Glioblastomas contains stem-like tumor cells that display differential metabolic profiles. Here the authors show that brain tumor initiating cells contain fragmented mitochondria owing to activation of the key mediator of mitochondrial fission, DRP1, controlled by a competitive CDK5–CAMK2 axis. Targeting DRP1 activity attenuates growth of stem-like tumor cells, and activated DRP1 informs poor patient prognosis. Brain tumor initiating cells (BTICs) co-opt the neuronal high affinity glucose transporter, GLUT3, to withstand metabolic stress. We investigated another mechanism critical to brain metabolism, mitochondrial morphology, in BTICs. BTIC mitochondria were fragmented relative to non-BTIC tumor cell mitochondria, suggesting that BTICs increase mitochondrial fission. The essential mediator of mitochondrial fission, dynamin-related protein 1 (DRP1), showed activating phosphorylation in BTICs and inhibitory phosphorylation in non-BTIC tumor cells. Targeting DRP1 using RNA interference or pharmacologic inhibition induced BTIC apoptosis and inhibited tumor growth. Downstream, DRP1 activity regulated the essential metabolic stress sensor, AMP-activated protein kinase (AMPK), and targeting AMPK rescued the effects of DRP1 disruption. Cyclin-dependent kinase 5 (CDK5) phosphorylated DRP1 to increase its activity in BTICs, whereas Ca2+-calmodulin-dependent protein kinase 2 (CAMK2) inhibited DRP1 in non-BTIC tumor cells, suggesting that tumor cell differentiation induces a regulatory switch in mitochondrial morphology. DRP1 activation correlated with poor prognosis in glioblastoma, suggesting that mitochondrial dynamics may represent a therapeutic target for BTICs.

Mitotic mitochondrial fission requires CDK1-mediated phosphorylation of DRP1. The Aurora A substrate RALA is found to concentrate DRP1 and RALBP1 at mitochondria to promote DRP1 phosphorylation and fission. Mitochondria exist as dynamic interconnected networks that are maintained through a balance of fusion and fission1. Equal distribution of mitochondria to daughter cells during mitosis requires fission2. Mitotic mitochondrial fission depends on both the relocalization of the large GTPase DRP1 to the outer mitochondrial membrane and phosphorylation of Ser 616 on DRP1 by the mitotic kinase cyclin B–CDK1 (ref. 2). We now report that these processes are mediated by the small Ras-like GTPase RALA and its effector RALBP1 (also known as RLIP76, RLIP1 or RIP1; refs 3, 4). Specifically, the mitotic kinase Aurora A phosphorylates Ser 194 of RALA, relocalizing it to the mitochondria, where it concentrates RALBP1 and DRP1. Furthermore, RALBP1 is associated with cyclin B–CDK1 kinase activity that leads to phosphorylation of DRP1 on Ser 616. Disrupting either RALA or RALBP1 leads to a loss of mitochondrial fission at mitosis, improper segregation of mitochondria during cytokinesis and a decrease in ATP levels and cell number. Thus, the two mitotic kinases Aurora A and cyclin B–CDK1 converge on RALA and RALBP1 to promote mitochondrial fission, the appropriate distribution of mitochondria to daughter cells and ultimately proper mitochondrial function.

Significance: Here, we present unequivocal evidence of physical association of AMPK holoenzymes with mitochondrial reticulum (mitoAMPK) across multiple mouse tissues with evidence of conservation in human skeletal muscle and heart. We demonstrate that mitoAMPK is activated heterogeneously across the mitochondrial reticulum by mitochondrial energetic stress. Finally, we present evidence that suggests activation of mitoAMPK in skeletal muscle is required for mitophagy. We propose that mitoAMPK responds to mitochondrial microenvironment cues to maintain energetic homeostasis through mitochondrial quality control.

The transcription factor NF-κB has been shown to be predominantly cytoplasmically localized in the absence of an inductive signal. Stimulation of cells with inflammatory cytokines such as tumor necrosis factor α or interleukin-1 induces the degradation of IκB, the inhibitor of NF-κB, allowing nuclear accumulation of NF-κB and regulation of specific gene expression. The degradation of IκB is controlled initially by phosphorylation induced by the IκB kinase, which leads to ubiquitination and subsequent proteolysis of the inhibitor by the proteasome. We report here that NF-κB and IκBα (but not IκBβ) are also localized in the mitochondria. Stimulation of cells with tumor necrosis factor α leads to the phosphorylation of mitochondrial IκBα and its subsequent degradation by a nonproteasome-dependent pathway. Interestingly, expression of the mitochondrially encoded cytochromec oxidase III and cytochrome bmRNAs were reduced by cytokine treatment of cells. Inhibition of activation of mitochondrial NF-κB by expression of the superrepressor form of IκBα inhibited the loss of expression of both cytochromec oxidase III and cytochrome b mRNA. These data indicate that the NF-κB regulatory pathway exists in mitochondria and that NF-κB can negatively regulate mitochondrial mRNA expression.