The surface‐exposed chaperone, Hsp60, is an agonist of the microglial TREM2 receptor

Abstract

The triggering receptor expressed in myeloid (TREM) cells 2, referred to with the acronym TREM2, is a single-spanning membrane receptor of an immunoglobulin/lectin-like family. The members of this family operate through the interaction with a common coupling protein, DNAX-activation protein of 12 kDa (DAP12) which upon phosphorylation induces the activation of various intracellular signalling pathways (tyrosine kinases, phospholipase Cc). Activation of other Received December 20, 2008; revised manuscript received February 11, 2009; accepted April 21, 2009. Abbreviations used: BSA, bovine serum albumin; DAP12, DNAXactivation protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hHsp60, human heat shock protein 60; HRP, horseradish peroxidase; Hsp60, heat shock protein 60; PBS, phosphate-buffered saline; PLOSL, polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy; rHsp60, rat heat shock protein 60; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; siRNA, short interfering RNA; TREM, triggering receptor expressed in myeloid cells. AbstractTriggering receptor expressed in myeloid (TREM) cells 2, a receptor expressed by myeloid cells, osteoclasts and microglia, is known to play a protective role in bones and brain. Mutations of the receptor (or of its coupling protein, DAP12) sustain in fact a genetic disease affecting the two organs, the polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy (PLOSL or Nasu-Hakola disease). So far, specific agonist(s) of TREM2 have not been identified and its (their) transduction mechanisms are largely unknown. Heat shock protein 60 (Hsp60) is a mitochondrial chaperone that can also be harboured at the cell surface. By using constructs including the extracellular domain of TREM2 and the Fc domain of IgGs we have identified Hsp60 as the only TREM2-binding protein exposed at the surface of neuroblastoma N2A cells and astrocytes, and lacking in U373 astrocytoma. Treatment with Hsp60 was found to stimulate the best known TREM2-dependent process, phagocytosis, however, only in the microglial N9 cells rich in the receptor. Upon TREM2 down-regulation, the Hsp60-induced stimulation of N9 phagocytosis was greatly attenuated. Hsp60 is also released by many cell types, segregated within exosomes or shedding vesicles which might then undergo dissolution. However, the affinity of its binding (K d = 3.8 lM) might be too low for the soluble chaperone released from the vesicles to the extracellular space to induce a significant activation of TREM2. It might in contrast be appropriate for the binding of TREM2 to Hsp60 exposed at the surface of cells closely interacting with microglia. The ensuing stimulation of phagocytosis could play protective effects on the brain. Keywords: receptor binding, surface-harboured agonist, western and far-western blotting, phagocytosis, polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy.

Microglial Activation in Stroke: Therapeutic Targets

“…117 It has been described on activated macrophages and microglia, 118 -120 and binds to one or more ill-defined ligands on eukaryotic cells including neurons and astrocytes. [121][122][123] More recent work suggests that one such ligand might be HSP60, 122 a mitochondrial stress protein that can move to the cell surface under appropriate conditions. 124 Stimulation of HSP60 stimulates phagocytic activity of TREM2 expressing microglia, but not of TREM2 deficient microglia.…”

“…124 Stimulation of HSP60 stimulates phagocytic activity of TREM2 expressing microglia, but not of TREM2 deficient microglia. 122 When bound to a ligand, TREM2 engages its adapter protein, DAP12, which then recruits and activates the tyrosine kinase Syk, 120,125,126 leading to downstream signaling through pathways including phosphatidylinositol 3-kinase (PI3K), phospholipase C␥1, and p44 -p42 extracellular signal regulated kinase (ERK), but not through classical inflammatory pathways such as nuclear factor B (NF-B) and the p38 stress-activated protein kinase. 127,128 TREM2 has been shown to mediate phagocytosis of apoptotic neurons.…”

Triggering Receptor Expressed on Myeloid Cells-2 Correlates to Hypothermic Neuroprotection in Ischemic Stroke

“…The triggering receptor expressed by myeloid cells-2 (TREM2) is a newly indentified molecule described on activated microglia and macrophage (Daws et al, 2001;Sessa et al, 2004;Takahashi et al, 2005), involved in innate immunity and is considered to play an important role in the phagocytosis of microbes (Daws et al, 2001). Its ligand has also been detected in the brain (Daws et al, 2003), and has been postulated to mediate phagocytosis of injured or apoptotic neurons (Daws et al, 2003;Takahashi et al, 2005;Hsieh et al, 2009;Stefano et al, 2009). In this study, we compared paradigms where hypothermia was protective and where hypothermia was not protected in a mouse stroke model to characterize the inflammatory response and TREM2 expression.…”

The Evolving Biology of Microglia in Alzheimer's Disease

“…Studies have yet to identify definitively the ligand of Trem2 and the cell type on which it is expressed. Stefano et al [122] used a Trem2-Fc construct to identify heat shock protein 60 (Hsp60) as potential ligand present on neuroblastoma cells. While Hsp60 is normally located in the mitochondria, it can be expressed on the cell surface during cell stress [123], potentially making it accessible to Trem2 in the context of neurodegeneration.…”