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. 2013 Dec 15;27(24):2642-7.
doi: 10.1101/gad.225169.113.

Roles of PINK1, mTORC2, and mitochondria in preserving brain tumor-forming stem cells in a noncanonical Notch signaling pathway

Kyu-Sun Lee  1 , Zhihao Wu, Yan Song, Siddhartha S Mitra, Abdullah H Feroze, Samuel H Cheshier, Bingwei Lu
Affiliations

Roles of PINK1, mTORC2, and mitochondria in preserving brain tumor-forming stem cells in a noncanonical Notch signaling pathway

Kyu-Sun Lee et al. Genes Dev. .

Abstract

The self-renewal versus differentiation choice of Drosophila and mammalian neural stem cells (NSCs) requires Notch (N) signaling. How N regulates NSC behavior is not well understood. Here we show that canonical N signaling cooperates with a noncanonical N signaling pathway to mediate N-directed NSC regulation. In the noncanonical pathway, N interacts with PTEN-induced kinase 1 (PINK1) to influence mitochondrial function, activating mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling. Importantly, attenuating noncanonical N signaling preferentially impaired the maintenance of Drosophila and human cancer stem cell-like tumor-forming cells. Our results emphasize the importance of mitochondria to N and NSC biology, with important implications for diseases associated with aberrant N signaling.

Keywords: Notch; PINK1; cancer stem cells; mTORC2/AKT signaling; mitochondria; neural stem cells.

