocytosis and Sensitizing Neurons to Glutamate.

Neuraminidase 1 (Neu1) hydrolyses terminal sialic acid residues from glycoproteins and glycolipids, and is normally located in lysosomes, but can be released onto the surface of activated myeloid cells and microglia. We report that endotoxin/lipopolysaccharide-activated microglia released Neu1 into culture medium, and knockdown of Neu1 in microglia reduced both Neu1 protein and neuraminidase activity in the culture medium. Release of Neu1 was reduced by inhibitors of lysosomal exocytosis, and accompanied by other lysosomal proteins, including protective protein/cathepsin A, known to keep Neu1 active. Extracellular neuraminidase or over-expression of Neu1 increased microglial phagocytosis, while knockdown of Neu1 decreased phagocytosis. Microglial activation caused desialylation of microglial phagocytic receptors Trem2 and MerTK, and increased binding to Trem2 ligand galectin-3. Culture media from activated microglia contained Neu1, and when incubated with neurons induced their desialylation, and increased the neuronal death induced by low levels of glutamate. Direct desialylation of neurons by adding sialidase or inhibiting sialyltransferases also increased glutamate-induced neuronal death. We conclude that activated microglia can release active Neu1, possibly by lysosomal exocytosis, and this can both increase microglial phagocytosis and sensitize neurons to glutamate, thus potentiating neuronal death.

The microglial P2Y6 receptor mediates neuronal loss and memory deficits in neurodegeneration.

Microglia are implicated in neurodegeneration, potentially by phagocytosing neurons, but it is unclear how to block the detrimental effects of microglia while preserving their beneficial roles. The microglial P2Y6 receptor (P2Y6R) - activated by extracellular UDP released by stressed neurons - is required for microglial phagocytosis of neurons. We show here that injection of amyloid beta (Aß) into mouse brain induces microglial phagocytosis of neurons, followed by neuronal and memory loss, and this is all prevented by knockout of P2Y6R. In a chronic tau model of neurodegeneration (P301S TAU mice), P2Y6R knockout prevented TAU-induced neuronal and memory loss. In vitro, P2Y6R knockout blocked microglial phagocytosis of live but not dead targets and reduced tau-, Aß-, and UDP-induced neuronal loss in glial-neuronal cultures. Thus, the P2Y6 receptor appears to mediate Aß- and tau-induced neuronal and memory loss via microglial phagocytosis of neurons, suggesting that blocking this receptor may be beneficial in the treatment of neurodegenerative diseases.

Wild-type sTREM2 blocks Aß aggregation and neurotoxicity, but the Alzheimer's R47H mutant increases Aß aggregation.

TREM2 is a pattern recognition receptor, expressed on microglia and myeloid cells, detecting lipids and Aß and inducing an innate immune response. Missense mutations (e.g., R47H) of TREM2 increase risk of Alzheimer's disease (AD). The soluble ectodomain of wild-type TREM2 (sTREM2) has been shown to protect against AD in vivo, but the underlying mechanisms are unclear. We show that Aß oligomers bind to cellular TREM2, inducing shedding of the sTREM2 domain. Wild-type sTREM2 bound to Aß oligomers (measured by single-molecule imaging, dot blots, and Bio-Layer Interferometry) inhibited Aß oligomerization and disaggregated preformed Aß oligomers and protofibrils (measured by transmission electron microscopy, dot blots, and size-exclusion chromatography). Wild-type sTREM2 also inhibited Aß fibrillization (measured by imaging and thioflavin T fluorescence) and blocked Aß-induced neurotoxicity (measured by permeabilization of artificial membranes and by loss of neurons in primary neuronal-glial cocultures). In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aß but rather promotes Aß protofibril formation and neurotoxicity. Thus, in addition to inducing an immune response, wild-type TREM2 may protect against amyloid pathology by the Aß-induced release of sTREM2, which blocks Aß aggregation and neurotoxicity. In contrast, R47H sTREM2 promotes Aß aggregation into protofibril that may be toxic to neurons. These findings may explain how wild-type sTREM2 apparently protects against AD in vivo and why a single copy of the R47H variant gene is associated with increased AD risk.

