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

Abstract

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.

Microglial dysfunction as a key pathological change in adrenomyeloneuropathy

“…During classical microglial activation, LPS can significantly reduce MFGE8 expression (Fig 4C,D) while increasing phagocytic activity (Fig 4F). This is consistent with other past reports performed under WT conditions 18, 19, 20…”

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

“…However, this has not previously been investigated in microglia. Previously, we have shown that BV‐2 microglia present a desialylating activity on their cell surface when stimulated with the TLR‐4 agonist LPS (Nomura et al, ). In this study, we show that primary microglia activated by LPS, Aβ, TAU or PMA have increased surface sialidase activity and a substantial desialylation of the surface.…”

“…This indicates that CD11b is involved in the removal of neurons from primary cultures by desialylated microglia. However, it is important to note that desialylation (of neurons or microglia) can activate microglial phagocytosis of neurons or neuronal parts (neurophagy) by multiple potential mechanisms, including: (a) increased deposition and activity of complement C1q and C3b (Linnnartz et al, ), (b) increased binding of opsonin galectin‐3 (Nomura et al, ), (c) decreased trans‐activation of inhibitory Siglec‐11 (Wang & Neumann, ), Siglec‐F (Wielgat & Braszko, ) and Siglec‐E (Claude, Linnartz‐Gerlach, Kudin, Kunz, & Neumann, ), and (d) possibly decreased cis‐activation of inhibitory Siglec‐2 (CD22; Pluvinage et al, ) and Siglec‐3 (CD33; Bradshaw et al, ).…”

“…Per well four microscopic fields were quantified for a single experiment with n = 2 wells per condition. We previously found that microglial BV-2 cells have increased sialidase activity on the cell surface when stimulated with bacterial LPS (Nomura et al, 2017), so we tested here whether primary rat microglia have increased sialidase activity when activated by a variety of inflammatory stimuli. We found that primary rat microglia incubated with LPS, PMA, fibrillar Aβ and TAU protein have increased sialidase activity on the microglial surface ( Figure 1a).…”

Modulation of three key innate immune pathways for the most common retinal degenerative diseases

“…Successive recruitment of the second messenger SHP1 dephosphorylates the intracellular immunoreceptor tyrosine‐based activation motif (ITAM), thus limiting a number of downstream pathways linked to the phagocytosis of neurites and NADPH oxidase (NOX2)‐mediated production of ROS (Fig ; Graham et al, ; Hamerman et al, ). Under pathological conditions, immune cells secrete neuraminidases which cleave sialic acid residues on neurons (Amith et al, ; Pshezhetsky & Hinek, ; Nomura et al, ). Desialylated neurons are consequently opsonized by complement component C1q, which is produced and secreted by microglia (Linnartz et al, ; Madeira et al, ).…”