Impaired phagocytic efficiency by beclin 1-deficient BV2 cells could also be rescued by recovering beclin GSK2656157 clinical trial 1 levels (Figure 2B). To confirm these findings in primary cells, we next isolated microglia from beclin 1 heterozygous knockout mice (beclin 1+/−) ( Figure 2C), which show a 40%–50% reduction in beclin 1 levels ( Pickford et al., 2008 and Qu et al., 2003). In agreement with our lentiviral approach,

beclin 1+/− microglia also showed impairments in phagocytic efficiency when analyzed by flow cytometry ( Figure 2D). To determine if impaired phagocytic efficiency in beclin 1-deficient cells resulted from beads stalling at the cell surface or from a disruption in the kinetics of phagocytosis, we used microscopy and live-cell imaging. We observed that while beads were initially phagocytosed at a similar rate ( Figure S2A), beclin 1-deficient BV2 cells were less able to phagocytose subsequent beads ( Figure S2B and Figure 2E). Quantification of cell migration confirmed that beclin 1-deficient cells have a similar migratory capacity as control cells, indicating that impaired movement Erastin is not responsible for phagocytic deficits ( Figure 2F). Instead, our data suggest that beclin 1 deficiency impairs the ability of cells to phagocytose subsequent

beads beyond the initial phagocytic event (see Figure 2G for representative live-cell images), resulting in overall reduced phagocytic uptake ( Figure S2C). Phagocytosis is initiated by numerous receptors that recognize molecular structures on extracellular substrates. Upon binding and internalization of substrates, phagocytic receptors are recycled back to the cell surface to be used again. Accordingly, disruptions in Adenosine phagocytic receptor recycling have dramatic consequences on phagocytic efficiency (Chen et al., 2010). To determine if reduced phagocytic efficiency seen in beclin 1-deficient cells is due to

changes in phagocytic receptor dynamics, we used an established receptor recycling assay (Mitchell et al., 2004) (Figure 3B). Indeed, beclin 1-deficient BV2 cells showed a prominent reduction in recycling of the phagocytic receptor CD36 (Figures 3C and 3D), a class B scavenger receptor involved in phagocytosing a wide range of substrates, including Aβ (El Khoury et al., 2003) and latex beads (Figure 3A). Primary microglia obtained from beclin 1+/− mice also showed a similar deficit in CD36 recycling ( Figures 3E and 3F). Importantly, flow cytometric analysis demonstrated that beclin 1 shRNA did not affect baseline cell surface expression of CD36 in the absence of ligand ( Figure 3G). Additionally, because the phagocytic receptor Trem2 has been reported to recycle ( Prada et al., 2006) and is a risk factor for AD ( Guerreiro et al., 2013 and Jonsson et al.