Az autofág gének szerepe az öregedési folyamat szabályozásában a fonálféreg Caenorhabditis elegansban = Coordinated regulation of ageing by autophagy genes in the nematode Caenorhabditis elegans

Meghatároztuk az élesztő autofág gének nematóda ortológjait. Számos C. elegans autofág gén funkcióvesztéses mutáns alléljét izoláltuk és/vagy specifikus RNS intereferencia klónját állítottuk elő. Autofágia defektív C. elegans törzseket genetikailag jellemeztük. Három C. elegans autofág gén esetében transzlációs fúziós riporter konstrukciót hoztunk létre és jellemeztük a gének expressziós mintázatát. Megállapítottuk, hogy az autofágia deficiens nematódák rövidebb ideig élnek a vad típusnál. Ezek az állatok gyorsabban halmoztak fel öregedési pigmenteket és hamarabb váltak paralizálttá, mint a vad típusú kontroll állatok. Autofágia hiányában tehát az állatok gyorsabban öregednek. Ezzel összhangban az inzulin/IGF-1 és TOR kináz útvonal deficiens mutáns fonalférgek, a csökkent mitokondriális respirációjú mutánsok és a kalorikusan csökkent tápanyagfelvételű mutáns állatok hosszú élettartamát az autofágia blokkolása szuppresszálta (az autofág mutációk episztatikusan hatottak). Az élethosszt szabályozó genetikai útvonalak tehát az autofág génkaszkádon konvergálódnak (az autofágia az öregedési folyamat központi szabályozó mechanizmusa). Kimutattuk, hogy az autofág gének és az apoptotikus génkaszkád redundánsan hatnak az embrionális fejlődés szabályozásában. Végül meghatároztunk egy myotubularin-szerű foszfatázt, amely negatívan szabályozza az autofágiát: ennek gátlásával az autofág folyamatot hiperaktiváltuk, és ez növelte az élettartamot és neuroprotektív hatású volt. | We identified the nematode orthologs of yeast autophagy genes. We then isolated loss-of-function mutations in some of these worm autophagy genes or generated their specific RNA interference clones. We performed a genetic analysis of autophagy deficient nematode strains. In the case of three autophagy genes, we also generated translational fusion reporter constructs to determine their developmental expression pattern. Nematodes defective for autophagy live significantly shorter than the wild-type. These mutants accumulate age pigments (lipofuscin) faster and become paralyzed earlier than normal animals. Thus, autophagy deficient nematodes are progeric as they exhibit an accelerated rate at which the cells and tissues age. Consistently, autophagy genes are required for lifespan extension in insulin/IGF-1 and TOR signaling mutants, in nematodes with decreased mitochondrial respiration and in calorically restricted worms; the corresponding double mutants are short-lived (autophagy mutations are epistatic to longevity mutations). Together, we can conclude that autophagy act as a central regulatory mechanism of animal aging. We also revealed that autophagy genes function redundantly with the apoptotic gene cascade to control embryonic development. Finally, we determined a myotubularin-like phosphatase that is able to downregulate the autophagic process. Inhibition of this phosphatase was capable of extending lifespan and protecting against neuronal damage

The maintenance of sex in bacteria is ensured by its potential to reload genes

Why sex is maintained in nature is a fundamental question in biology. Natural genetic transformation (NGT) is a sexual process by which bacteria actively take up exogenous DNA and use it to replace homologous chromosomal sequences. As it has been demonstrated, the role of NGT in repairing deleterious mutations under constant selection is insufficient for its survival, and the lack of other viable explanations have left no alternative except that DNA uptake provides nucleotides for food. Here we develop a novel simulation approach for the long-term dynamics of genome organization (involving the loss and acquisition of genes) in a bacterial species consisting of a large number of spatially distinct populations subject to independently fluctuating ecological conditions. Our results show that in the presence of weak inter-population migration NGT is able to subsist as a mechanism to reload locally lost, intermittently selected genes from the collective gene pool of the species through DNA uptake from migrants. Reloading genes and combining them with those in locally adapted genomes allow individual cells to re-adapt faster to environmental changes. The machinery of transformation survives under a wide range of model parameters readily encompassing real-world biological conditions. These findings imply that the primary role of NGT is not to serve the cell with food, but to provide homologous sequences for restoring genes that have disappeared from or become degraded in the local population.Comment: 16 pages with 3 color figures. Manuscript accepted for publication in Genetics (www.genetics.org

