Microglia are brain-resident macrophages and play pivotal roles in central nervous system (CNS) development, homeostasis and pathology. Calreticulin and LRPAP-1 are ubiquitously expressed protein chaperones that aid in protein folding and processing within the endoplasmic reticulum (ER). Both proteins might also be released into the extracellular space, but, if so, it is unclear whether and how they affect microglial functions when present extracellularly.

Microglia are the primary innate cells of the CNS and one of the first cell types to respond to signs of injury or inflammation. However, the mechanisms that mediate and regulate this early immune response are unclear. In this work, I show stressed microglia (inflamed, ER-stressed or apoptotic) and neurons (crushed) release calreticulin into the extracellular culture media, where it reaches nanomolar levels. Applications of nanomolar calreticulin activated microglia in culture to release pro-inflammatory cytokines and chemokines, and inhibited microglial proliferation. Nanomolar calreticulin also upregulated surface MHC-II and upregulated the expression and release of APOE, but did not change the expression of 11 other genes associated with disease-associated microglia. Microglia also migrated towards media containing extracellular calreticulin. Overall, this suggests that calreticulin can be released from stressed brain cells, and this released calreticulin can act as an alarmin to recruit and activate microglia.

Calreticulin apparently activated microglia by stimulating toll-like receptor 4 (TLR4) signalling, as nanomolar calreticulin could not activate microglia or a TLR4 reporter line when i) intracellular TLR4 signalling was blocked, ii) binding to TLR4 was blocked with function blocking antibodies, or iii) the hydrophobic binding pocket formed between TLR4 and its co-receptor MD2 was blocked. Microglial activation was also inhibited when calreticulin was pretreated with sugars, so TLR4 activation may require calreticulin’s carbohydrate-binding domain. Calreticulin partially oligomerised under the same conditions used to activate microglia, so oligomeric calreticulin might contribute to activation, but this remains unclear. Thus, calreticulin is a microglial alarmin, and activates microglia by activating TLR4.

LRP-1 and related LDL family receptors mediate many cell functions, and these receptors are inhibitable by extracellular LRPAP-1. However, it is not known whether extracellular LRPAP-1 is a physiological (or pathological) regulator of these receptors, because it is not known whether LRPAP-1 is released extracellularly in physiological conditions and concentrations sufficient to inhibit these receptors. In this work, I found that microglia activated with LPS or ER-stressed with tunicamycin released nanomolar levels of LRPAP-1. Released LRPAP-1 was detected on the surface of microglia, and anti-LRPAP-1 antibodies induced internalisation to peri-nuclear compartments, consistent with LRPAP-1 being bound to endocytic LDL family receptors. Extracellular LRPAP-1, applied at levels released by stressed microglia, did not activate microglia, nor did it prevent LPS neurotoxicity in mixed neuronal-glial cultures. However, extracellular LRPAP-1 did inhibit microglial phagocytosis of dead cells and isolated synapses.

Amyloid beta (Ab) is implicated in Alzheimer’s disease (AD), and LRPAP-1 can bind and regulate Ab uptake in a variety of cells. I show extracellular LRPAP-1 regulates microglial uptake of Ab in a serum- and concentration dependent manner. Extracellular LRPAP-1 inhibited Ab fibrillization. In mixed neuronal-glial cultures, extracellular LRPAP-1 increased Ab bound and/or internalised by neurons but reduced Ab neurotoxicity. Thus. LRPAP-1 can be released by stressed microglia to inhibit microglial phagocytosis, inhibit Ab fibrillization and inhibit Ab neurotoxicity. More generally, this work supports the novel concept that released LRPAP-1 may be an extracellular regulator of LRP-1 and related LDL family receptors and their multiple functions.

Taken together, these findings indicate that calreticulin and LRPAP-1 are secretable regulators of microglial function and are extracellular chaperones.