Mitochondria and lifespan extension in Saccharomyces cerevisiae: the longevity mutations sch9Δ and rei1Δ contribute to mitochondrial DNA stability

L’invecchiamento cellulare e la progressione di patologie umane associate all’invecchiamento sono accompagnati da un progressivo declino della funzionalità mitocondriale, con riduzione dell’attività respiratoria, alterazioni morfologiche e accumulo di mutazioni sul DNA mitocondriale. I mitocondri sono coinvolti nella regolazione di diversi processi cellulari: oltre al loro ruolo nella produzione di ATP, i mitocondri partecipano alla trasduzione di svariati segnali intracellulari, avviano il processo apoptotico e rappresentano il sito primario di produzione di specie reattive dell’ossigeno (ROS), molecole dannose che promuovono la disfunzione mitocondriale e l’invecchiamento cellulare. Tuttavia recenti evidenze suggeriscono che i ROS agiscano anche da segnalatori molecolari nella regolazione di processi come la crescita cellulare, l’immunità e la longevità. E’ ormai nota una lunga lista di geni la cui delezione o mutazione porta ad un incremento della chronological lifespan (CLS): mutazioni che riducono l’attività dei pathway che integrano segnali nutrizionali TOR/SCH9 e RAS/PKA, aumentano la lifespan in diversi organismi modello. Per studiare la relazione tra il mantenimento di un’efficiente funzionalità mitocondriale e l’invecchiamento abbiamo analizzato nel lievito S. cerevisiae alcuni mutanti longevi con l’obiettivo di valutare se l’aumentata CLS correlasse o meno con una maggiore stabilità del genoma mitocondriale. Solo due dei mutanti longevi analizzati, sch9∆ e rei1∆, hanno mostrato un minore accumulo di mutazioni estese, ma non puntiformi, del mtDNA. Questo risultato suggerisce che l’accumulo di mutazioni estese sul mtDNA possano avere un ruolo primario nell’avvio del processo di senescenza; infatti grosse delezioni/inserzioni sul mtDNA comportano inevitabilmente la perdita di alcune funzioni mitocondriali cruciali per il mantenimento dell’attività respiratoria ed è ormai dimostrato come la presenza di una buona attività respiratoria sia indispensabile per sostenere un incremento del lifespan. Inoltre l’evidenza che non tutti i mutanti longevi mostrano una maggiore stabilità del mtDNA indica che la ridotta mutabilità sia uno dei fattori coinvolti nella regolazione della lifespan ma non il fattore chiave. Entrambi i mutanti longevi mostrano inoltre un incremento dell’attività respiratoria rispetto ad un ceppo parentale ma agiscono attraverso meccanismi di segnalazione differenti. La delezione di SCH9 porta ad un aumento della quantità di ROS durante le fasi precoci di crescita con lo scopo di promuovere un segnale adattativo che stimola l’estensione del lifespan e riduca il danno ossidativo delle cellule in fase stazionaria, attraverso l’attivazione di un programma di risposta allo stress mediato dall’overespressione di Sod2p. REI1 appartiene al gruppo dei geni RiBi e l’aumento della stabilità del mtDNA in seguito alla sua delezione è stata osservata anche in altri geni coinvolti nella biogenesi ribosomiale. Inoltre abbiamo dimostrato che l’aumento dell’attività respiratoria e della stabilità del DNA mitocondriale nel mutante rei1∆ dipende da un meccanismo di segnalazione differente rispetto a quello osservato nel mutante sch9∆, che comporta un effetto diretto sulla stabilizzazione del DNA mitocondriale attraverso l’overespressione di alcune componenti del nucleoide.Age related mitochondrial decline has been reported in different organisms, and is accompanied by a reduction in mitochondrial respiratory activity, morphological alterations and progressive accumulation of mutations. Besides their role in ATP production, mitochondria take also part in the transduction of intracellular signals, in cell death and they are the main sites of production of oxygen reactive species (ROS), detrimental molecules that promote mitochondrial dysfunction and ageing. Recent evidence showed that ROS may act also as signaling molecules to regulate cell growth, immune response and lifespan extension and that mutations that reduce the activity of the nutritional signaling pathways TOR/SCH9 and RAS/PKA induce an increase in chronological lifespan (CLS) extension in different model organisms, redirecting cells towards a respiratory metabolism and promoting stress resistance. In order to assess the relationship between mitochondrial function and ageing we investigated whether the increased CLS of some well-known longevity mutants correlated with an increase in mitochondrial DNA stability. Among the different longevity mutations analyzed only two, sch9∆ and rei1∆, accumulate large-scale rearrangements on mtDNA at lower rate compared to the parental strain, while there is no difference in the accumulation of point-mutations. This result suggests that large-scale rearrangements on mtDNA may have a primary role in the senescence process as mtDNA integrity is strictly important to maintain proper mitochondrial function and respiratory activity, a key determinant of lifespan extension. Moreover, the evidence that only two of the eight longevity mutations analyzed were able to increase mitochondrial DNA stability indicates that maintenance of mtDNA is only one of the actors involved in the regulation of the ageing process and that different mechanisms cooperate to regulate lifespan extension in yeast. Sch9∆ and rei1∆ longevity mutants show high rate of respiratory activity accompanied by no significant difference in mtDNA amount, suggesting that the increased respiratory rate is imputable to an increased OXPHOS complexes content. We have been able to identify two different mechanisms through which the two longevity mutations promote lifespan extension and mtDNA stability. Deletion of SCH9 leads to an increase of ROS production early during growth to promote an adaptive signal that stimulates lifespan extension and reduces oxidative damage in stationary cells, activating a stress response program mediated by Sod2p overexpression. REI1 belongs to the RiBi genes regulon and the increase in mitochondrial DNA stability upon REI1 deletion was observed also in other genes involved in ribosome biogenesis. In addition, we showed that reduced mtDNA rearrangements and increased respiratory activity in rei1∆ longevity mutant depend on a mechanism completely different from the adaptive response observed in sch9∆ mutant, and appear to rely on a direct stabilization of mitochondrial DNA through overexpression of nucleoid components

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3′-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T≥4 stretch within 50 bp of 3′-flanking region. In vitro analysis of tDNAs with a distanced T≥4 revealed the existence of non-canonical terminators resembling degenerate T≥5 elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3′ trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3′-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3′-trailer sequences with the potential to contribute novel functional ncRNA

EXOSC8 mutations alter mRNA metabolism and cause hypomyelination with spinal muscular atrophy and cerebellar hypoplasia.

