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The crucial role of Atg5 in cortical neurogenesis during early brain development.

Lv X, Jiang H, Li B, Liang Q, Wang S, Zhao Q, Jiao J - Sci Rep (2014)

Bottom Line: The β-Catenin phosphorylation level decreased when Atg5 was blocked.Atg5 cooperated with β-Catenin to modulate cortical NPCs differentiation and proliferation.Our results revealed that Atg5 has a crucial role in cortical neurogenesis during early embryonic brain development, which may contribute to the understanding of neurodevelopmental disorders caused by autophagy dysregulation.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China [2].

ABSTRACT
Autophagy plays an important role in the central nervous system. However, it is unknown how autophagy regulates cortical neurogenesis during early brain development. Here, we report that autophagy-related gene 5 (Atg5) expression increased with cortical development and differentiation. The suppression of Atg5 expression by knockdown led to inhibited differentiation and increased proliferation of cortical neural progenitor cells (NPCs). Additionally, Atg5 suppression impaired cortical neuronal cell morphology. We lastly observed that Atg5 was involved in the regulation of the β-Catenin signaling pathway. The β-Catenin phosphorylation level decreased when Atg5 was blocked. Atg5 cooperated with β-Catenin to modulate cortical NPCs differentiation and proliferation. Our results revealed that Atg5 has a crucial role in cortical neurogenesis during early embryonic brain development, which may contribute to the understanding of neurodevelopmental disorders caused by autophagy dysregulation.

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Atg5 regulates the β-Catenin signaling pathway.(a). The effect of Atg5 on the expression of β-Catenin. HEK293FT cells were transfected with Atg5 shRNA, Atg5-expression or control plasmids. Ctrl, Atg5-expression plasmids plus control shRNA plasmids; Atg5 KD, Atg5-expression plasmids plus Atg5 shRNA plasmids; Ctrl', Atg5-expression vector plasmids; Atg5 OE, Atg5-expression plasmids. A western blot was used to analyze the β-Catenin protein levels 3 days later. β-actin was used as a control. Blot images were cropped for comparison. (b). Co-immunostaining shows that the expression of non-pS33/37/T41 β-Catenin increases after Atg5 knockdown. Control or Atg5 shRNA plasmids were transfected into HEK 293FT cells. (c). The expression of LC3 decreases after Atg5 knockdown. Control or Atg5 shRNA plasmids were transfected into HEK 293FT cells. (d). Co-immunostaining shows the expression of pS33/37/T41 β-Catenin and GFP-LC3 in 293FT cells under normal or starvation conditions (in HBSS solution) for 6 hr. (e). LC3 interacts with β-Catenin. Control or LC3 plasmids were transfected into HEK 293FT cells. The transfected cell lysates were subjected to co-immunoprecipitation (co-IP) using an anti-LC3 antibody and immunoblotted with anti-pS33/37/T41 β-Catenin and anti-total β-Catenin antibodies. Input lysates are shown. β-actin was used as a control. Blot images were cropped for comparison. (f). Effects of Atg5 on stability of β-Catenin protein. Flag-Atg5 or empty vector was overexpressed in primary neural stem cells under differentiation conditions. Cycloheximide (CHX, 100 μg/mL) was added after 48 hours. The cells lysates were harvested at the indicated times and immunoblotted with anti-total β-Catenin, anti-flag, and anti-β-actin antibodies. (g). The graph shows the relative band intensity of total β-Catenin relative to the level of β-actin at each time point, compared with the level of β-Catenin at time zero, taken as 1. (h, i, j). Atg5 regulates the downstream target genes (cyclin D1, c-Myc, and neurogenin 2 (Ngn2)) expression of β-Catenin. Primary neural stem cells were infected with control and Atg5 shRNA lentivirus. The cells were harvested after 3 days for real-time PCR. Scale bars, 2 μm (b–d).
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f4: Atg5 regulates the β-Catenin signaling pathway.(a). The effect of Atg5 on the expression of β-Catenin. HEK293FT cells were transfected with Atg5 shRNA, Atg5-expression or control plasmids. Ctrl, Atg5-expression plasmids plus control shRNA plasmids; Atg5 KD, Atg5-expression plasmids plus Atg5 shRNA plasmids; Ctrl', Atg5-expression vector plasmids; Atg5 OE, Atg5-expression plasmids. A western blot was used to analyze the β-Catenin protein levels 3 days later. β-actin was used as a control. Blot images were cropped for comparison. (b). Co-immunostaining shows that the expression of non-pS33/37/T41 β-Catenin increases after Atg5 knockdown. Control or Atg5 shRNA plasmids were transfected into HEK 293FT cells. (c). The expression of LC3 decreases after Atg5 knockdown. Control or Atg5 shRNA plasmids were transfected into HEK 293FT cells. (d). Co-immunostaining shows the expression of pS33/37/T41 β-Catenin and GFP-LC3 in 293FT cells under normal or starvation conditions (in HBSS solution) for 6 hr. (e). LC3 interacts with β-Catenin. Control or LC3 plasmids were transfected into HEK 293FT cells. The transfected cell lysates were subjected to co-immunoprecipitation (co-IP) using an anti-LC3 antibody and immunoblotted with anti-pS33/37/T41 β-Catenin and anti-total β-Catenin antibodies. Input lysates are shown. β-actin was used as a control. Blot images were cropped for comparison. (f). Effects of Atg5 on stability of β-Catenin protein. Flag-Atg5 or empty vector was overexpressed in primary neural stem cells under differentiation conditions. Cycloheximide (CHX, 100 μg/mL) was added after 48 hours. The cells lysates were harvested at the indicated times and immunoblotted with anti-total β-Catenin, anti-flag, and anti-β-actin antibodies. (g). The graph shows the relative band intensity of total β-Catenin relative to the level of β-actin at each time point, compared with the level of β-Catenin at time zero, taken as 1. (h, i, j). Atg5 regulates the downstream target genes (cyclin D1, c-Myc, and neurogenin 2 (Ngn2)) expression of β-Catenin. Primary neural stem cells were infected with control and Atg5 shRNA lentivirus. The cells were harvested after 3 days for real-time PCR. Scale bars, 2 μm (b–d).