Autophagy in zebrafish

From a hitherto underappreciated phenomenon, autophagy has become one of the most intensively studied cellular processes in recent years. Its role in cellular homeostasis, development and disease is supported by a fast growing body of evidence. Surprisingly, only a small fraction of new observations regarding the physiological functions of cellular "self-digestion" comes from zebrafish, one of the most popular vertebrate model organisms. Here we review the existing information about autophagy reporter lines, genetic knock-down assays and small molecular reagents that have been tested in this system. As we argue, some of these tools have to be used carefully due to possible pleiotropic effects. However, when applied rigorously, in combination with novel mutant strains and genome editing techniques, they could also transform zebrafish into an important animal model of autophagy research

The genome loading model for the origin and maintenance of sex in eukaryotes

Understanding why sexual reproduction—which involves syngamy (union of gametes) and meiosis—emerged and how it has subsisted for millions of years remains a fundamental problem in biology. Considered as the essence of sex, meiotic recombination is initiated by a DNA double-strand break (DSB) that forms on one of the pairing homologous chromosomes. This DNA lesion is subsequently repaired by gene conversion, the non-reciprocal transfer of genetic information from the intact homolog. A major issue is which of the pairing homologs undergoes DSB formation. Accumulating evidence shows that chromosomal sites where the pairing homologs locally differ in size, i.e., are heterozygous for an insertion or deletion, often display disparity in gene conversion. Biased conversion tends to duplicate insertions and lose deletions. This suggests that DSB is preferentially formed on the “shorter” homologous region, which thereby acts as the recipient for DNA transfer. Thus, sex primarily functions as a genome (re)loading mechanism. It ensures the restoration of formerly lost DNA sequences (deletions) and allows the efficient copying and, mainly in eukaryotes, subsequent spreading of newly emerged sequences (insertions) arising initially in an individual genome, even if they confer no advantage to the host. In this way, sex simultaneously repairs deletions and increases genetic variability underlying adaptation. The model explains a remarkable increase in DNA content during the evolution of eukaryotic genomes

The C. elegans Hox gene ceh-13 regulates cell migration and fusion in a non-colinear way. Implications for the early evolution of Hox clusters

Background: Hox genes play a central role in axial patterning during animal development. They are clustered in the genome and specify cell fate in sequential domains along the anteroposterior (A-P) body axis in a conserved order that is co-linear with their relative genomic position. In the soil worm Caenorhabditis elegans, this striking rule of co-linearity is broken by the anterior Hox gene ceh-13, which is located between the two middle Hox paralogs, lin-39 and mab-5, within the loosely organized nematode Hox cluster. Despite its evolutionary and developmental significance, the functional consequence of this unusual genomic organization remains unresolved.Results: In this study we have investigated the role of ceh-13 in different developmental processes, and found that its expression and function are not restricted to the anterior body part. We show that ceh-13 affects cell migration and fusion as well as tissue patterning in the middle and posterior body regions too. These data reveal novel roles for ceh-13 in developmental processes known to be under the control of middle Hox paralogs. Consistently, enhanced activity of lin-39 and mab-5 can suppress developmental arrest and morphologic malformation in ceh-13 deficient animals.Conclusion: Our findings presented here show that, unlike other Hox genes in C. elegans which display region-specific accumulation and function along the A-P axis, the expression and functional domain of the anterior Hox paralog ceh-13 extends beyond the anterior region of the worm. Furthermore, ceh-13 and the middle Hox paralogs share several developmental functions. Together, these results suggest the emergence of the middle-group Hox genes from a ceh-13-like primordial Hox ancestor

Heat shock factor-1 intertwines insulin/IGF-1, TGF-beta and cGMP signaling to control development and aging.