Microglial phagocytosis of neurons in neurodegeneration, and its regulation.

There is growing evidence that excessive microglial phagocytosis of neurons and synapses contributes to multiple brain pathologies. RNA-seq and genome-wide association (GWAS) studies have linked multiple phagocytic genes to neurodegenerative diseases, and knock-out of phagocytic genes has been found to protect against neurodegeneration in animal models, suggesting that excessive microglial phagocytosis contributes to neurodegeneration. Here, we review recent evidence that microglial phagocytosis of live neurons and synapses causes neurodegeneration in animal models of Alzheimer's disease and other tauopathies, Parkinson's disease, frontotemporal dementias, multiple sclerosis, retinal degeneration and neurodegeneration induced by ischaemia, infection or ageing. We also review factors regulating microglial phagocytosis of neurons, including: nucleotides, frackalkine, phosphatidylserine, calreticulin, UDP, CD47, sialylation, complement, galectin-3, Apolipoprotein E, phagocytic receptors, Siglec receptors, cytokines, microglial epigenetics and expression profile. Some of these factors may be potential treatment targets to prevent neurodegeneration mediated by excessive microglial phagocytosis of live neurons and synapses.

Sialylation acts as a checkpoint for innate immune responses in the central nervous system.

Sialic acids are monosaccharides that normally terminate the glycan chains of cell surface glyco-proteins and -lipids in mammals, and are highly enriched in the central nervous tissue. Sialic acids are conjugated to proteins and lipids (termed "sialylation") by specific sialyltransferases, and are removed ("desialylation") by neuraminidases. Cell surface sialic acids are sensed by complement factor H (FH) to inhibit complement activation or by sialic acid-binding immunoglobulin-like lectin (SIGLEC) receptors to inhibit microglial activation, phagocytosis, and oxidative burst. In contrast, desialylation of cells enables binding of the opsonins C1, calreticulin, galectin-3, and collectins, stimulating phagocytosis of such cells. Hypersialylation is used by bacteria and cancers as camouflage to escape immune recognition, while polysialylation of neurons protects synapses and neurogenesis. Insufficient lysosomal cleavage of sialylated molecules can lead to lysosomal accumulation of lipids and aggregated proteins, which if excessive may be expelled into the extracellular space. On the other hand, desialylation of immune receptors can activate them or trigger removal of proteins. Loss of inhibitory SIGLECs or FH triggers reduced clearance of aggregates, oxidative brain damage and complement-mediated retinal damage. Thus, cell surface sialylation recognized by FH, SIGLEC, and other immune-related receptors acts as a major checkpoint inhibitor of innate immune responses in the central nervous system, while excessive cleavage of sialic acid residues and consequently removing this checkpoint inhibitor may trigger lipid accumulation, protein aggregation, inflammation, and neurodegeneration.

Sialylation and Galectin-3 in Microglia-Mediated Neuroinflammation and Neurodegeneration.

Microglia are brain macrophages that mediate neuroinflammation and contribute to and protect against neurodegeneration. The terminal sugar residue of all glycoproteins and glycolipids on the surface of mammalian cells is normally sialic acid, and addition of this negatively charged residue is known as "sialylation," whereas removal by sialidases is known as "desialylation." High sialylation of the neuronal cell surface inhibits microglial phagocytosis of such neurons, via: (i) activating sialic acid receptors (Siglecs) on microglia that inhibit phagocytosis and (ii) inhibiting binding of opsonins C1q, C3, and galectin-3. Microglial sialylation inhibits inflammatory activation of microglia via: (i) activating Siglec receptors CD22 and CD33 on microglia that inhibit phagocytosis and (ii) inhibiting Toll-like receptor 4 (TLR4), complement receptor 3 (CR3), and other microglial receptors. When activated, microglia release a sialidase activity that desialylates both microglia and neurons, activating the microglia and rendering the neurons susceptible to phagocytosis. Activated microglia also release galectin-3 (Gal-3), which: (i) further activates microglia via binding to TLR4 and TREM2, (ii) binds to desialylated neurons opsonizing them for phagocytosis via Mer tyrosine kinase, and (iii) promotes Aß aggregation and toxicity in vivo. Gal-3 and desialylation may increase in a variety of brain pathologies. Thus, Gal-3 and sialidases are potential treatment targets to prevent neuroinflammation and neurodegeneration.