Mentions: The important function of β-Catenin in NPCs proliferation and differentiation during brain development has been reported192021. The regulatory roles of β-Catenin in neurogenesis are developmental stage-specific2223. Previous research has demonstrated that β-Catenin overexpression causes the number of cortical precursor expansions, whereas β-Catenin elimination leads to premature neuronal differentiation24. Therefore, Atg5 possibly affects neurogenesis through β-Catenin regulation in cortical development. We studied whether the gain or loss of function of Atg5 had similar effects on the expression of β-Catenin using western blotting. We observed that Non-pS33/37/T41 (active) β-Catenin levels decreased when Atg5 was overexpressed, whereas the levels increased with Atg5 knockdown (Fig. 4a). Similar results were reported by previous research25. The results suggested that Atg5 might regulate β-Catenin stability. To test this possibility, we performed in vitro immunostaining experiments. The results showed an increased expression of Non-pS33/37/T41 β-Catenin after Atg5 knockdown (Fig. 4b). These results revealed the regulatory role of Atg5 in β-Catenin stability. The above results, together with the regulatory roles of autophagy in β-Catenin degradation26, indicated that Atg5 activating autophagy might regulate β-Catenin degradation. To investigate this possibility, we initially demonstrated the important role of Atg5 in autophagy. The immunostaining results showed reduced microtubule-associated protein 1 light chain 3 (LC3) expression after Atg5 knockdown (Fig. 4c). Importantly, LC3 serves not only as a marker of autophagosomes but also is important in autophagosome formation2728. To further test whether Atg5 was essential for autophagy, we next demonstrated that autophagy levels could not be enhanced by rapamycin when Atg5 was knocked down in primary NPCs (data not shown). We also observed that LC3 gathered and primarily co-localized with pS33/37/T41 β-Catenin under starvation conditions, whereas LC3 was dispersed under normal conditions (Fig. 4d). The co-localization of LC3 with pS33/37/T41 β-Catenin suggested that they may interact. We subsequently tested this possibility using co-immunoprecipitation experiments, observing that pS33/37/T41-catenin associated with LC3 (Fig. 4e). To confirm whether Atg5 affects β-Catenin degradation directly, we followed up the residual level of β-Catenin after cycloheximide (CHX) addition. Data showed that the degradation rate of β-Catenin was accelerated by Atg5 overexpression (Fig. 4f, g). These results indicated that Atg5 regulated β-Catenin stability by activating autophagy. To further study the crucial roles of Atg5 in the β-Catenin signaling pathway, we used a lentivirus system to infect cells and examined changes in the downstream target gene expression of β-Catenin after Atg5 knockdown in NPCs. We observed that the expression of cyclin D1 and c-Myc, downstream target genes of β-Catenin2930, significantly increased after Atg5 knockdown (Fig. 4h, i). By contrast, we observed the reduced expression of neurogenin 2 (Ngn2), which is a neuronal determination gene31 (Fig. 4j). All of these results indicated that Atg5 regulated the β-Catenin signaling pathway.


