Autophagy and UPS pathway contribute to nicotine-induced protection effect in Parkinson's disease.

The gradual nature of age-related neurodegeneration causes Parkinson's disease (PD) and impairs movement, memory, intellectual ability, and social interaction. One of the most prevalent neurodegenerative conditions affecting the central nervous system (CNS) among the elderly is PD. PD affects both motor and cognitive functions. Degeneration of dopaminergic (DA) neurons and buildup of the protein α-synuclein (α-Syn) in the substantia nigra pars compacta (SNpc) are two major causes of this disorder. Both UPS and ALS systems serve to eliminate α-Syn. Autophagy and UPS deficits, shortened life duration, and lipofuscin buildup accelerate PD. This sickness has no cure. Innovative therapies are halting PD progression. Bioactive phytochemicals may provide older individuals with a natural substitute to help delay the onset of neurodegenerative illnesses. This study examines whether nicotine helps transgenic C. elegans PD models. According to numerous studies, nicotine enhances synaptic plasticity and dopaminergic neuronal survival. Upgrades UPS pathways, increases autophagy, and decreases oxidative stress and mitochondrial dysfunction. At 100, 150, and 200 µM nicotine levels, worms showed reduced α-Syn aggregation, repaired DA neurotoxicity after 6-OHDA intoxication, increased lifetime, and reduced lipofuscin accumulation. Furthermore, nicotine triggered autophagy and UPS. We revealed nicotine's potential as a UPS and autophagy activator to prevent PD and other neurodegenerative diseases.

Structural Basis of the Differential Function of the Two C. elegans Atg8 Homologs, LGG-1 and LGG-2, in Autophagy.

Multicellular organisms have multiple homologs of the yeast ATG8 gene, but the differential roles of these homologs in autophagy during development remain largely unknown. Here we investigated structure/function relationships in the two C. elegans Atg8 homologs, LGG-1 and LGG-2. lgg-1 is essential for degradation of protein aggregates, while lgg-2 has cargo-specific and developmental-stage-specific roles in aggregate degradation. Crystallography revealed that the N-terminal tails of LGG-1 and LGG-2 adopt the closed and open form, respectively. LGG-1 and LGG-2 interact differentially with autophagy substrates and Atg proteins, many of which carry a LIR motif. LGG-1 and LGG-2 have structurally distinct substrate binding pockets that prefer different residues in the interacting LIR motif, thus influencing binding specificity. Lipidated LGG-1 and LGG-2 possess distinct membrane tethering and fusion activities, which may result from the N-terminal differences. Our study reveals the differential function of two ATG8 homologs in autophagy during C. elegans development.

Autophagosomal Sperm Organelle Clearance and mtDNA Inheritance in C. elegans.

The nematode C. elegans represents a powerful experimental system with key properties and advantages to study the mechanisms underlying mitochondrial DNA maternal inheritance and paternal components sorting. First, the transmission is uniparental and maternal as in many animal species; second, at fertilization sperm cells contain both mitochondria and mtDNA; and third, the worm allows powerful genetics and cell biology approaches to characterize the mechanisms underlying the uniparental and maternal transmission of mtDNA. Fertilization of C. elegans oocyte occurs inside the transparent body when the mature oocyte resumes meiosis I and passes through the spermatheca. One amoeboid sperm cell fuses with the oocyte and delivers its whole content. Among the structures entering the embryo, the sperm mitochondria and a fraction of the nematode-specific membranous organelles are rapidly degraded, whereas others like centrioles and sperm genomic DNA are transmitted. In this chapter, we will review the knowledge acquired on sperm inherited organelles clearance during the recent years using C. elegans.

The crystal structure of titanium dioxide nanoparticles influences immune activity in vitro and in vivo.

BACKGROUND: The use of engineered nanoparticles (NP) is widespread and still increasing. There is a great need to assess their safety. Newly engineered NP enter the market in a large variety; therefore safety evaluation should preferably be in a high-throughput fashion. In vitro screening is suitable for this purpose. TiO 2 NP exist in a large variety (crystal structure, coating and size), but information on their relative toxicities is scarce. TiO 2 NP may be inhaled by workers in e.g. paint production and application. In mice, inhalation of TiO 2 NP increases allergic reactions. Dendritic cells (DC) form an important part of the lung immune system, and are essential in adjuvant activity. The present study aimed to establish the effect of a variety of TiO 2 NP on DC maturation in vitro. Two NP of different crystal structure but similar in size, uncoated and from the same supplier, were evaluated for their adjuvant activity in vivo. METHODS: Immature DC were differentiated in vitro from human peripheral blood monocytes. Exposure effects of a series of fourteen TiO 2 NP on cell viability, CD83 and CD86 expression, and IL-12p40 and TNF-α production were measured. BALB/c mice were intranasally sensitized with ovalbumin (OVA) alone, OVA plus anatase TiO 2 NP, OVA plus rutile TiO 2 NP, and OVA plus Carbon Black (CB; positive control). The mice were intranasally challenged with OVA. OVA-specific IgE and IgG1 in serum, cellular inflammation in bronchoalveolar lavage fluid (BALF) and IL-4 and IL-5 production in draining bronchial lymph nodes were evaluated. RESULTS: All NP dispersions contained NP aggregates. The anatase NP and anatase/rutile mixture NP induced a higher CD83 and CD86 expression and a higher IL-12p40 production in vitro than the rutile NP (including coated rutile NP and a rutile NP of a 10-fold larger primary diameter). OVA-specific serum IgE and IgG1 were increased by anatase NP, rutile NP, and CB, in the order rutile

