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Translational errors frequently arise during protein synthesis, producing misfolded and dysfunctional proteins. Chronic stress resulting from translation errors may be particularly relevant in tissues that must synthesize and secrete large amounts of secretory proteins. Here, we studied the proteostasis networks in the liver of mice that express the Rps2-A226Y ribosomal ambiguity (ram) mutation to increase the translation error rate across all proteins. We found that Rps2-A226Y mice lack activation of the eIF2 kinase/ATF4 pathway, the main component of the integrated stress response (ISR), as well as the IRE1 and ATF6 pathways of the ER unfolded protein response (ER-UPR). Instead, we found downregulation of chronic ER stress responses, as indicated by reduced gene expression for lipogenic pathways and acute phase proteins, possibly via upregulation of Sirtuin-1. In parallel, we observed activation of alternative proteostasis responses, including the proteasome and the formation of stress granules. Together, our results point to a concerted response to error-prone translation to alleviate ER stress in favor of activating alternative proteostasis mechanisms, most likely to avoid cell damage and apoptotic pathways, which would result from persistent activation of the ER and integrated stress responses.

Summary Stress adaptation is essential for neuronal health. While the fundamental role of mitochondria in neuronal development has been demonstrated, it is still not clear how adult neurons respond to alterations in mitochondrial function and how neurons sense, signal, and respond to dysfunction of mitochondria and their interacting organelles. Here, we show that neuron-specific, inducible in vivo ablation of the mitochondrial fission protein Drp1 causes ER stress, resulting in activation of the integrated stress response to culminate in neuronal expression of the cytokine Fgf21. Neuron-derived Fgf21 induction occurs also in murine models of tauopathy and prion disease, highlighting the potential of this cytokine as an early biomarker for latent neurodegenerative conditions., Graphical Abstract, Highlights • Neuronal Drp1 ablation is sensed by branches of the integrated stress response (ISR) • Activation of the ISR induces catabolic cytokine Fgf21 in the brain • Brain Fgf21 induced in neurodegeneration models may be a potential biomarker, Restelli et al. show that deletion of mitochondrial fission protein Drp1 in adult mouse neurons activates multiple stress-sensing pathways. These converge on the integrated stress response, resulting in neuron-specific expression of metabolic cytokine Fgf21. Cerebral induction of Fgf21 also occurs in mechanistically independent mouse models of protein misfolding-associated neurodegeneration.

During apoptosis mitochondria undergo cristae remodeling and fragmentation, but how the latter relates to outer membrane permeabilization and downstream caspase activation is unclear. Here we show that the mitochondrial fission protein Dynamin Related Protein (Drp) 1 participates in cytochrome c release by selected intrinsic death stimuli. While Bax, Bak double deficient (DKO) and Apaf1(-/-) mouse embryonic fibroblasts (MEFs) were less susceptible to apoptosis by Bcl-2 family member BID, H(2)O(2), staurosporine and thapsigargin, Drp1(-/-) MEFs were protected only from BID and H(2)O(2). Resistance to cell death of Drp1(-/-) and DKO MEFs correlated with blunted cytochrome c release, whereas mitochondrial fragmentation occurred in all cell lines in response to all tested stimuli, indicating that other mechanisms accounted for the reduced cytochrome c release. Indeed, cristae remodeling was reduced in Drp1(-/-) cells, potentially explaining their resistance to apoptosis. Our results indicate that caspase-independent mitochondrial fission and Drp1-dependent cristae remodeling amplify apoptosis. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

The 1555 A to G substitution in mitochondrial 12S A‐site rRNA is associated with maternally transmitted deafness of variable penetrance in the absence of otherwise overt disease. Here, we recapitulate the suggested A1555G‐mediated pathomechanism in an experimental model of mitoribosomal mistranslation by directed mutagenesis of mitoribosomal protein MRPS5. We first establish that the ratio of cysteine/methionine incorporation and read‐through of mtDNA‐encoded MT‐CO1 protein constitute reliable measures of mitoribosomal misreading. Next, we demonstrate that human HEK293 cells expressing mutant V336Y MRPS5 show increased mitoribosomal mistranslation. As for immortalized lymphocytes of individuals with the pathogenic A1555G mutation, we find little changes in the transcriptome of mutant V336Y MRPS5 HEK cells, except for a coordinated upregulation of transcripts for cytoplasmic ribosomal proteins. Homozygous knock‐in mutant Mrps5 V338Y mice show impaired mitochondrial function and a phenotype composed of enhanced susceptibility to noise‐induced hearing damage and anxiety‐related behavioral alterations. The experimental data in V338Y mutant mice point to a key role of mitochondrial translation and function in stress‐related behavioral and physiological adaptations.