The crucial role of Atg5 in cortical neurogenesis during early brain development.

Lv X, Jiang H, Li B, Liang Q, Wang S, Zhao Q, Jiao J - Sci Rep (2014)

Atg5 regulates the β-Catenin signaling pathway.(a). The effect of Atg5 on the expression of β-Catenin. HEK293FT cells were transfected with Atg5 shRNA, Atg5-expression or control plasmids. Ctrl, Atg5-expression plasmids plus control shRNA plasmids; Atg5 KD, Atg5-expression plasmids plus Atg5 shRNA plasmids; Ctrl', Atg5-expression vector plasmids; Atg5 OE, Atg5-expression plasmids. A western blot was used to analyze the β-Catenin protein levels 3 days later. β-actin was used as a control. Blot images were cropped for comparison. (b). Co-immunostaining shows that the expression of non-pS33/37/T41 β-Catenin increases after Atg5 knockdown. Control or Atg5 shRNA plasmids were transfected into HEK 293FT cells. (c). The expression of LC3 decreases after Atg5 knockdown. Control or Atg5 shRNA plasmids were transfected into HEK 293FT cells. (d). Co-immunostaining shows the expression of pS33/37/T41 β-Catenin and GFP-LC3 in 293FT cells under normal or starvation conditions (in HBSS solution) for 6 hr. (e). LC3 interacts with β-Catenin. Control or LC3 plasmids were transfected into HEK 293FT cells. The transfected cell lysates were subjected to co-immunoprecipitation (co-IP) using an anti-LC3 antibody and immunoblotted with anti-pS33/37/T41 β-Catenin and anti-total β-Catenin antibodies. Input lysates are shown. β-actin was used as a control. Blot images were cropped for comparison. (f). Effects of Atg5 on stability of β-Catenin protein. Flag-Atg5 or empty vector was overexpressed in primary neural stem cells under differentiation conditions. Cycloheximide (CHX, 100 μg/mL) was added after 48 hours. The cells lysates were harvested at the indicated times and immunoblotted with anti-total β-Catenin, anti-flag, and anti-β-actin antibodies. (g). The graph shows the relative band intensity of total β-Catenin relative to the level of β-actin at each time point, compared with the level of β-Catenin at time zero, taken as 1. (h, i, j). Atg5 regulates the downstream target genes (cyclin D1, c-Myc, and neurogenin 2 (Ngn2)) expression of β-Catenin. Primary neural stem cells were infected with control and Atg5 shRNA lentivirus. The cells were harvested after 3 days for real-time PCR. Scale bars, 2 μm (b–d).
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f4: Atg5 regulates the β-Catenin signaling pathway.(a). The effect of Atg5 on the expression of β-Catenin. HEK293FT cells were transfected with Atg5 shRNA, Atg5-expression or control plasmids. Ctrl, Atg5-expression plasmids plus control shRNA plasmids; Atg5 KD, Atg5-expression plasmids plus Atg5 shRNA plasmids; Ctrl', Atg5-expression vector plasmids; Atg5 OE, Atg5-expression plasmids. A western blot was used to analyze the β-Catenin protein levels 3 days later. β-actin was used as a control. Blot images were cropped for comparison. (b). Co-immunostaining shows that the expression of non-pS33/37/T41 β-Catenin increases after Atg5 knockdown. Control or Atg5 shRNA plasmids were transfected into HEK 293FT cells. (c). The expression of LC3 decreases after Atg5 knockdown. Control or Atg5 shRNA plasmids were transfected into HEK 293FT cells. (d). Co-immunostaining shows the expression of pS33/37/T41 β-Catenin and GFP-LC3 in 293FT cells under normal or starvation conditions (in HBSS solution) for 6 hr. (e). LC3 interacts with β-Catenin. Control or LC3 plasmids were transfected into HEK 293FT cells. The transfected cell lysates were subjected to co-immunoprecipitation (co-IP) using an anti-LC3 antibody and immunoblotted with anti-pS33/37/T41 β-Catenin and anti-total β-Catenin antibodies. Input lysates are shown. β-actin was used as a control. Blot images were cropped for comparison. (f). Effects of Atg5 on stability of β-Catenin protein. Flag-Atg5 or empty vector was overexpressed in primary neural stem cells under differentiation conditions. Cycloheximide (CHX, 100 μg/mL) was added after 48 hours. The cells lysates were harvested at the indicated times and immunoblotted with anti-total β-Catenin, anti-flag, and anti-β-actin antibodies. (g). The graph shows the relative band intensity of total β-Catenin relative to the level of β-actin at each time point, compared with the level of β-Catenin at time zero, taken as 1. (h, i, j). Atg5 regulates the downstream target genes (cyclin D1, c-Myc, and neurogenin 2 (Ngn2)) expression of β-Catenin. Primary neural stem cells were infected with control and Atg5 shRNA lentivirus. The cells were harvested after 3 days for real-time PCR. Scale bars, 2 μm (b–d).
Mentions: The important function of β-Catenin in NPCs proliferation and differentiation during brain development has been reported192021. The regulatory roles of β-Catenin in neurogenesis are developmental stage-specific2223. Previous research has demonstrated that β-Catenin overexpression causes the number of cortical precursor expansions, whereas β-Catenin elimination leads to premature neuronal differentiation24. Therefore, Atg5 possibly affects neurogenesis through β-Catenin regulation in cortical development. We studied whether the gain or loss of function of Atg5 had similar effects on the expression of β-Catenin using western blotting. We observed that Non-pS33/37/T41 (active) β-Catenin levels decreased when Atg5 was overexpressed, whereas the levels increased with Atg5 knockdown (Fig. 4a). Similar results were reported by previous research25. The results suggested that Atg5 might regulate β-Catenin stability. To test this possibility, we performed in vitro immunostaining experiments. The results showed an increased expression of Non-pS33/37/T41 β-Catenin after Atg5 knockdown (Fig. 4b). These results revealed the regulatory role of Atg5 in β-Catenin stability. The above results, together with the regulatory roles of autophagy in β-Catenin degradation26, indicated that Atg5 activating autophagy might regulate β-Catenin degradation. To investigate this possibility, we initially demonstrated the important role of Atg5 in autophagy. The immunostaining results showed reduced microtubule-associated protein 1 light chain 3 (LC3) expression after Atg5 knockdown (Fig. 4c). Importantly, LC3 serves not only as a marker of autophagosomes but also is important in autophagosome formation2728. To further test whether Atg5 was essential for autophagy, we next demonstrated that autophagy levels could not be enhanced by rapamycin when Atg5 was knocked down in primary NPCs (data not shown). We also observed that LC3 gathered and primarily co-localized with pS33/37/T41 β-Catenin under starvation conditions, whereas LC3 was dispersed under normal conditions (Fig. 4d). The co-localization of LC3 with pS33/37/T41 β-Catenin suggested that they may interact. We subsequently tested this possibility using co-immunoprecipitation experiments, observing that pS33/37/T41-catenin associated with LC3 (Fig. 4e). To confirm whether Atg5 affects β-Catenin degradation directly, we followed up the residual level of β-Catenin after cycloheximide (CHX) addition. Data showed that the degradation rate of β-Catenin was accelerated by Atg5 overexpression (Fig. 4f, g). These results indicated that Atg5 regulated β-Catenin stability by activating autophagy. To further study the crucial roles of Atg5 in the β-Catenin signaling pathway, we used a lentivirus system to infect cells and examined changes in the downstream target gene expression of β-Catenin after Atg5 knockdown in NPCs. We observed that the expression of cyclin D1 and c-Myc, downstream target genes of β-Catenin2930, significantly increased after Atg5 knockdown (Fig. 4h, i). By contrast, we observed the reduced expression of neurogenin 2 (Ngn2), which is a neuronal determination gene31 (Fig. 4j). All of these results indicated that Atg5 regulated the β-Catenin signaling pathway.

