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1.
Nat Commun ; 12(1): 4795, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373460

RESUMO

Chemogenetic and optogenetic tools have transformed the field of neuroscience by facilitating the examination and manipulation of existing circuits. Yet, the field lacks tools that enable rational rewiring of circuits via the creation or modification of synaptic relationships. Here we report the development of HySyn, a system designed to reconnect neural circuits in vivo by reconstituting synthetic modulatory neurotransmission. We demonstrate that genetically targeted expression of the two HySyn components, a Hydra-derived neuropeptide and its receptor, creates de novo neuromodulatory transmission in a mammalian neuronal tissue culture model and functionally rewires a behavioral circuit in vivo in the nematode Caenorhabditis elegans. HySyn can interface with existing optogenetic, chemogenetic and pharmacological approaches to functionally probe synaptic transmission, dissect neuropeptide signaling, or achieve targeted modulation of specific neural circuits and behaviors.


Assuntos
Neurônios/fisiologia , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia , Animais , Comportamento Animal/fisiologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Cálcio , Expressão Gênica , Técnicas Genéticas , Hydra/genética , Hydra/fisiologia , Vias Neurais/fisiologia , Neuropeptídeos , Optogenética , Transdução de Sinais
2.
FEBS J ; 288(15): 4435-4438, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34342148

RESUMO

In this special interview series, we profile members of The FEBS Journal editorial board to highlight their research focus and perspectives on the journal and future directions in their field. Brent Derry is Professor at the Department of Molecular Genetics of University of Toronto and Senior Scientist of the Developmental & Stem Cell Biology Program at The Hospital for Sick Children (Toronto, Canada). He has served as an editorial board member of The FEBS Journal since 2017.


Assuntos
Biologia Molecular/história , Animais , Caenorhabditis elegans/genética , Canadá , História do Século XX , História do Século XXI
3.
Molecules ; 26(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34443430

RESUMO

Parkinson's disease (PD) is a currently incurable neurodegenerative disorder characterized by the loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta and α-synuclein aggregation. Accumulated evidence indicates that the saponins, especially from ginseng, have neuroprotective effects against neurodegenerative disorders. Interestingly, saponin can also be found in marine organisms such as the sea cucumber, but little is known about its effect in neurodegenerative disease, including PD. In this study, we investigated the anti-Parkinson effects of frondoside A (FA) from Cucumaria frondosa and ginsenoside Rg3 (Rg3) from Panax notoginseng in C. elegans PD model. Both saponins were tested for toxicity and optimal concentration by food clearance assay and used to treat 6-OHDA-induced BZ555 and transgenic α-synuclein NL5901 strains in C. elegans. Treatment with FA and Rg3 significantly attenuated DAergic neurodegeneration induced by 6-OHDA in BZ555 strain, improved basal slowing rate, and prolonged lifespan in the 6-OHDA-induced wild-type strain with downregulation of the apoptosis mediators, egl-1 and ced-3, and upregulation of sod-3 and cat-2. Interestingly, only FA reduced α-synuclein aggregation, rescued lifespan in NL5901, and upregulated the protein degradation regulators, including ubh-4, hsf-1, hsp-16.1 and hsp-16.2. This study indicates that both FA and Rg3 possess beneficial effects in rescuing DAergic neurodegeneration in the 6-OHDA-induced C. elegans model through suppressing apoptosis mediators and stimulating antioxidant enzymes. In addition, FA could attenuate α-synuclein aggregation through the protein degradation process.


