Brain Cells Release Calreticulin That Attracts and Activates Microglia, and Inhibits Amyloid Beta Aggregation and Neurotoxicity.

Calreticulin is a chaperone, normally found in the endoplasmic reticulum, but can be released by macrophages into the extracellular medium. It is also found in cerebrospinal fluid bound to amyloid beta (Aß). We investigated whether brain cells release calreticulin, and whether extracellular calreticulin had any effects on microglia and neurons relevant to neuroinflammation and neurodegeneration. We found that microglia release nanomolar levels of calreticulin when inflammatory-activated with lipopolysaccharide, when endoplasmic reticulum stress was induced by tunicamycin, or when cell death was induced by staurosporine, and that neurons release calreticulin when crushed. Addition of nanomolar levels of extracellular calreticulin was found to chemoattract microglia, and activate microglia to release cytokines TNF-α, IL-6 and IL-1ß, as well as chemokine (C-C motif) ligand 2. Calreticulin blocked Aß fibrillization and modified Aß oligomerization, as measured by thioflavin T fluorescence and transmission electron microscopy. Extracellular calreticulin also altered microglial morphology and proliferation, and prevented Aß-induced neuronal loss in primary neuron-glial cultures. Thus, calreticulin is released by microglia and neurons, and acts: as an alarmin to recruit and activate microglia, as an extracellular chaperone to prevent Aß aggregation, and as a neuroprotectant against Aß neurotoxicity.

The microglial P2Y6 receptor mediates neuronal loss and memory deficits in neurodegeneration.

Microglia are implicated in neurodegeneration, potentially by phagocytosing neurons, but it is unclear how to block the detrimental effects of microglia while preserving their beneficial roles. The microglial P2Y6 receptor (P2Y6R) - activated by extracellular UDP released by stressed neurons - is required for microglial phagocytosis of neurons. We show here that injection of amyloid beta (Aß) into mouse brain induces microglial phagocytosis of neurons, followed by neuronal and memory loss, and this is all prevented by knockout of P2Y6R. In a chronic tau model of neurodegeneration (P301S TAU mice), P2Y6R knockout prevented TAU-induced neuronal and memory loss. In vitro, P2Y6R knockout blocked microglial phagocytosis of live but not dead targets and reduced tau-, Aß-, and UDP-induced neuronal loss in glial-neuronal cultures. Thus, the P2Y6 receptor appears to mediate Aß- and tau-induced neuronal and memory loss via microglial phagocytosis of neurons, suggesting that blocking this receptor may be beneficial in the treatment of neurodegenerative diseases.

The Phagocytic Code Regulating Phagocytosis of Mammalian Cells.

Mammalian phagocytes can phagocytose (i.e. eat) other mammalian cells in the body if they display certain signals, and this phagocytosis plays fundamental roles in development, cell turnover, tissue homeostasis and disease prevention. To phagocytose the correct cells, phagocytes must discriminate which cells to eat using a 'phagocytic code' - a set of over 50 known phagocytic signals determining whether a cell is eaten or not - comprising find-me signals, eat-me signals, don't-eat-me signals and opsonins. Most opsonins require binding to eat-me signals - for example, the opsonins galectin-3, calreticulin and C1q bind asialoglycan eat-me signals on target cells - to induce phagocytosis. Some proteins act as 'self-opsonins', while others are 'negative opsonins' or 'phagocyte suppressants', inhibiting phagocytosis. We review known phagocytic signals here, both established and novel, and how they integrate to regulate phagocytosis of several mammalian targets - including excess cells in development, senescent and aged cells, infected cells, cancer cells, dead or dying cells, cell debris and neuronal synapses. Understanding the phagocytic code, and how it goes wrong, may enable novel therapies for multiple pathologies with too much or too little phagocytosis, such as: infectious disease, cancer, neurodegeneration, psychiatric disease, cardiovascular disease, ageing and auto-immune disease.

Calreticulin and Galectin-3 Opsonise Bacteria for Phagocytosis by Microglia.

Opsonins are soluble, extracellular proteins, released by activated immune cells, and when bound to a target cell, can induce phagocytes to phagocytose the target cell. There are three known classes of opsonin: antibodies, complement factors and secreted pattern recognition receptors, but these have limited access to the brain. We identify here two novel opsonins of bacteria, calreticulin, and galectin-3 (both lectins that can bind lipopolysaccharide), which were released by microglia (brain-resident macrophages) when activated by bacterial lipopolysaccharide. Calreticulin and galectin-3 both bound to Escherichia coli, and when bound increased phagocytosis of these bacteria by microglia. Furthermore, lipopolysaccharide-induced microglial phagocytosis of E. coli bacteria was partially inhibited by: sugars, an anti-calreticulin antibody, a blocker of the calreticulin phagocytic receptor LRP1, a blocker of the galectin-3 phagocytic receptor MerTK, or simply removing factors released from the microglia, indicating this phagocytosis is dependent on extracellular calreticulin and galectin-3. Thus, calreticulin and galectin-3 are opsonins, released by activated microglia to promote clearance of bacteria. This innate immune response of microglia may help clear bacterial infections of the brain.