The exosome is a multi-protein complex, required for the degradation of AU-rich element (ARE) containing messenger RNAs (mRNAs). EXOSC8 is an essential protein of the exosome core, as its depletion causes a severe growth defect in yeast. Here we show that homozygous missense mutations in EXOSC8 cause progressive and lethal neurological disease in 22 infants from three independent pedigrees. Affected individuals have cerebellar and corpus callosum hypoplasia, abnormal myelination of the central nervous system or spinal motor neuron disease. Experimental downregulation of EXOSC8 in human oligodendroglia cells and in zebrafish induce a specific increase in ARE mRNAs encoding myelin proteins, showing that the imbalanced supply of myelin proteins causes the disruption of myelin, and explaining the clinical presentation. These findings show the central role of the exosomal pathway in neurodegenerative disease

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3′-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T≥4 stretch within 50 bp of 3′-flanking region. In vitro analysis of tDNAs with a distanced T≥4 revealed the existence of non-canonical terminators resembling degenerate T≥5 elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3′ trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3′-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3′-trailer sequences with the potential to contribute novel functional ncRNAs

Structural deciphering of the NG2/CSPG4 proteoglycan multifunctionality

The chondroitin sulfate proteoglycan 4 (CSPG4) gene encodes a transmembrane proteoglycan (PG) constituting the largest and most structurally complex macromolecule of the human surfaceome. Its transcript shows an extensive evolutionary conservation and, due to the elaborated intracellular processing of the translated protein, it generates an array of glycoforms with the potential to exert variant-specific functions. CSPG4-mediated molecular events are articulated through the interaction with more than 40 putative ligands and the concurrent involvement of the ectodomain and cytoplasmic tail. Alternating inside-out and outside-in signal transductions may thereby be elicited through a tight functional connection of the PG with the cytoskeleton and its regulators. The potential of CSPG4 to influence both types of signaling mechanisms is also asserted by its lateral mobility along the plasma membrane and its intersection with microdomain-restricted internalization and endocytic trafficking. Owing to the multitude of molecular interplays that CSPG4 may engage, and thanks to a differential phosphorylation of its intracellular domain accounted by crosstalking signaling pathways, the PG stands out for its unique capability to affect numerous cellular phenomena, including those purporting pathologic conditions. We discuss here the progresses made in advancing our understanding about the structural-functional bases for the ability of CSPG4 to widely impact on cell behavior, such as to highlight how its multivalency may be exploited to interfere with disease progression.Tamburini, E., Dallatomasina, A., Quartararo, J., Cortelazzi, B., Mangieri, D., Lazzaretti, M., Perris, R. Structural deciphering of the NG2/CSPG4 proteoglycan multifunctionality

The impairment of HCCS leads to MLS syndrome by activating a non-canonical cell death pathway in the brain and eyes.

Mitochondrial-dependent (intrinsic) programmed cell death (PCD) is an essential homoeostatic mechanism that selects bioenergetically proficient cells suitable for tissue/organ development. However, the link between mitochondrial dysfunction, intrinsic apoptosis and developmental anomalies has not been demonstrated to date. Now we provide the evidence that non-canonical mitochondrial-dependent apoptosis explains the phenotype of microphthalmia with linear skin lesions (MLS), an X-linked developmental disorder caused by mutations in the holo-cytochrome c-type synthase (HCCS) gene. By taking advantage of a medaka model that recapitulates the MLS phenotype we demonstrate that downregulation of hccs, an essential player of the mitochondrial respiratory chain (MRC), causes increased cell death via an apoptosome-independent caspase-9 activation in brain and eyes. We also show that the unconventional activation of caspase-9 occurs in the mitochondria and is triggered by MRC impairment and overproduction of reactive oxygen species (ROS). We thus propose that HCCS plays a key role in central nervous system (CNS) development by modulating a novel non-canonical start-up of cell death and provide the first experimental evidence for a mechanistic link between mitochondrial dysfunction, intrinsic apoptosis and developmental disorders

EXOSC8 mutations alter mRNA metabolism and cause hypomyelination with spinal muscular atrophy and cerebellar hypoplasia

The exosome is a multi-protein complex, required for the degradation of AU-rich element (ARE) containing messenger RNAs (mRNAs). EXOSC8 is an essential protein of the exosome core, as its depletion causes a severe growth defect in yeast. Here we show that homozygous missense mutations in EXOSC8 cause progressive and lethal neurological disease in 22 infants from three independent pedigrees. Affected individuals have cerebellar and corpus callosum hypoplasia, abnormal myelination of the central nervous system or spinal motor neuron disease. Experimental downregulation of EXOSC8 in human oligodendroglia cells and in zebrafish induce a specific increase in ARE mRNAs encoding myelin proteins, showing that the imbalanced supply of myelin proteins causes the disruption of myelin, and explaining the clinical presentation. These findings show the central role of the exosomal pathway in neurodegenerative disease