Assuntos
Caenorhabditis elegans/fisiologia , Ginsenosídeos/farmacologia , Glicosídeos/farmacologia , Doença de Parkinson/patologia , Triterpenos/farmacologia , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ginsenosídeos/química , Ginsenosídeos/toxicidade , Glicosídeos/química , Glicosídeos/toxicidade , Longevidade/efeitos dos fármacos , Degeneração Neural/complicações , Degeneração Neural/patologia , Oxidopamina , Doença de Parkinson/complicações , Proteólise/efeitos dos fármacos , Triterpenos/química , Triterpenos/toxicidade , alfa-Sinucleína/metabolismo
4.
Science ; 373(6558): 984-991, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34446600

RESUMO

Protein kinase activity must be precisely regulated, but how a cell governs hyperactive kinases remains unclear. In this study, we generated a constitutively active mitogen-activated protein kinase DYF-5 (DYF-5CA) in Caenorhabditis elegans that disrupted sensory cilia. Genetic suppressor screens identified that mutations of ADR-2, an RNA adenosine deaminase, rescued ciliary phenotypes of dyf-5CA We found that dyf-5CA animals abnormally transcribed antisense RNAs that pair with dyf-5CA messenger RNA (mRNA) to form double-stranded RNA, recruiting ADR-2 to edit the region ectopically. RNA editing impaired dyf-5CA mRNA splicing, and the resultant intron retentions blocked DYF-5CA protein translation and activated nonsense-mediated dyf-5CA mRNA decay. The kinase RNA editing requires kinase hyperactivity. The similar RNA editing-dependent feedback regulation restricted the other ciliary kinases NEKL-4/NEK10 and DYF-18/CCRK, which suggests a widespread mechanism that underlies kinase regulation.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Cílios/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Edição de RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Caenorhabditis elegans/genética , Núcleo Celular/metabolismo , Cílios/enzimologia , Ativação Enzimática , Fenótipo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA , Estabilidade de RNA , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Transcrição Genética
5.
6.
BMC Res Notes ; 14(1): 311, 2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34391474

RESUMO

OBJECTIVE: RNA-binding proteins (RBPs) are important regulators of gene expression that influence mRNA splicing, stability, localization, transport, and translational control. In particular, RBPs play an important role in neurons, which have a complex morphology. Previously, we showed that there are many RBPs that play a conserved role in dendrite development in Drosophila dendritic arborization neurons and Caenorhabditis elegans (C. elegans) PVD neurons including the cytoplasmic polyadenylation element binding proteins (CPEBs), Orb in Drosophila and CPB-3 in C. elegans, and the DEAD box RNA helicases, Me31B in Drosophila and CGH-1 in C. elegans. During these studies, we observed that fluorescently-labeled CPB-3 and CGH-1 localize to cytoplasmic particles that are motile, and our research aims to further characterize these RBP-containing particles in live neurons. RESULTS: Here we extend on previous work to show that CPB-3 and CGH-1 localize to motile particles within dendrites that move at a speed consistent with microtubule-based transport. This is consistent with a model in which CPB-3 and CGH-1 influence dendrite development through the transport and localization of their mRNA targets. Moreover, CPB-3 and CGH-1 rarely localize to the same particles suggesting that these RBPs function in discrete ribonucleoprotein particles (RNPs) that may regulate distinct mRNAs.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dendritos , RNA Nucleotidiltransferases , Proteínas de Ligação a RNA/genética , Células Receptoras Sensoriais/metabolismo
7.
Nutrients ; 13(7)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34371921

RESUMO

The study of different natural products can provide a wealth of bioactive compounds, and more interestingly, their combination can exert a new strategy for several neurodegenerative diseases with major public health importance, such as Alzheimer's disease (AD). Here, we investigated the synergistic neuroprotective effects of a mixed extract composed of docosahexaenoic acid, Ginkgo biloba, D-pinitol, and ursolic acid in several transgenic Caenorhabditis elegans (C. elegans) and a senescence-accelerated prone mice 8 (SAMP8) model. First, we found a significantly higher survival percentage in the C. elegans group treated with the natural product mixture compared to the single extract-treated groups. Likewise, we found a significantly increased lifespan in group of C. elegans treated with the natural product mixture compared to the other groups, suggesting synergistic effects. Remarkably, we determined a significant reduction in Aß plaque accumulation in the group of C. elegans treated with the natural product mixture compared to the other groups, confirming synergy. Finally, we demonstrated better cognitive performance in the group treated with the natural product mixture in both AD models (neuronal Aß C. elegans strain CL2355 and the SAMP8 mice model), confirming the molecular results and unraveling the synergist effects of this combination. Therefore, our results proved the potential of this new natural product mixture for AD therapeutic strategies.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Comportamento Animal/efeitos dos fármacos , Produtos Biológicos/farmacologia , Encéfalo/efeitos dos fármacos , Cognição/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Encéfalo/patologia , Caenorhabditis elegans/genética , Modelos Animais de Doenças , Longevidade , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Placa Amiloide
8.
Chemosphere ; 277: 130359, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34384190

