Shedding of membrane-associated LDL receptor-related protein-1 from microglia amplifies and sustains neuroinflammation

Abstract

In the CNS, microglia are activated in response to injury or infection and in neurodegenerative diseases. The endocytic and cell signaling receptor, LDL receptor-related protein-1 (LRP1), is reported to suppress innate immunity in macrophages and oppose microglial activation. The goal of this study was to identify novel mechanisms by which LRP1 may regulate microglial activation. Using primary cultures of microglia isolated from mouse brains, we demonstrated that gene silencing increases expression of proinflammatory mediators; however, the observed response was modest. By contrast, the LRP1 ligand, receptor-associated protein (RAP), robustly activated microglia, and its activity was attenuated in LRP1-deficient cells. An important element of the mechanism by which RAP activated microglia was its ability to cause LRP1 shedding from the plasma membrane. This process eliminated cellular LRP1, which is anti-inflammatory, and generated a soluble product, shed LRP1 (sLRP1), which is potently proinflammatory. Purified sLRP1 induced expression of multiple proinflammatory cytokines and the mRNA encoding inducible nitric-oxide synthase in both LRP1-expressing and -deficient microglia. LPS also stimulated LRP1 shedding, as did the heat-shock protein and LRP1 ligand, calreticulin. Other LRP1 ligands, including α-macroglobulin and tissue-type plasminogen activator, failed to cause LRP1 shedding. Treatment of microglia with a metalloproteinase inhibitor inhibited LRP1 shedding and significantly attenuated RAP-induced cytokine expression. RAP and sLRP1 both caused neuroinflammation when administered by stereotaxic injection into mouse spinal cords. Collectively, these results suggest that LRP1 shedding from microglia may amplify and sustain neuroinflammation in response to proinflammatory stimuli.

LRP1 deficiency in microglia blocks neuro‐inflammation in the spinal dorsal horn and neuropathic pain processing

“…Because, in cells of myeloid origin, membrane-anchored LRP1 expresses anti-inflammatory activity (Brifault et al, 2017;Gorovoy et al, 2010;Mantuano et al, 2016;May et al, 2013;Overton et al, 2007;Pocivavsek, Burns, & Rebeck, 2009;Pocivavsek, Mikhailenko, et al, 2009;Staudt et al, 2013;Yang et al, 2016), proteolytic shedding of LRP1 serves as a key reaction that converts the activity of LRP1 from anti-inflammatory to pro-inflammatory (Brifault et al, 2017;Gorovoy et al, 2010). However, to date, in in vivo model systems, only the anti-inflammatory activity of LRP1 has been observed (Chuang et al, 2016;Mantuano et al, 2016;Overton et al, 2007;Staudt et al, 2013).…”

“…The microglia were then isolated by magnetic cell sorting using CD11b-Microbeads (Miltenyi Biotec). Cultures of brain-derived microglia were established as previously described (Brifault et al, 2017). Cultures of microglia from adult mouse spinal cords were established as described by Yip et al (Yip, Kaan, Fenesan, & Malcangio, 2009).…”

“…RAP ligand blotting was performed to detect sLRP1 in conditioned medium (CM). Samples of CM were analyzed without concentrating as previously described (Brifault et al, 2017). CM was subjected to SDS-PAGE and electrotransferred to PVDF membranes, which were blocked and then incubated with 100 nM GST-RAP in 5% nonfat milk for 1 hr at 22 C. The membranes were then washed three times and incubated with GST-specific antibody coupled to horseradish peroxidase (84-814; Genesee Scientific).…”

“…Purified sLRP1 is a potent activator of brain-derived microglia in culture, which also induces neuro-inflammation when injected directly into the SDH (Brifault et al, 2017). By contrast, membrane-anchored microglial LRP1 is reported to express anti-inflammatory activity (Pocivavsek, Burns, & Rebeck, 2009;Pocivavsek, Mikhailenko, et al, 2009;Yang et al, 2016).…”

Activation of stimulator of interferon genes (STING) induces ADAM17-mediated shedding of the immune semaphorin SEMA4D

“…LRPAP1, also known as RAP, interacts with members of the LDL receptor family such as LRP1 within the ER to assist the folding of such receptor proteins (46). Because of its high-affinity binding, recombinant LRPAP1 protein is used for LRP1 ligand and induces proinflammatory cytokine expression (47). However, whether ER chaperone protein LRPAP1 is secreted under physiological conditions was unclear.…”