Tau is a microtubule-associated protein, found at high levels in neurons, and the aggregation of which is associated with neurodegeneration. Recently it was found that tau can be actively secreted from neurons, but the effects of extracellular tau on neuronal viability are not clear. In this study, we investigated whether extracellular tau 2N4R can cause neurotoxicity in primary cultures of rat brain neurons and glial cells. Cell cultures were treated with recombinant fulllength tau 2N4R isoform, and neuronal numbers, death and phosphatidylserine exposure, as well as microglial phagocytosis were analysed by fluorescent microscopy. Aggregation of tau 2N4R was assests by atomic force microscopy. We found that extracellular addition of tau induced a gradual loss of neurons over 1-2 days, without neuronal necrosis or apoptosis, but accompanied by proliferation of microglia in the neuronal-glial co-cultures. Tau addition caused exposure of the "eat-me" signal phosphatidylserine on the neuronal cell surface (without apparent apoptosis or necrosis), and this was prevented by elimination of the microglia or by inhibition of neutral sphingomyelinase. Tau also increased the phagocytic activity of pure microglia, and this was blocked by inhibitors of neutral sphingomyelinase or protein kinase C. The neuronal loss induced by tau was prevented by inhibitors of neutral sphingomyelinase, protein kinase C or the phagocytic receptor MerTK, or by eliminating microglia from the cultures. The data suggest that extracellular tau induces primary phagocytosis of stressed neurons by activated microglia, and identifies multiple ways in which the neuronal loss induced by tau can be prevented.

Highlights d P2Y 6 R knockout prevents microglial phagocytosis of stressed-but-viable cells d P2Y 6 R knockout does not alter microglial phagocytosis of healthy or dead cells d P2Y 6 R knockout prevents microglial phagocytosis of neurons induced by Ab in vivo d P2Y 6 R knockout reduces neuronal loss and memory deficits

Stroke is the second leading cause of death worldwide. Treatment options for ischemic stroke are limited, and the development of new therapeutic agents or combined therapies is imperative. Growing evidence suggests that metformin treatment, due to its anti-inflammatory action, exerts a neuroprotective effect against ischemia/reperfusion-induced brain damage. Experimental assessment has typically been performed in models of cerebral transient ischemia followed by long-term reperfusion. The aim of this study was to evaluate the neuroprotective effect of metformin treatment after permanent middle cerebral artery occlusion (pMCAO) without reperfusion in rats. Neurological deficits were assessed using the Longa scale, which offers a graded scale on body movement following pMCAO. Both infarct size and brain oedema area were measured by staining with 2,3,5-triphenyltetrazolium chloride. The number of neurons and total and activated microglia, as well as interleukin 10 (IL-10) production, in brain sections were evaluated by immunohistochemical staining. Our results show that metformin treatment improves the neurological state and reduces infarct size after 120 h of pMCAO. Metformin also prevents neuronal loss in the ischemic cortex but not in the striatum after 48 h of pMCAO. Moreover, post-stroke treatment with metformin significantly decreases the number of total and activated microglia at 48 h. The anti-inflammatory effect of metformin is associated with increased IL-10 production at 48 h after pMCAO. The results of the present study suggest that post-stroke treatment with metformin exerts anti-inflammatory and neuroprotective effects in a pMCAO model.

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