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

Abstract

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.

Microglia Mediate Contact-Independent Neuronal Network Remodeling via Secreted Neuraminidase-3 Associated with Extracellular Vesicles

“…Therefore, NEU3 KO cells are still able to secrete inflammatory signals. TNFα secretion in both cell lines was inhibited by the pan-sialidase inhibitor deoxy-2,3-anhydroneuraminic acid (DANA), consistent with previous observations with Neu1. , Given that the BV-2 microglia are still capable of secreting inflammatory molecules, Neu3-mediated autodesialylation is not a prerequisite for inflammatory activity in the manner as has been reported for Neu1 . Moreover, these data implicate Neu3 as the secreted sialidase, rather than an upstream component.…”

“…As prior studies have implicated Neu1 translocation and desialylation in cis as a critical component of microglial activation, ,, we sought to determine whether the loss of secreted sialidase activity in NEU3 KO BV-2 cells was a consequence of impaired inflammatory activity. We observed that NEU3 KO cells had impaired autodesialylation in response to LPS treatment compared to WT as measured by periodate labeling of sialic acids ( p = 0.51 and = 0.038, respectively), but that both WT and NEU3 KO cells upregulated TNFα to similar levels in (WT, p = 0.002; NEU3 KO, p = 0.02) (Figure S2).…”

“…Neuroinflammation has been correlated with changes in the glycocalyxthe coating of sugars on cell surfacesof both neurons and microglia. − Sialic acids are a particular subset of bioactive sugars in the glycocalyx. They are known to modulate neuronal excitability and plasticity, , and changes in the sialylation state are associated with neuroinflammation and microglial activation. − Upon exposure to inflammatory stimuli, microglia have been observed to release sialidase activity into the surrounding media, which effects desialylation, ,, deposition of opsonizing factors, , microglial activation, , and phagocytosis of neurons. , Additionally, our lab has recently identified the sialylation state as a critical factor in maintaining neuronal excitability and network integration .…”

“…Neuroinflammation has been correlated with changes in the glycocalyx—the coating of sugars on cell surfaces—of both neurons and microglia. 11 − 15 Sialic acids are a particular subset of bioactive sugars in the glycocalyx. They are known to modulate neuronal excitability and plasticity, 7 , 16 and changes in the sialylation state are associated with neuroinflammation and microglial activation.…”

Galectin-3 (MAC-2) controls phagocytosis and macropinocytosis through intracellular and extracellular mechanisms

“…Moreover, sialic acid often binds galactose residues on cell surface phagocytic receptors (e.g., MerTK) and phagocytic targets as does Gal-3, thus impeding phagocytosis by blocking Gal-3 binding/crosslinking MerTK and targets (reviewed in Puigdellivol et al, 2020). Nomura et al (2017) and Allendorf and Brown (2022) showed in this context that the LPS-treated microglia increased the release of Neuraminidase 1 (Neu1), which through desialylation exposed galactose residues on MerTK and targets, thus leading to augmented phagocytosis by enabling Gal-3 binding and so crosslinking MerTK and targets. Taken altogether, efficient MerTK-mediated phagocytosis of Gal-3-opsonized targets depended on inflammatory stimuli activating microglia to upregulate MerTK expression and further increase Gal-3 and neuraminidase secretion.…”