Well-balanced mitochondrial fission and fusion processes are essential for nervous system development. Loss of function of the main mitochondrial fission mediator, dynamin-related protein 1 (Drp1), is lethal early during embryonic development or around birth, but the role of mitochondrial fission in adult neurons remains unclear. Here we show that inducible Drp1 ablation in neurons of the adult mouse forebrain results in progressive, neuronal subtype-specific alterations of mitochondrial morphology in the hippocampus that are marginally responsive to antioxidant treatment. Furthermore, DRP1 loss affects synaptic transmission and memory function. Although these changes culminate in hippocampal atrophy, they are not sufficient to cause neuronal cell death within 10 weeks of genetic Drp1 ablation. Collectively, our in vivo observations clarify the role of mitochondrial fission in neurons, demonstrating that Drp1 ablation in adult forebrain neurons compromises critical neuronal functions without causing overt neurodegeneration.

Antibiotics targeting the bacterial ribosome typically bind to highly conserved rRNA regions with only minor phylogenetic sequence variations. It is unclear whether these sequence variations affect antibiotic susceptibility or resistance development. To address this question, we have investigated the drug binding pockets of aminoglycosides and macrolides/ketolides. The binding site of aminoglycosides is located within helix 44 of the 16S rRNA (A site); macrolides/ketolides bind to domain V of the 23S rRNA (peptidyltransferase center). We have used mutagenesis of rRNA sequences in Mycobacterium smegmatis ribosomes to reconstruct the different bacterial drug binding sites and to study the effects of rRNA sequence variations on drug activity. Our results provide a rationale for differences in species-specific drug susceptibility patterns and species-specific resistance phenotypes associated with mutational alterations in the drug binding pocket.