Activated microglia desialylate their surface, stimulating complement receptor 3-mediated phagocytosis of neurons.

The glycoproteins and glycolipids of the cell surface have sugar chains that normally terminate in a sialic acid residue, but inflammatory activation of myeloid cells can cause sialidase enzymes to remove these residues, resulting in desialylation and altered activity of surface receptors, such as the phagocytic complement receptor 3 (CR3). We found that activation of microglia with lipopolysaccharide (LPS), fibrillar amyloid beta (Aß), Tau or phorbol myristate acetate resulted in increased surface sialidase activity and desialylation of the microglial surface. Desialylation of microglia by adding sialidase, stimulated microglial phagocytosis of beads, but this was prevented by siRNA knockdown of CD11b or a blocking antibody to CD11b (a component of CR3). Desialylation of microglia by a sialyl-transferase inhibitor (3FAx-peracetyl-Neu5Ac) also stimulated microglial phagocytosis of beads. Desialylation of primary glial-neuronal co-cultures by adding sialidase or the sialyl-transferase inhibitor resulted in neuronal loss that was prevented by inhibiting phagocytosis with cytochalasin D or the blocking antibody to CD11b. Adding desialylated microglia to glial-neuronal cultures, in the absence of neuronal desialylation, also caused neuronal loss prevented by CD11b blocking antibody. Adding LPS or Aß to primary glial-neuronal co-cultures caused neuronal loss, and this was prevented by inhibiting endogenous sialidase activity with N-acetyl-2,3-dehydro-2-deoxyneuraminic acid or blockage of CD11b. Thus, activated microglia release a sialidase activity that desialylates the cell surface, stimulating CR3-mediated phagocytosis of neurons, making extracellular sialidase and CR3 potential treatment targets to prevent inflammatory loss of neurons.

Activated Microglia Desialylate and Phagocytose Cells via Neuraminidase, Galectin-3, and Mer Tyrosine Kinase.

Activated microglia can phagocytose dying, stressed, or excess neurons and synapses via the phagocytic receptor Mer tyrosine kinase (MerTK). Galectin-3 (Gal-3) can cross-link surface glycoproteins by binding galactose residues that are normally hidden below terminal sialic acid residues. Gal-3 was recently reported to opsonize cells via activating MerTK. We found that LPS-activated BV-2 microglia rapidly released Gal-3, which was blocked by calcineurin inhibitors. Gal-3 bound to MerTK on microglia and to stressed PC12 (neuron-like) cells, and it increased microglial phagocytosis of PC12 cells or primary neurons, which was blocked by inhibition of MerTK. LPS-activated microglia exhibited a sialidase activity that desialylated PC12 cells and could be inhibited by Tamiflu, a neuraminidase (sialidase) inhibitor. Sialidase treatment of PC12 cells enabled Gal-3 to bind and opsonize the live cells for phagocytosis by microglia. LPS-induced microglial phagocytosis of PC12 was prevented by small interfering RNA knockdown of Gal-3 in microglia, lactose inhibition of Gal-3 binding, inhibition of neuraminidase with Tamiflu, or inhibition of MerTK by UNC569. LPS-induced phagocytosis of primary neurons by primary microglia was also blocked by inhibition of MerTK. We conclude that activated microglia release Gal-3 and a neuraminidase that desialylates microglial and PC12 surfaces, enabling Gal-3 binding to PC12 cells and their phagocytosis via MerTK. Thus, Gal-3 acts as an opsonin of desialylated surfaces, and inflammatory loss of neurons or synapses may potentially be blocked by inhibiting neuraminidases, Gal-3, or MerTK.