Consistent with lack of effect on basal activation, minocycline did not affect normal differentiation of NSCs measured by DCX (neuroblasts), GFAP (astrocytes), and NeuN (olfactory bulb neurons; Fig. Our results suggest that autophagy plays a crucial role in regulating neurogenesis and restricting local immune response in postnatal NSCs through nonCcell autonomous mechanisms. Introduction Postnatal neural stem cells (NSCs)/progenitor cells reside in the subventricular zone (SVZ) of the lateral ventricle and subgranular zone of dentate gyrus in the hippocampus of rodent brain (Gage, 2000; Kriegstein and Alvarez-Buylla, 2009). The self-renewal and differentiation of NSCs are regulated by cellCcell and cellCmatrix 20(S)-Hydroxycholesterol interactions and diffusible signals from other cells, such as endothelial cells (Shen et al., 2004) and microglia 20(S)-Hydroxycholesterol (Sierra et al., 2010). Microglia are the resident immune cells in the central nervous system. They defend against pathogens and foreign bodies and clear dead cells and debris (Kettenmann et al., 2011). Microglia also regulate neurogenesis in the early postnatal SVZ (Shigemoto-Mogami et al., 2014) and adult subgranular zone (Sierra et al., 2010). Soluble factors secreted by microglia at injury sites direct NSC migration to these sites and NSC differentiation (Aarum et al., 2003). Embryonic ventricular zone/SVZ basal progenitors recruit microglia for cerebral cortex development (Arn et al., 2014). Postnatal NSCs secreting VEGF enhance the proliferation and function of resident microglia (Mosher et al., 2012). However, our understanding of the cross-regulation of NSCs and microglia is still very limited. Autophagy is a highly conserved lysosomal-dependent degradation pathway that clears damaged organelles and protein aggregates (Klionsky et al., 2016). Autophagy plays important roles, balancing the effects of immunity and inflammation in cancer, infection, and autoimmune diseases (Levine et al., 2011; Shibutani et al., 2015). For example, carriers of the T300A mutation in Atg16L1, an autophagy-related gene, have a higher incidence of Crohn disease (Hampe et al., 2007) and show increased infiltration of inflammatory cells (Adolph et al., 2013). Our 20(S)-Hydroxycholesterol previous studies showed that ablation of FIP200, an essential component of the ULK1CAtg13CFIP200CAtg101 autophagy induction complex, resulted in increased infiltration of immune cells to the skin and tumor mass of mammary tumors (Wei et al., 2009, 2011). These findings suggest that autophagy may function nonCcell autonomously during inflammation and oncogenic transformation. Increasing data show crucial functions of autophagy in self-renewal and differentiation of stem cells, including postnatal NSCs (Guan et al., 2013; Wang et al., 2013), but the underlying mechanisms are not GADD45BETA well understood. Whether autophagy plays a role in cross talk between NSCs and microglia in the SVZ has not been investigated. We used unique mouse models to explore the contributions and mechanisms of microglia as a factor in the regulation of NSCs by autophagy. Results Increased microglia infiltration in the SVZ upon FIP200 deletion in NSCs We first examined the number of microglia in different regions of the brain from conditional knockout (FIP cKO) mice (Wang et al., 2013) 20(S)-Hydroxycholesterol using Iba1 as a marker, as described previously (Ito et al., 1998). The number of microglia significantly increased in the SVZ (Fig. 1, A and B) and rostral migratory stream (RMS; Fig. 1 C) of postnatal day 28 (P28) FIP cKO mice compared with control (Ctrl) mice. Time-course analysis showed that the number of microglia in the SVZ of FIP cKO and Ctrl mice was similar at P0 but gradually increased 20(S)-Hydroxycholesterol in FIP cKO mice compared with Ctrl mice at P7, P14, and P28 (Fig. 1, B and C; and Fig. S1 A). The number of microglia in the striatum and cerebral cortex (Fig. S1, BCD) was comparable between Ctrl and FIP cKO mice. Proliferation of microglia was low in the SVZ and comparable between groups (Figs. 1 D and S1 A), suggesting that the increased number of microglia was not caused by proliferation. Open in a separate window Figure 1. Increased infiltration of microglia in FIP200-deficient SVZ. (A) Immunofluorescence of Iba1 and DAPI in the SVZ (three mice each) in Ctrl, FIP cKO, 2cKO and p53 cKO mice at P28. Arrows indicate Iba1+ microglia. Bar, 40 m. (B and C) Number of Iba1+ cells per SVZ (B) and RMS (C) section in Ctrl and FIP cKO mice at P0, P7, P14, and P28 (mean SEM; six mice per time point). (D) Percentage of Ki67+ microglia (Iba1+).
Consistent with lack of effect on basal activation, minocycline did not affect normal differentiation of NSCs measured by DCX (neuroblasts), GFAP (astrocytes), and NeuN (olfactory bulb neurons; Fig