RESUMO

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant that might induce disorders in fatty acid (FA) metabolism, but the underlying mechanisms remain unresolved. Caenorhabditis elegans (C. elegans) as a model organism can synthesize polyunsaturated FAs de novo via the polyunsaturated FA synthesis pathway. In this study, synchronized L1 C. elegans were exposed to 0, 0.01, 0.1, 0.5 and 1 µM PFOS for 72 h. Gas chromatography-mass spectrometry (GC-MS) was used to establish a sensitive and reliable analysis method for PFASs in exposed nematode, the instrument detection limits of nine fatty acid methyl esters examined ranged between 1.11 and 27.6 ng/mL, with satisfactory reproducibility (RSD < 10%) observed. Methyl pentadecanoate (C15:0) was used as an internal standard, the linearity of the calibration (0.1-10 µg/mL) nine FAs from the nematode were quantitatively analyzed. Comparing with the control group, PFOS exposure caused significantly decreased levels of C18:0 while significantly increased levels of C18:3n6. A decrease in the C18:3n6: C18:2n6 ratio was observed. Consistently, expression of the FA desaturation gene fat-3 was significantly down-regulated. These findings suggest that the FA disorder is associated with decrease in mRNA expression of Δ6-desaturase genes in C. elegans. Simultaneously, the disorders in FA metabolism were found to disrupt mitochondrial function with a reduction in ATP synthesis, as determined by the luciferase method. In summary, the results of the study provide insights into the adverse effects of PFOS on FA metabolism in living organisms.


Assuntos
Ácidos Alcanossulfônicos , Fluorcarbonetos , Ácidos Alcanossulfônicos/toxicidade , Animais , Caenorhabditis elegans/genética , Ácidos Graxos , Fluorcarbonetos/toxicidade , Cromatografia Gasosa-Espectrometria de Massas , Reprodutibilidade dos Testes
9.
BMC Genomics ; 22(1): 586, 2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344317

RESUMO

BACKGROUND: Circular RNAs (circRNAs) play diverse roles in different biological and physiological environments and are always expressed in a tissue-specific manner. Especially, circRNAs are enriched in the brain tissues of almost all investigated species, including humans, mice, Drosophila, etc. Although circRNAs were found in C. elegans, the neuron-specific circRNA data is not available yet. Exon-skipping is found to be correlated to circRNA formation, but the mechanisms that link them together are not clear. RESULTS: Here, through large-scale neuron isolation from the first larval (L1) stage of C. elegans followed by RNA sequencing with ribosomal RNA depletion, the neuronal circRNA data in C. elegans were obtained. Hundreds of novel circRNAs were annotated with high accuracy. circRNAs were highly expressed in the neurons of C. elegans and were positively correlated to the levels of their cognate linear mRNAs. Disruption of reverse complementary match (RCM) sequences in circRNA flanking introns effectively abolished circRNA formation. In the zip-2 gene, deletion of either upstream or downstream RCMs almost eliminated the production of both the circular and the skipped transcript. Interestingly, the 13-nt RCM in zip-2 is highly conserved across five nematode ortholog genes, which show conserved exon-skipping patterns. Finally, through in vivo one-by-one mutagenesis of all the splicing sites and branch points required for exon-skipping and back-splicing in the zip-2 gene, I showed that back-splicing still happened without exon-skipping, and vice versa. CONCLUSIONS: Through protocol optimization, total RNA obtained from sorted neurons is increased to hundreds of nanograms. circRNAs highly expressed in the neurons of C. elegans are more likely to be derived from genes also highly expressed in the neurons. RCMs are abundant in circRNA flanking introns, and RCM-deletion is an efficient way to knockout circRNAs. More importantly, these RCMs are not only required for back-splicing but also promote the skipping of exon(s) to be circularized. Finally, RCMs in circRNA flanking introns can directly promote both exon-skipping and back-splicing, providing a new explanation for the correlation between them.


