Bcl-2 family proteins regulate the release of proteins like cytochrome c from mitochondria during apoptosis. We used cell-free systems and ultimately a vesicular reconstitution from defined molecules to show that outer membrane permeabilization by Bcl-2 family proteins requires neither the mitochondrial matrix, the inner membrane, nor other proteins. Bid, or its BH3-domain peptide, activated monomeric Bax to produce membrane openings that allowed the passage of very large (2 megadalton) dextran molecules, explaining the translocation of large mitochondrial proteins during apoptosis. This process required cardiolipin and was inhibited by antiapoptotic Bcl-x(L). We conclude that mitochondrial protein release in apoptosis can be mediated by supramolecular openings in the outer mitochondrial membrane, promoted by BH3/Bax/lipid interaction and directly inhibited by Bcl-x(L).

PREFACE Mitochondria are remarkably dynamic organelles that migrate, divide and fuse. Cycles of mitochondrial fission and fusion ensure metabolite and mitochondrial DNA (mtDNA) mixing and dictate organelle shape, number and bioenergetic functionality. There is mounting evidence that mitochondrial dysfunction is an early and causal event in neurodegeneration. Mutations in mitochondrial fusion GTPases (mitofusin-2 and optic atrophy-1), neurotoxins and oxidative stress all disrupt the cable-like morphology of functional mitochondria. This results in impaired bioenergetics and mitochondrial migration and can trigger neurodegeneration. These findings suggest potential new treatment avenues for neurodegenerative diseases.

Summary NF-κB, a key activator of inflammation primes the NLRP3-inflammasome for activation by inducing pro-IL-1β and NLRP3 expression. NF-κB, however, also prevents excessive inflammation and restrains NLRP3-inflammasome activation through a poorly defined mechanism. We now show that NF-κB exerts its anti-inflammatory activity by inducing delayed accumulation of the autophagy receptor p62/SQSTM1. External NLRP3-activating stimuli trigger a form of mitochondrial (mt) damage that is caspase-1- and NLRP3-independent and causes release of direct NLRP3-inflammasome activators, including mtDNA and mtROS. Damaged mitochondria undergo Parkin-dependent ubiquitin conjugation and are specifically recognized by p62, which induces their mitophagic clearance. Macrophage-specific p62 ablation causes pronounced accumulation of damaged mitochondria and excessive IL-1β-dependent inflammation, enhancing macrophage death. Therefore, the “NF-κB-p62-mitophagy” pathway is a macrophage-intrinsic regulatory loop through which NF-κB restrains its own inflammation-promoting activity and orchestrates a self-limiting host response that maintains homeostasis and favors tissue repair.

Sestrins are conserved proteins that accumulate in cells exposed to stress and potentiate adenosine monophosphate-activated protein kinase (AMPK) and inhibit activation of target of rapamycin (TOR). We show that abundance of Drosophila Sestrin (dSesn) is increased upon chronic TOR activation through accumulation of reactive oxygen species (ROS) that cause activation of c-Jun N-terminal kinase (JNK) and transcription factor FoxO (Forkhead box O). Loss of dSesn resulted in age-associated pathologies including triglyceride accumulation, mitochondrial dysfunction, muscle degeneration and cardiac malfunction, which were prevented by pharmacological activation of AMPK or inhibition of TOR. Hence, dSesn appears to be a negative feedback regulator of TOR that integrates metabolic and stress inputs and prevents pathologies caused by chronic TOR activation, that may result from diminished autophagic clearance of damaged mitochondria, protein aggregates, or lipids.TOR (target of rapamycin) is a key protein kinase that regulates cell growth and metabolism to maintain cellular and organismal homeostasis (1-3). Insulin (Ins) and insulin-like growth factors (IGF) are major TOR activators that operate through phosphoinositide 3-kinase (PI3K) and the protein kinase AKT (2). Conversely, adenosine monophosphate activated protein kinase (AMPK), which is activated upon energy depletion, caloric restriction (CR), or genotoxic damage, is a stress-responsive inhibitor of TOR activation (2,4). TOR stimulates cell growth and anabolism by increasing protein and lipid synthesis through p70 S6 kinase (S6K), eukaryotic translation initiation factor 4E-binding protein (4E-BP), and sterol response element binding protein (SREBP) (1-3,5) and by decreasing autophagic † To whom correspondence should be addressed. karinoffice@ucsd.edu. Prolonged TOR signaling induces dSesnPersistent TOR activation in wing discs by a constitutively active form of insulin receptor (InR CA ) resulted in prominent dSesn protein accumulation, not seen in a dSesn-null larvae (Fig. 1, A to C). InR CA also induced accumulation of dSesn RNA (Fig. 1, D to F), indicating that dSesn accumulation is due to increased transcription or mRNA stabilization. As dSesn accumulation was restricted to cells in which TOR was activated, the response is likely to be cell autonomous. dSesn was also induced when TOR was chronically activated by overexpression of the small guanine triphosphatase Rheb (Fig. 1G), or clonal loss of PTEN (phosphatase and tensin homolog) or TSC1 (tuberous sclerosis complex 1) (Fig. 1, H TOR signaling generates ROS to induce dSesnIn mammals, transcription of Sesn genes is increased in cells exposed to oxidative stress (9,11) and we observed ROS accumulation, detected by oxidation of dihydroethidium (DHE), in the same region of the imaginal discs in which InR CA or Rheb were expressed (Fig. 2, A (Fig. 2F).FoxO and p53 are ROS-activated transcription factors that control mammalian Sesn genes (9-12,14). The dSesn locus contains 8 perfect FoxO-response elemen...

Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant-negative Dynamin related protein 1 (Drp1 K38A ) inhibits mitochondrial fission induced by NO, rotenone and Amyloid-b peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO-induced neuronal cell death was mitigated by Mfn1 and Drp1 K38A . Thus, persistent mitochondrial fission may play a causal role in NO-mediated neurotoxicity.