EMBO J 30 14, 2779–2792 (2011); published online June142011 [PMC free article] [PubMed] Apoptosis is a type of programmed cell death (PCD) conserved among metazoans and crucial for the development and homeostasis of tissues in multicellular organisms. Lower unicellular eukaryotes like yeast would thus have no need for it and they apparently lack the key regulators of mitochondria-dependent apoptosis. In this issue, Buttner et al (2011) challenge this view, discovering Ybh3p, a yeast protein homologous to the proapoptotic ‘BH3-only' members of the mammalian Bcl-2 death regulators. Ybh3p kills yeast and mammalian cells impinging on inner mitochondrial membrane (IMM) proteins. Thus, mitochondria-dependent PCD emerges as an ancestral response of all eukaryotes, including unicellular ones. Ever since cells interacted to form multicellular organisms, a controlled and programmed form of cellular death also known as ‘apoptosis' arose, for the benefit of the organism and of its offspring. In its basic mechanisms, apoptosis is conserved in metazoans where it is required for elimination of unneeded cells during development or of cells that are potentially harmful to developed organs, without eliciting inflammation, like necrosis does (Danial and Korsmeyer, 2004). The need of such a process for unicellular organisms is debatable: on one hand, yeast could benefit from the targeted removal of aged cells from their colony, thereby preserving nutrients and hindering genetic conservatism; on the other, the evidence for yeast PCD is controversial (Mazzoni and Falcone, 2008). In metazoans, the hallmark event of apoptosis is the activation of caspases, proteases that execute death by degrading cytoskeleton, DNA and other cellular components. Caspases can be directly activated by extrinsic stimuli that engage the ‘death receptors' in the plasma membrane, or by a variety of intrinsic stimuli converging on gateways that control and amplify the signal (Danial and Korsmeyer, 2004). This intrinsic pathway must be tightly regulated to avoid unwanted death and simultaneously to efficiently trigger caspase activation when needed. These key regulators belong to the family of Bcl-2 proteins. The founders of this family are the antiapoptotic proteins Bcl-2 and Bcl-xL, bearing four Bcl-2 homology (BH) domains (BH1–4). Bcl-2 and Bcl-xL are mainly located in the outer mitochondrial membrane (OMM) where they regulate the permeabilization of the organelle followed by the release of proapoptotic factors like cytochrome c from the mitochondrial intermembrane space. Once in the cytosol, cytochrome c triggers the activation of caspases by forming a multimolecular complex with the adaptor protein Apaf-1 and pro-caspase 9 (Wasilewski and Scorrano, 2009). Key mediators of OMM permeabilization are the ‘multidomain proapoptotics' Bax and Bak that harbour BH domains 1–3. Activation of Bax and Bak occurs directly in the OMM by another subset of proapoptotic Bcl-2 family members, known as ‘BH3-only' proteins that possess only the BH3 domain. In turn, the antiapoptotic Bcl-2-like molecules regulate the whole process by sequestering ‘BH3-only' proteins (Wasilewski and Scorrano, 2009). Whether yeast can undergo apoptosis has been a matter of intense debate. Yeast cells lack ‘classical' mammalian-like caspases. They do, however, express Yca1p, which belongs to the family of metacaspases found throughout the plant and fungi kingdoms (Madeo et al, 2002). Yca1p is activated following toxic (oxidative) as well as physiological stress (low concentrations of mating pheromone, chronological lifespan), and greatly influences yeast death although its targets or mode of activation remain unknown at present. For example, the role of OMM permeabilization in yeast apoptosis is controversial (Mazzoni and Falcone, 2008), especially since yeast lack Bcl-2-like proteins. Similarities between the death pathways in metazoans and yeast have, however, also emerged. For example, mitochondrial fragmentation, a hallmark of mammalian apoptosis (Wasilewski and Scorrano, 2009), occurs also in yeast and accordingly physiological death is reduced in strains lacking the key fission protein Dnm1p (Fannjiang et al, 2004). The emerging consensus is that physiological and morphological changes of mitochondria are key for yeast death, but how these organelles could be recruited in the apoptotic cascade in the absence of proximal BH3-only-like sensors of cellular damage remained unclear. In the current issue of The EMBO Journal, Madeo and colleagues close this gap, by discovering Ybh3p, a proapoptotic yeast BH3-only protein. Ybh3p is a cytoplasmic protein whose overexpression facilitates yeast apoptosis induced by a variety of stimuli, whereas its ablation inhibits death and increases replicative lifespan. In analogy to certain mammalian BH3-only proteins, during apoptosis Ybh3p translocates to mitochondria, triggering their depolarization and cytochrome c release. In this respect, Ybh3p resembles more mammalian Bax that can autonomously permeabilize mitochondria, whereas BH3-only proteins require Bax and Bak to release cytochrome c (Wei et al, 2001), but not to remodel the IMM (Scorrano et al, 2002). Ybh3p can permeabilize murine mitochondria and kill a human lung cancer cell line like a typical mammalian BH3-only protein. Taken together, these data suggest that perhaps BH3-only proteins are the prototypes of the Bcl-2 family regulators of cell death. Antiapoptotic as well as multidomain members could have emerged later in evolution to tighten up the control on mitochondrial damage and apoptosis. Madeo and colleagues identify two mitochondrial interactors of Ybh3p essential for its killing action: Mir1p and Cor1p, the yeast orthologues of the mitochondrial phosphate carrier and of a core subunit of the ubiquinol-cytochrome c reductase (complex III). The link between these two IMM proteins and Ybh3p suggests that perhaps the most ancestral function of the BH3-like proteins is to trigger changes in function and/or topology of the IMM. In this respect, it would be interesting to explore whether Ybh3p and the fission protein Dnm1p participate in the same pathway of yeast death. Also, it would be worth studying the role of Mir1p and Cor1p in mitochondrial Ybh3p translocation. Notably, the mechanism of killing by Ybh3p seems somehow conserved, since the ablation of the orthologues of Mir1p and Cor1p can blunt apoptosis in human osteosarcoma cells, further pointing to a role for the IMM in mammalian apoptosis (Figure 1). Figure 1 The role of mitochondria in yeast and mammalian apoptosis. The cartoon depicts the mitochondrial involvement in the amplification of apoptosis in yeast and mammals. In response to BH3-only proteins, the multidomains Bax and Bak permeabilize mammalian ... Taken together, the results by Madeo and colleagues unveil the existence of an ancestral, proapoptotic, BH3-only gene in lower eukaryotes that later in evolution maybe replicated and diversified into multiple genes with one or more BH3 domains to serve specialized functions and to fine-tune death. In this respect, the striking genetic interaction between Ybh3p and IMM components suggests that OMM permeabilization could have emerged later in evolution. Changes in function and morphology of the IMM that are also observed in mammalian cells could then represent the remnant of the ancestral programme of death, which has subsequently been ‘coated' by evolution with an outer membrane to provide a further layer of control to death signalling.

14 páginas, 10 figuras. PMID:21984814[PubMed], The apoptotic protease activating factor 1 (Apaf1) is the main component of the apoptosome, and a crucial factor in the mitochondria-dependent death pathway. Here we show that Apaf1 plays a role in regulating centrosome maturation. By analyzing Apaf1-depleted cells, we have found that Apaf1 loss induces centrosome defects that impair centrosomal microtubule nucleation and cytoskeleton organization. This, in turn, affects several cellular processes such as mitotic spindle formation, cell migration and mitochondrial network regulation. As a consequence, Apaf1-depleted cells are more fragile and have a lower threshold to stress than wild-type cells. In fact, we found that they exhibit low Bcl-2 and Bcl-X(L) expression and, under apoptotic treatment, rapidly release cytochrome c. We also show that Apaf1 acts by regulating the recruitment of HCA66, with which it interacts, to the centrosome. This function of Apaf1 is carried out during the cell life and is not related to its apoptotic role. Therefore, Apaf1 might also be considered a pro-survival molecule, whose absence impairs cell performance and causes a higher responsiveness to stressful conditions, This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC) [grant no. IG10081], the Telethon Foundation [grant no. S99038TELC], the Health and the Italian Ministry of University and Research [grant nos RF07.96M2, 020903305]. D. De Zio is a FILAS-Regione Lazio fellow.