Interestingly, the processes of resting microglia make frequent contact with synapses in a neuronal activity-dependent manner, suggesting that they constantly monitor the functional status of synapses (54C56). Microglia are the resident phagocytes in the central nervous system (CNS) and are responsible for the maintenance of CNS homeostasis (1, 2). They take up invasive microorganisms, apoptotic/necrotic cellular debris, and the aberrant protein depositions in progressive neurodegenerative disorders, including the amyloid beta (A) peptides in Alzheimer’s disease (AD) (3). Understanding the mechanisms of endocytosis in these cells is usually thus relevant to understanding their functions. Phagocytosis and pinocytosis are two clathrin-independent endocytic processes that occur in phagocytes, and both create large endocytic vacuolar compartments ( 0.2 m) through organized membrane movements and actin polymerization (4). Nevertheless, distinct models and molecular mechanisms have been suggested for the formation of phagosomes and pinosomes (4). The well-studied Fc receptor-mediated phagocytosis is usually guided by a zipper-like progression of receptor-initiated membrane invagination that is shaped by the geometry of the internalized particle, whereas pinosomes, which may vary from 0.2 to 10 m in diameter, are suggested to form spontaneously or in response to growth Triphendiol (NV-196) factor receptor stimulation from membrane ruffles that close at their distal margins to engulf extracellular fluid without strict guidance from receptors (4, 5). Phagocytosis in phagocytes is known to be brought on by eat me signals expressed around the cell surface of dying cells (6). In addition, UDP leakage from damaged neurons has been suggested to function as a diffusible eat me signal to induce phagocytic activity in microglia through activation of P2Y6 receptors (7). Pinocytosis by phagocytes is usually involved in many physiological and pathological processes, including development, innate immunity, and the entry of pathogens into host cells (4, 5). However, the mechanisms underlying the regulation of pinocytosis in microglia are not clear. In the present study, we found that ATP brought on microglial pinocytosis through activation of P2Y4 purinergic receptors. The phosphatidylinositol 3-kinase (PI3K)/Akt cascade was shown to be the downstream pathway of the ATP-induced pinocytosis. Interestingly, soluble A itself induced pinocytosis, which is an ATP/P2Y4-dependent process, indicating that microglial pinocytosis of A is usually a nucleotide-regulated active process, rather than a constitutive, passive activity. Moreover, either P2Y4 knockdown by RNA interference or ATP deprivation by the ATP-degradation enzyme apyrase decreased the spontaneous pinocytosis of A by microglia. Thus, in addition to the previously identified P2Y12 receptor-mediated find me signal (8C10) and the P2Y6 receptor-mediated eat me signal (7), our study demonstrates that nucleotides also function as an autocrine drink me signal for microglia and mediate the uptake of soluble A through activation of P2Y4 receptors. MATERIALS AND METHODS Animals. The use and care of animals followed the guidelines of the Shanghai Institutes for Biological Sciences Animal Research Advisory Committee (Shanghai, China) and the Animal Advisory Committee at Zhejiang University, which approved the protocols. APPswe/PS1dE9 transgenic mice on a C3H background were obtained from the Jackson Laboratory (Bar Triphendiol (NV-196) Harbor, ME). Cell culture. Primary cultured microglia were harvested according to a method described previously (11). In brief, a mixed glial culture was prepared from the cortices of neonatal Sprague-Dawley rats (either sex) and maintained for 7 to 10 days in minimum essential medium (MEM; Gibco, Grand Island, NY) made up of 10% fetal bovine serum (FBS; Gibco). The cells floating over the Triphendiol (NV-196) mixed glial culture were collected by gentle shaking and transferred to appropriate glass coverslips. Microglia were obtained as rapidly attached cells and maintained in low-FBS medium (2% FBS in MEM) for 12 to 24 h before use. Mixed glial cultures can also give rise to purified secondary astrocytes. As Triphendiol (NV-196) previously described (12), mixed glial cultures were shaken at 200 rpm overnight at 37C Rabbit Polyclonal to KCY to dislodge other glial cells attached to the astrocyte layer. After medium alternative, astrocytes were obtained by trypsinization (0.125% trypsin, 5 min, 37C) and replated at a low density. When the cell density reached 70%, they were used for drug treatment or transfection experiments. Primary real neuronal cultures were prepared as described previously (13) with some modifications. In brief, embryonic day 18 (E18) rat (either sex) hippocampi were dissected, dissociated with 0.125% trypsin, and plated.