Assuntos
Caenorhabditis elegans , RNA , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Éxons , Camundongos , RNA/metabolismo , Splicing de RNA , RNA Circular
10.
Nat Commun ; 12(1): 4802, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376665

RESUMO

During meiosis, gene expression is silenced in aberrantly unsynapsed chromatin and in heterogametic sex chromosomes. Initiation of sex chromosome silencing is disrupted in meiocytes with sex chromosome-autosome translocations. To determine whether this is due to aberrant synapsis or loss of continuity of sex chromosomes, we engineered Caenorhabditis elegans nematodes with non-translocated, bisected X chromosomes. In early meiocytes of mutant males and hermaphrodites, X segments are enriched with euchromatin assembly markers and active RNA polymerase II staining, indicating active transcription. Analysis of RNA-seq data showed that genes from the X chromosome are upregulated in gonads of mutant worms. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, our data indicate that unsynapsed X segments are transcribed. Therefore, our results suggest that sex chromosome chromatin has a unique character that facilitates its meiotic expression when its continuity is lost, regardless of whether or not it is synapsed.


Assuntos
Caenorhabditis elegans/genética , Inativação Gênica , Meiose/genética , Cromossomo X/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Pareamento Cromossômico/genética , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Histonas/metabolismo , Hibridização in Situ Fluorescente , Masculino , Microscopia de Fluorescência , Transcrição Genética
11.
Nat Commun ; 12(1): 4898, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385431

RESUMO

Hedgehog (Hh) signaling is essential during development and in organ physiology. In the canonical pathway, Hh binding to Patched (PTCH) relieves the inhibition of Smoothened (SMO). Yet, PTCH may also perform SMO-independent functions. While the PTCH homolog PTC-3 is essential in C. elegans, worms lack SMO, providing an excellent model to probe non-canonical PTCH function. Here, we show that PTC-3 is a cholesterol transporter. ptc-3(RNAi) leads to accumulation of intracellular cholesterol and defects in ER structure and lipid droplet formation. These phenotypes were accompanied by a reduction in acyl chain (FA) length and desaturation. ptc-3(RNAi)-induced lethality, fat content and ER morphology defects were rescued by reducing dietary cholesterol. We provide evidence that cholesterol accumulation modulates the function of nuclear hormone receptors such as of the PPARα homolog NHR-49 and NHR-181, and affects FA composition. Our data uncover a role for PTCH in organelle structure maintenance and fat metabolism.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Colesterol/metabolismo , Homeostase/genética , Metabolismo dos Lipídeos/genética , Receptor Patched-1/genética , Animais , Western Blotting , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestrutura , Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica , Microscopia Eletrônica de Transmissão , Receptor Patched-1/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa
12.
Nat Commun ; 12(1): 4902, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385461

RESUMO

Efficient and precise base editors (BEs) for C-to-G transversion are highly desirable. However, the sequence context affecting editing outcome largely remains unclear. Here we report engineered C-to-G BEs of high efficiency and fidelity, with the sequence context predictable via machine-learning methods. By changing the species origin and relative position of uracil-DNA glycosylase and deaminase, together with codon optimization, we obtain optimized C-to-G BEs (OPTI-CGBEs) for efficient C-to-G transversion. The motif preference of OPTI-CGBEs for editing 100 endogenous sites is determined in HEK293T cells. Using a sgRNA library comprising 41,388 sequences, we develop a deep-learning model that accurately predicts the OPTI-CGBE editing outcome for targeted sites with specific sequence context. These OPTI-CGBEs are further shown to be capable of efficient base editing in mouse embryos for generating Tyr-edited offspring. Thus, these engineered CGBEs are useful for efficient and precise base editing, with outcome predictable based on sequence context of targeted sites.