A fluorescent reporter for rapid assessment of autophagic flux reveals unique autophagy signatures during C. elegans post-embryonic development and identifies compounds that modulate autophagy.

Autophagy is important for many physiological processes; and disordered autophagy can contribute to the pathogenesis of a broad range of systemic disorders. C. elegans is a useful model organism for studying the genetics of autophagy, however, current methods for studying autophagy are labor-intensive and not readily amenable to high-throughput procedures. Here we describe a fluorescent reporter, GFP::LGG-1::mKate2, which is useful for monitoring autophagic flux in live animals. In the intestine, the fusion protein is processed by endogenous ATG-4 to generate GFP::LGG-1 and mKate2 proteins. We provide data indicating that the GFP:mKate ratio is a suitable readout for measuring cellular autophagic flux. Using this reporter, we measured autophagic flux in L1 larvae to day 7 adult animals. We show that basal autophagic flux is relatively low during larval development but increases markedly in reproductive adults before decreasing with age. Furthermore, we show that wild-type, eat-2, and daf-2 mutant animals have distinct autophagic flux profiles through post-embryonic development. Finally, we demonstrate the utility of this reporter by performing a high-content small molecule screen to identify compounds that alter autophagic flux in C. elegans.

In Vivo Monitoring of Nucleophagy in Caenorhabditis elegans.

The autophagy-lysosomal pathway enables the controlled degradation of cellular contents. Nucleophagy is the selective autophagic recycling of nuclear components upon delivery to the lysosome. Although methods to monitor and quantify autophagy as well as selective types of autophagy have been developed and implemented in cells and in vivo, methods monitoring nucleophagy remain scarce. Here, we describe a procedure to monitor the autophagic engagement of an endogenous nuclear envelope component, i.e., ANC-1, the nematode homologue of the mammalian Nesprins in vivo, utilizing super-resolution microscopy.

MON-2, a Golgi protein, promotes longevity by upregulating autophagy through mediating inter-organelle communications.

The Golgi apparatus regulates the process of modification and subcellular localization of macromolecules, including proteins and lipids. Aberrant protein sorting caused by defects in the Golgi leads to various diseases in mammals. However, the role of the Golgi apparatus in organismal longevity remained largely unknown. By employing a quantitative proteomic approach, we demonstrated that MON-2, an evolutionarily conserved Arf-GEF protein implicated in Golgi-to-endosome trafficking, promotes longevity via upregulating macroautophagy/autophagy in C. elegans. Our data using cultured mammalian cells indicate that MON2 translocates from the Golgi to the endosome under starvation conditions, subsequently increasing autophagic flux by binding LGG-1/GABARAPL2. Thus, Golgi-to-endosome trafficking appears to be an evolutionarily conserved process for the upregulation of autophagy, which contributes to organismal longevity.

Monitoring the Recruitment and Fusion of Autophagosomes to Phagosomes During the Clearance of Apoptotic Cells in the Nematode Caenorhabditis elegans.

During an animal's development, a large number of cells undergo apoptosis, a suicidal form of death. These cells are promptly phagocytosed by other cells and degraded inside phagosomes. The recognition, engulfment, and degradation of apoptotic cells is an evolutionarily conserved process occurring in all metazoans. Recently, we discovered a novel event in the nematode Caenorhabditis elegans: the double-membrane autophagosomes are recruited to the surface of phagosomes; subsequently, the outer membrane of an autophagosome fuses with the phagosomal membrane, allowing the inner vesicle to enter the phagosomal lumen and accumulate there over time. This event facilitates the degradation of the apoptotic cell inside the phagosome. During this study, we developed a real-time imaging protocol monitoring the recruitment and fusion of autophagosomes to phagosomes over two hours during embryonic development. This protocol uses a deconvolution-based microscopic imaging system with an optimized setting to minimize photodamage of the embryo during the recording period for high-resolution images. Furthermore, acid-resistant fluorescent reporters are chosen to label autophagosomes, allowing the inner vesicles of an autophagosome to remain visible after entering the acidic phagosomal lumen. The methods described here, which enable high sensitivity, quantitative measurement of each step of the dynamic incorporation in developing embryos, are novel since the incorporation of autophagosomes to phagosomes has not been reported previously. In addition to studying the degradation of apoptotic cells, this protocol can be applied to study the degradation of non-apoptotic cell cargos inside phagosomes, as well as the fusion between other types of intracellular organelles in living C. elegans embryos. Furthermore, its principle of detecting the membrane fusion event can be adapted to study the relationship between autophagosomes and phagosomes or other intracellular organelles in any biological system in which real-time imaging can be conducted. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.72466.