ABSTRACT: BACKGROUND: Temperature affects virtually all cellular processes. A quick increase in temperature challenges the cells to undergo a heat shock response to maintain cellular homeostasis. Heat shock factor-1 (HSF-1) functions as a major player in this response as it activates the transcription of genes coding for molecular chaperones (also called heat shock proteins) that maintain structural integrity of proteins. However, the mechanisms by which HSF-1 adjusts fundamental cellular processes such as growth, proliferation, differentiation and aging to the ambient temperature remain largely unknown. RESULTS: We demonstrate here that in Caenorhabditis elegans HSF-1 represses the expression of daf-7 encoding a TGF-beta (transforming growth factor-beta) ligand, to induce young larvae to enter the dauer stage, a developmentally arrested, non-feeding, highly stress-resistant, long-lived larval form triggered by crowding and starvation. Under favorable conditions, HSF-1 is inhibited by crowding pheromone-sensitive guanylate cyclase/cGMP (cyclic guanosine monophosphate) and systemic nutrient-sensing insulin/IGF-1 (insulin-like growth factor-1) signaling; loss of HSF-1 activity allows DAF-7 to promote reproductive growth. Thus, HSF-1 interconnects the insulin/IGF-1, TGF-beta and cGMP neuroendocrine systems to control development and longevity in response to diverse environmental stimuli. Furthermore, HSF-1 upregulates another TGF-beta pathway-interacting gene, daf-9/cytochrome P450, thereby fine-tuning the decision between normal growth and dauer formation. CONCLUSION: Together, these results provide mechanistic insight into how temperature, nutrient availability and population density coordinately influence development, lifespan, behavior and stress response through HSF-1

Az autofagocitózis transzkripcionális szabályozása C. elegansban = Transcriptional control of autophagy in C. elegans

C. elegansban genetikai és farmakológiai hatások masszív sejtpusztulást váltanak ki mind apoptotikus, mind autofág jellegzetességekkel. Ezek redundánsan vannak jelen és biztosítják a normális fejlődést. A CES-2-szerű leucin-zipper (bZip) transzkripciós faktor, az ATF-2, az apoptotikus sejthalál központi útvonalának upstream modulátora direkt módon regulálja legalább két autofág gén (bec-1/ATG6 és lgg-1/ATG8) expresszióját. A két sejthalál mechanizmusnak vannak közös transzkripciós elemei. Azonosítottunk négy új metazoa specifikus autofágia gént. Az epg-2,-3,-4, és-5 genetikai analízise felderítette, hogy ezek az autofág útvonal diszkrét genetikai lépéseit jelentik. Az epg-2 egy coiled-coil proteint kódol amely a specifikus kargo felismerésben szerepel. Az EPG-3/VMP1, EPG-4/EI24, és az EPG-5/mEPG5 emlős homológjai eszenciálisak az éhezési autofágiához. A VMP1 az autofagoszóma képződést szabályozza az omegaszómák élettartama révén. Az epg-6 egy eszenciális autofágia gén amely egy PtdIns(3)P-kötő WD-40 repeat proteint kódol. Az EPG-6 direkt módon interakcióba lép az ATG-2-vel. Az epg-6 és az atg-2 regulálja az omegasomák hozzájárulását az autofagoszóma képződéshez. Lf mutációik korai autofág struktúrák felhalmozódásához vezetnek. Egy másik WD40 repeat PtdIns(3)P effektor, az ATG-18, eltérő szerepet játszik az autofagoszóma képződésében. Az Unc-51/Atg-1 komplexum, az EPG-8/Atg14, és a lipidált LGG-1 protein aggregátumokhoz való kötődése szükséges az omegaszóma képződéshez. | We have shown in C. elegans, that various genetic and pharmacologic interventions trigger massive cell death response that has both autophagic and apoptotic features. The two degradation processes are also redundantly required for normal development and viability in this organism. Furthermore, the CES-2-like basic region leucine-zipper (bZip) transcription factor ATF-2, an upstream modulator of the core apoptotic cell death pathway, is able to directly regulate the expression of at least two key autophagy-related genes, bec-1/ATG6 and lgg-1/ATG8. Thus, the two cell death mechanisms share a common method of transcriptional regulation. We identified four metazoan-specific autophagy genes, named epg-2, -3, -4, and -5. Genetic analysis revealed that epg-2, -3, -4, and -5 define discrete genetic steps of the autophagy pathway. epg-2 encodes a coiled-coil protein that functions in specific autophagic cargo recognition. Mammalian homologs of EPG-3/VMP1, EPG-4/EI24, and EPG-5/mEPG5 are essential for starvation-induced autophagy. VMP1 regulates autophagosome formation by controlling the duration of omegasomes. C. elegans epg-6 encodes a WD40 repeat-containing protein with PtdIns(3)P-binding activity. EPG-6 directly interacts with ATG-2. epg-6 and atg-2 regulate progression of omegasomes to autophagosomes, and their loss of function causes accumulation of enlarged early autophagic structures

Systems-Level Feedbacks of NRF2 Controlling Autophagy upon Oxidative Stress Response