Assuntos
Sistemas CRISPR-Cas , Citidina Desaminase/metabolismo , Edição de Genes/métodos , Aprendizado de Máquina , Uracila-DNA Glicosidase/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Códon/genética , Citidina Desaminase/genética , Escherichia coli/genética , Feminino , Biblioteca Gênica , Células HEK293 , Humanos , Camundongos , Reprodutibilidade dos Testes , Uracila-DNA Glicosidase/genética
13.
Nat Commun ; 12(1): 4912, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389721

RESUMO

Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between these multimodular megaenzymes. In the canal-associated neurons (CANs) of Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides biosynthesized by animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional enzymes expressed in the CANs that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes and discover a mechanism for trafficking intermediates between a PKS and an NRPS. Specifically, the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking mechanism provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.


Assuntos
Vias Biossintéticas/genética , Proteínas de Caenorhabditis elegans/genética , Peptídeo Sintases/genética , Peptídeos/metabolismo , Policetídeo Sintases/genética , Policetídeos/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografia Líquida/métodos , Enzimas/genética , Enzimas/metabolismo , Expressão Gênica , Espectrometria de Massas/métodos , Estrutura Molecular , Mutação , Neurônios/metabolismo , Peptídeo Sintases/metabolismo , Peptídeos/química , Policetídeo Sintases/metabolismo , Policetídeos/química
14.
Nat Commun ; 12(1): 4239, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244495

RESUMO

Stable epigenetic changes appear uncommon, suggesting that changes typically dissipate or are repaired. Changes that stably alter gene expression across generations presumably require particular conditions that are currently unknown. Here we report that a minimal combination of cis-regulatory sequences can support permanent RNA silencing of a single-copy transgene and its derivatives in C. elegans simply upon mating. Mating disrupts competing RNA-based mechanisms to initiate silencing that can last for >300 generations. This stable silencing requires components of the small RNA pathway and can silence homologous sequences in trans. While animals do not recover from mating-induced silencing, they often recover from and become resistant to trans silencing. Recovery is also observed in most cases when double-stranded RNA is used to silence the same coding sequence in different regulatory contexts that drive germline expression. Therefore, we propose that regulatory features can evolve to oppose permanent and potentially maladaptive responses to transient change.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/genética , Animais , Animais Geneticamente Modificados , Feminino , Masculino , Elementos Reguladores de Transcrição , Reprodução/genética
15.
Nat Commun ; 12(1): 4212, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244496

RESUMO

CSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs targeting most germline-expressed genes. Here we show that the two isoforms of CSR-1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Interferência de RNA , Espermatogênese/genética , Animais , Animais Geneticamente Modificados , Arginina/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Éxons/genética , Feminino , Masculino , Metilação , Oogênese/genética , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Processos de Determinação Sexual/genética
16.
Nat Commun ; 12(1): 4527, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312384

RESUMO

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.


Assuntos
Membrana Celular/fisiologia , Opsinas/metabolismo , Optogenética/métodos , Células Piramidais/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Furões/genética , Furões/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Potenciais da Membrana/fisiologia , Camundongos Transgênicos , Opsinas/genética , Técnicas de Patch-Clamp/métodos , Células Piramidais/citologia , Células Piramidais/metabolismo , Ratos Wistar , Reprodutibilidade dos Testes
17.
Nucleic Acids Res ; 49(15): 8836-8865, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34329465