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Figures

Figure 1.
Figure 1.
Notch regulates the mTORC2/AKT pathway in NBs. (A) Diagram of Drosophila larval CNS showing type I and type II NBs in the central brain area (left) and the lineage hierarchy of type II NBs (right). (B) The effects of NB-specific overexpression of canonical Notch pathway components Su(H), Mam, and dMyc. Larval brains at 120 h after larval hatching (ALH) were stained for Dpn (NBs), Pros (differentiated cells), and F-actin (cell cortex). Type II NB lineages are marked with fine white lines. In this and all subsequent figures, the central brain area is outlined with a bold white dashed line, and the Dpn+ NBs within this area are quantified. (C) Quantification of data from B. (D) pS505-dAKT staining of GFP-marked NB flip-out clones overexpressing full-length N (N-v5). NBs within clones (circled in yellow) show an increased pS505-AKT signal compared with those outside of clones (circled in red). (E) Assay of mTORC2 activity by pS505-dAKT Western blot analysis of larva brain extracts after RNAi (RI) or dominant-negative (DN) transgene expression. Total dAKT was used as a loading control. (F) N-induced NB expansion is blocked by inhibition of key components of the mTORC2/AKT pathway but not Trc-DN, and the effect of Rictor inhibition is restored by AKT-S505D. (G) Quantification of data from E. (*) P < 0.0003 (vs. 1407>N-V5/+); (**) P < 0.002 (vs. 1407>N-V5; rictor-RI) in Student's t-test; n = 10. Bars: A,E, 100 μm; C, 20 μm.
Figure 2.
Figure 2.
Involvement of PINK1 and other mitochondria-related genes in N-induced NB overproliferation. (A) N-induced NB expansion is blocked by inhibiting genes involved in mitochondrial regulation. Larval brains at 120 h ALH were immunostained. (B) Quantification of data from A. (*) P<0.0001 versus 1407>N-V5/+; n = 10. Complex-III RNAi served as a specificity control. (C) Clonal analysis of NBs in the Drp1 mutant (Drp12), Drp12; Nact, and Nact backgrounds. MARCM clones are marked with GFP and outlined with white dashed lines. (D) Quantification of data from C. (NS) Not significant; (*) P<0.001; n = 5. (E) N-induced NB expansion is attenuated by Mdivi-1 treatment. (F) Quantification of data from E. (*) P<0.001 versus PntP1-Gal4/+; (**) P < 0.005 versus PntP1>N-V5; n = 5. (G,H) Primary (G) and secondary (H) neurosphere-forming activity of human GBM CSCs and normal fetal NSCs after chemical inhibition of Drp1 (Mdivi-1) or mTORC2 (Ku-0063794). (*) P < 0.0001 versus DMSO control; (**) P < 0.001 human NSC versus human GBM. (I) Effects of lentiviral delivery of PINK1 shRNA (either singularly or with two shRNAs combined) on the proliferation of human NSCs and GBM cells. (*) P < 0.001 versus scrambled shRNA control in Student's t-test. Bars: A,E, 100 μm; C, 20 μm.
Figure 3.
Figure 3.
Genetic and biochemical interactions between N and PINK1. (A,B) Effects of N overexpression or N RNAi on PINK1 LOF-induced abnormal wing posture (A) and aggregation of muscle mitochondria (B). (*) P < 0.0001 versus w− control in Student's t-test. (C) Effects of N LOF on muscle mitochondrial morphology monitored with mito-GFP. The uneven and enhanced mito-GFP signals suggest mitochondrial aggregation. (D) Effects of N mutations on mitochondrial membrane potential (indicated by JC-1 signal at 575–625 nm). The PINK1 mutant was used as a control. (E) Transmission electron microscopy analysis of mitochondrial cristae morphology in N mutants. Bar, 100 nm. N[55e11] refers to N[55e11]/+ heterozygous females in C–E. (F) Blue native gel (BNG) and Western blot analyses of RCC assembly in PINK1 and N LOF and GOF backgrounds. Actin and the complex IV subunit 1 served as loading controls. (G) Effects of N GOF on RCC assembly and pS505-dAKT level changes caused by PINK1 LOF. (H,I) Fractionation assay showing enrichment of full-length N at mitochondria (H) and increased mitochondrial N in the PINK1 overexpression condition (I). (J) Coimmunoprecipitation assays using NiGFP fly head tissue to demonstrate N interaction with PINK1 and complex I 30 kD. The complex III subunit served as a negative control. (K) The interaction between hNotch1 and PINK1 in the mitochondria of human NSCs and GBM cells. The complex III subunit served as a negative control. GBM cells show elevated expression of PINK1 and both the full-length and the intracellular domain of Notch1.
Figure 4.
Figure 4.
The interaction between canonical and noncanonical N signaling on NB homeostasis. (A) The effects of combined RNAi of Su(H) and PINK1 on N GOF-induced NB expansion. (B) Quantification of data from A. (*) P < 0.0002 versus 1407>N-V5/+; (**) P < 0.0005 versus 1407>N-V5; Su(H)-RI or 1407>N-V5; dPINK1-RI; n = 5. (C) The effects of combined RNAi of Myc and PINK1 on normal NB number. (D) Quantification of data from C. (*) P < 0.001 versus 1407>dMyc-RI or 1407>dPINK1-RI; n = 5. (E) The effects of coexpression of PINK1 and Rictor on N GOF-induced NB expansion. (F) Quantification of data from E. (*) P < 0.0001 versus PntP1-Gal4/+ control; (**) P < 0.05 versus PntP1>N-V5/+; n = 5. (G) Synergy between dMyc overexpression and Marf-RI in rescuing N LOF-induced type II NB loss. (H) Quantification of data from G. (*) P < 0.005 versus 1407>N-RI/+; (**) P < 0.05 versus 1407>N-RI;dMyc or 1407>N-RI;Marf-RI; n = 5. Parkin or GFP coexpression served as a specificity control. (I) The effects of coexpression of Su(H) and dAKT-S505D in mature IPs (driven by Erm-gal4). The ectopic NBs dedifferentiated from mature IPs are identified by marker expression (Dpn+ and Ase−) and marked by white dashed circles. (J) Quantification of data from I. (*) P < 0.0002 versus Erm-Gal4/+ control in Student's t-test; n = 5. Bars, 100 μm.
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