Although the primary role of autophagy-dependent cellular self-eating is cytoprotective upon various stress events (such as starvation, oxidative stress, and high temperatures), sustained autophagy might lead to cell death. A transcription factor called NRF2 (nuclear factor erythroid-related factor 2) seems to be essential in maintaining cellular homeostasis in the presence of either reactive oxygen or nitrogen species generated by internal metabolism or external exposure. Accumulating experimental evidence reveals that oxidative stress also influences the balance of the 5′ AMP-activated protein kinase (AMPK)/rapamycin (mammalian kinase target of rapamycin or mTOR) signaling pathway, thereby inducing autophagy. Based on computational modeling here we propose that the regulatory triangle of AMPK, NRF2 and mTOR guaranties a precise oxidative stress response mechanism comprising of autophagy. We suggest that under conditions of oxidative stress, AMPK is crucial for autophagy induction via mTOR down-regulation, while NRF2 fine-tunes the process of autophagy according to the level of oxidative stress. We claim that the cellular oxidative stress response mechanism achieves an incoherently amplified negative feedback loop involving NRF2, mTOR and AMPK. The mTOR-NRF2 double negative feedback generates bistability, supporting the proper separation of two alternative steady states, called autophagy-dependent survival (at low stress) and cell death (at high stress). In addition, an AMPK-mTOR-NRF2 negative feedback loop suggests an oscillatory characteristic of autophagy upon prolonged intermediate levels of oxidative stress, resulting in new rounds of autophagy stimulation until the stress events cannot be dissolved. Our results indicate that AMPK-, NRF2- and mTOR-controlled autophagy induction provides a dynamic adaptation to altering environmental conditions, assuming their new frontier in biomedicine

Starvation-response may not involve Atg1-dependent autophagy induction in non-unikont parasites

Autophagy, the lysosome-mediated self-degradation process, is implicated in survival during starvation in yeast, Dictyostelium and animals. In these eukaryotic taxa (collectively called Unikonts), autophagy is induced primarily through the Atg1/ULK1 complex in response to nutrient depletion. Autophagy has also been well-studied in non-unikont parasites, such as Trypanosoma and Plasmodium, and found important in their life-cycle transitions. However, how autophagy is induced in non-unikonts remains largely unrevealed. Using a bioinformatics approach, we examined the presence of Atg1 and of its complex in the genomes of 40 non-unikonts. We found that these genomes do not encode typical Atg1 proteins: BLAST and HMMER queries matched only with the kinase domain of Atg1, while other segments responsible for regulation and protein-binding were missing. Non-unikonts also lacked other components of the Atg1-inducing complex. Orthologs of an alternative autophagy inducer, Atg6 were found only in the half of the species, indicating that the other half may possess other inducing mechanisms. As key autophagy genes have differential expression patterns during life-cycle, we raise the possibility that autophagy in these protists is induced mainly at the post-transcriptional level. Understanding Atg1-independent autophagy induction mechanisms in these parasites may lead to novel pharmacological interventions, not affecting human Atg1-dependent autophagy

Rab2 is a potent new target for enhancing autophagy in the treatment of Parkinson's disease

Macroautophagy is a lysosomal-dependent degradational pathway of eukaryotic cells, during which toxic, unnecessary, and damaged intracellular components are broken down. Autophagic activity declines with age, and this change could contribute to the accumulation of intracellular damage at advanced ages, causing cells to lose their functionality and vitality. This could be particularly problematic in post-mitotic cells include neurons, the mass destruction of which leads to different neurodegenerative diseases. We aim to discover new regulation points where autophagy could be specifically activated, and test these potential drug targets in Drosophila neurodegenerative disease models. One possible way to activate autophagy is through the enhancement of autophagosome-lysosome fusion to become autolysosome. This fusion is regulated by HOPS (homotypic fusion and protein sorting) and SNARE (Snap receptor) complexes. The HOPS complex forms a bridge between lysosome and autophagosome with the assistance of small GTPase Rab (Ras-associated binding) proteins. Thus, Rab proteins are essential for autolysosome maturation, and among Rab proteins, Rab2 is required for the degradation of autophagic cargo. Our results revealed that GTP-locked (constitutively active) Rab2 (Rab2 CA) expression reduces the levels of the autophagic substrate p62/Ref2P in dopaminergic neurons, and improved the climbing ability of animals during aging. The expression of Rab2 CA also increased lifespan in a Parkinson’s disease model (human mutant alpha-synuclein [A53T] overexpressed animals). In these animals, Rab2 CA expression significantly increased autophagic degradation as compared to control. These results may reveal a new, more specific drug target for autophagic activation treating today’s incurable neurodegenerative disease