RESUMO

The Caenorhabditis elegans genome encodes nineteen functional Argonaute proteins that use 22G-RNAs, 26G-RNAs, miRNAs or piRNAs to regulate target transcripts. Only one Argonaute is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied widely, nearly all studies have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR-1a. Using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG into the long (CSR-1a) and short (CSR-1b) isoforms, we found that CSR-1a is expressed during spermatogenesis and in several somatic tissues, including the intestine. CSR-1b is expressed constitutively in the germline. small RNA sequencing of CSR-1 complexes shows that they interact with partly overlapping sets of 22G-RNAs. Phenotypic analyses reveal that the essential functions of csr-1 described in the literature coincide with CSR-1b, while CSR-1a plays tissue specific functions. During spermatogenesis, CSR-1a integrates into an sRNA regulatory network including ALG-3, ALG-4 and WAGO-10 that is necessary for fertility at 25°C. In the intestine, CSR-1a silences immunity and pathogen-responsive genes, and its loss results in improved survival from the pathogen Pseudomonas aeruginosa. Our findings functionally distinguish the CSR-1 isoforms and highlight the importance of studying each AGO isoform independently.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Espermatogênese/genética , Alelos , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Feminino , Fertilidade , Expressão Gênica , Masculino , Mutação , Oócitos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Pequeno RNA não Traduzido/metabolismo , Espermatozoides/metabolismo
18.
Cell Death Dis ; 12(7): 711, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267182

RESUMO

Mitochondria are the main oxygen consumers in cells and as such are the primary organelle affected by hypoxia. All hypoxia pathology presumably derives from the initial mitochondrial dysfunction. An early event in hypoxic pathology in C. elegans is disruption of mitochondrial proteostasis with induction of the mitochondrial unfolded protein response (UPRmt) and mitochondrial protein aggregation. Here in C. elegans, we screen through RNAis and mutants that confer either strong resistance to hypoxic cell death or strong induction of the UPRmt to determine the relationship between hypoxic cell death, UPRmt activation, and hypoxia-induced mitochondrial protein aggregation (HIMPA). We find that resistance to hypoxic cell death invariantly mitigated HIMPA. We also find that UPRmt activation invariantly mitigated HIMPA. However, UPRmt activation was neither necessary nor sufficient for resistance to hypoxic death and vice versa. We conclude that UPRmt is not necessarily hypoxia protective against cell death but does protect from mitochondrial protein aggregation, one of the early hypoxic pathologies in C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Resposta a Proteínas não Dobradas , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Hipóxia Celular , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Agregados Proteicos , Agregação Patológica de Proteínas
19.
Nature ; 596(7871): 281-284, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34290409

RESUMO

The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels1. Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B-GATOR2-mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer.


Assuntos
Leucina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transdução de Sinais , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Humanos , Leucina/deficiência , Longevidade/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/agonistas , Camundongos , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/deficiência , Proteínas Monoméricas de Ligação ao GTP/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Ecotoxicol Environ Saf ; 222: 112494, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34265532

RESUMO

Copper (Cu) pollution in water and agricultural soil has always been a worldwide concern. This research aims to investigate the health effects of copper exposure on Caenorhabditis elegans (C. elegans) under the existing environmental quality standards (1 mg/L and 2 mg/L) via lifespan, reproduction, biological markers and transcriptome analysis. The results showed that copper of these two environmental standards shorten the lifespan of nematodes, reduced the brood size, reduced the frequency of pharyngeal pumps and prolonged defecation time as aging-related behaviors, and increased the levels of aging-related markers ROS, MDA and H2O2. There was a certain effect trend for the two exposure concentrations. Further, the possible molecular mechanism of copper-induced aging and reproductive effects on C. elegans was explored. Differential gene expression analysis was performed, and 2332 genes (567 up- and 1765 down-regulated genes) in the 1 mg/L group, 2449 DEGs (724 up- and 1725 down-regulated genes) in the 2 mg/L group in response to copper treatment. The top 20 regulated genes were vit (vit-1, vit-3, vit-4) genes, col genes (col-35, col-72, col-114, col-123, col-164, col-183, col-185), eea-1, him-18 and grl-20, which suggested that cuticle collagen synthesis and yolk expression were disrupted by copper. Analysis of KEGG pathway showed copper exposure widely affects longevity regulation pathways, thereby promoting aging. In summary, the sequencing results extensively and deeply reveal the health hazards of environmentally relevant doses of copper exposure to C. elegans, and behavioral testing verified that copper promoted aging of C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Escala de Avaliação Comportamental , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cobre/toxicidade , Peróxido de Hidrogênio , Longevidade , Masculino , Transcriptoma
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