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, 28 (2), 229-246

Autophagy Mediates Astrogenesis in Adult Hippocampal Neural Stem Cells

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Autophagy Mediates Astrogenesis in Adult Hippocampal Neural Stem Cells

Shinwon Ha et al. Exp Neurobiol.

Abstract

Neural stem cells (NSCs) have the ability to self-renew and differentiate into neurons, oligodendrocytes, and astrocytes. Highly dynamic nature of NSC differentiation requires the intimate involvement of catabolic processes such as autophagy. Autophagy is a major intracellular degradation pathway necessary for cellular homeostasis and remodeling. Autophagy is important for mammalian development and its role in neurogenesis has recently drawn much attention. However, little is known about how autophagy is associated with differentiation of NSCs into other neural lineages. Here, we report that autophagy plays a critical role in differentiation of adult rat hippocampal neural stem (HCN) cells into astrocytes. During differentiation, autophagy flux peaked at early time points, and remained high. Pharmacological or genetic suppression of autophagy by stable knockdown of Atg7, LC3 or CRISPR-Cas9-mediated knockout (KO) of p62 impaired astrogenesis, while reintroduction of p62 recovered astrogenesis in p62 KO HCN cells. Taken together, our findings suggest that autophagy plays a key role in astrogenesis in adult NSCs.

Keywords: Adult stem cells; Astrocytes; Autophagy; Autophagy-related protein 7; Cell differentiation; Neural stem cells; Sequestosome-1 protein.

Figures

Fig. 1
Fig. 1. Differentiation of HCN cells into neurons, oligodendrocytes, and astrocytes. (A) A schematic timeline for differentiation experiments. (B) Undifferentiated and differentiated HCN cells were stained for Nestin, MAP2, RIP, and GFAP (green) with Hoechst 33342 (blue), and imaged by confocal microscopy. Scale bar, 25 µm. (C) Sox2 mRNA levels in undifferentiated HCN cells (Con) and HCN cells differentiated into neurons (Neuron), oligodendrocytes (Oligo), or astrocytes (Astro). (D) Changes in NeuN, GalC and Gfap mRNA levels after differentiation. *p<0.05, **p<0.01, and ***p<0.001. n≥3.
Fig. 2
Fig. 2. Time course analyses of HCN cell differentiation into astrocytes. (A) A schematic timeline for astrogenesis experiments. D, day. (B) Images of HCN cells stained with nestin and GFAP antibodies at D0 and D4 during astrogenesis. Scale bar, 50 µm. (C) Astrocyte morphology examined with GFAP staining. Scale bar, 25 µm. (D) GFAP and Sox2 protein levels analyzed by Western blotting. (E) Gfap mRNA levels. (F) Tnc mRNA levels. (G) mRNA levels of other neural cell markers, NeuN and GalC . ns, not significant. **p<0.01, and ***p<0.001. n≥3.
Fig. 3
Fig. 3. An increase in autophagy flux from early time points during astrogenesis. (A) Analysis of autophagy flux using mRFP-GFP-LC3 stable HCN cells. Scale bar, 15 µm. (B) After Baf.A1 treatment, autophagy flux was measured in mRFP-GFP-LC3 stable HCN cells at D2. Baf.A1 (20 nM) was added 1 h before harvesting. Scale bar, 15 µm. (C) Time course analyses of LC3-II by Western blotting. (D) Time course analyses of autophagy flux by Western blotting of LC3-II after Baf.A1 treatment. Baf.A1 (20 nM) was added 1 h before harvesting. (E) Increased autophagy flux at D2. Baf.A1 (20 nM) or PepA/E64d (10 µg/ml for each) was treated 1 h before harvesting. *p<0.05, **p<0.01, and ***p<0.001. n≥3.
Fig. 4
Fig. 4. Impaired astrogenesis by the suppression of autophagy in HCN cells. (A) Verification of Atg7 knockdown by Western blotting analysis. (B, C) Decrease in GFAP protein (B) and mRNA (C) levels in Sh-Atg7 cells compared with Sh-Con cells. (D) Impaired astrocyte morphology in Sh-Atg7 cells compared with Sh-Con cells at D4. (E) Verification of LC3 knockdown by Western blotting analysis. (F) Decrease in Gfap mRNA level in Sh-LC3 cells. (G) Reduction of Gfap and Tnc mRNA levels by Baf.A1 treatment. Baf.A1 (0.5 nM) was added at D0 and D2 and half of media was changed at D1 and D3. Scale bar, 25 µm. *p<0.05, **p<0.01, and ***p<0.001. n≥5.
Fig. 5
Fig. 5. Impaired astrogenesis in p62 knockout HCN cells. (A) Genetic deficiency of p62 abrogated an increase in GFAP expression in differentiated p62 knockout HCN cells (Sg-p62) in comparison with control cells (Sg-Con) at D4. (B) A dramatic decrease in Gfap mRNA expression level in Sg-p62 cells in comparison with Sg-Con cells at D4. (C) Impaired astrocyte morphology in Sg-p62 cells in comparison with Sg-Con cells at D4. Scale bar, 25 µm. (D) The absence of induction of cell death during astrogenesis. (E~G) Recovery of GFAP protein (E), mRNA (F) levels, and astrocyte morphology (G) by expression of GFP-p62, but not GFP-EV (empty vector) in Sg-p62 cells at D4. Scale bar, 25 µm. *p<0.05, **p<0.01, and ***p<0.001. n≥3.
Fig. 6
Fig. 6. A schematic diagram illustrating the involvement of autophagy in astrogenesis.

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