Bottom Line: The β-Catenin phosphorylation level decreased when Atg5 was blocked.Atg5 cooperated with β-Catenin to modulate cortical NPCs differentiation and proliferation.Our results revealed that Atg5 has a crucial role in cortical neurogenesis during early embryonic brain development, which may contribute to the understanding of neurodevelopmental disorders caused by autophagy dysregulation.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China [2].

ABSTRACT
Autophagy plays an important role in the central nervous system. However, it is unknown how autophagy regulates cortical neurogenesis during early brain development. Here, we report that autophagy-related gene 5 (Atg5) expression increased with cortical development and differentiation. The suppression of Atg5 expression by knockdown led to inhibited differentiation and increased proliferation of cortical neural progenitor cells (NPCs). Additionally, Atg5 suppression impaired cortical neuronal cell morphology. We lastly observed that Atg5 was involved in the regulation of the β-Catenin signaling pathway. The β-Catenin phosphorylation level decreased when Atg5 was blocked. Atg5 cooperated with β-Catenin to modulate cortical NPCs differentiation and proliferation. Our results revealed that Atg5 has a crucial role in cortical neurogenesis during early embryonic brain development, which may contribute to the understanding of neurodevelopmental disorders caused by autophagy dysregulation.

Show MeSH
Related in: MedlinePlus