A novel crosstalk between autophagosomes and phagosomes that facilitates the clearance of apoptotic cells.

During an animal's life, many cells undergo apoptosis, a form of genetically programmed cell death. These cells are swiftly engulfed by other cells through phagocytosis and subsequently degraded inside phagosomes. Phagocytosis and macroautophagy/autophagy are two different cellular events: whereas phagocytosis is a cell-eat-cell event, autophagy, or "self-eating", occurs within one cell, resulting in the enveloping of protein aggregates or damaged organelles within double-membrane autophagosomes. Despite this critical difference, these two events share common features: (1) both are means of safe garbage disposal; (2) both phagosomes and autophagosomes fuse to lysosomes, which drive the degradation of their contents; and (3) both events facilitate the recycling of biological materials. Previously, whether autophagosomes per se directly participate in the degradation of apoptotic cells was unknown, although autophagy proteins were implicated in apoptotic cell clearance. We recently discovered that autophagosomes fuse with phagosomes and contribute to the degradation of apoptotic cells.

Correlative Light and Electron Microscopy to Analyze LC3 Proteins in Caenorhabditis elegans Embryo.

In this chapter, we present a protocol to perform correlative light and electron microscopy (CLEM) on Caenorhabditis elegans embryos. We use a specific fixation method which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins, then by transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows to determine the subcellular localization of the fluorescent protein. We have used this method to characterize the roles of autophagy in the phagocytosis of apoptotic cells in C. elegans embryos. We analyzed in apoptotic cell and phagocytic cell the localization of the two homologs of LC3/GABARAP proteins, namely, LGG-1 and LGG-2.

ATG-18 and EPG-6 are Both Required for Autophagy but Differentially Contribute to Lifespan Control in Caenorhabditis elegans.

During macroautophagy, the human WIPI (WD-repeat protein interacting with phosphoinositides) proteins (WIPI1⁻4) function as phosphatidylinositol 3-phosphate effectors at the nascent autophagosome. Likewise, the two WIPI homologues in Caenorhabditis elegans, ATG-18 and EPG-6, play important roles in autophagy, whereby ATG-18 is considered to act upstream of EPG-6 at the onset of autophagy. Due to its essential role in autophagy, ATG-18 was found to be also essential for lifespan extension in Caenorhabditis elegans; however, this has not yet been addressed with regard to EPG-6. Here, we wished to address this point and generated mutant strains that expressed the autophagy marker GFP::LGG-1 (GFP-LC3 in mammals) and harbored functional deletions of either atg-18 (atg18(gk378)), epg-6 (epg-6(bp242)) or both (atg-18(gk378);epg-6(bp242)). Using quantitative fluorescence microscopy, Western blotting, and lifespan assessments, we provide evidence that in the absence of either ATG-18 or EPG-6 autophagy was impaired, and while atg-18 mutant animals showed a short-lived phenotype, lifespan was significantly increased in epg-6 mutant animals. We speculate that the long-lived phenotype of epg-6 mutant animals points towards an autophagy-independent function of EPG-6 in lifespan control that warrants further mechanistic investigations in future studies.

Western blot analysis of the autophagosomal membrane protein LGG-1/LC3 in Caenorhabditis elegans.

Autophagy is being studied intensively in Caenorhabditis elegans in the context of protein homeostasis and aging. However, in contrast to the yeast and mammalian autophagosomal membrane proteins Atg8 and LC3, lipidation of the C. elegans ortholog LGG-1 with phosphatidylethanolamine has rarely been investigated by western blotting. We attribute this shortcoming to technical problems with separating the nonlipidated from the lipidated LGG-1 protein by gel electrophoresis. Our new protocol for Western blot analysis is applicable for both the detection of transgenic and endogenous LGG-1 proteins and provides a quantifiable method to assess autophagic flux. As a proof of principle, we use this assay to analyze the role of the transcriptional master regulator HLH-30/TFEB in starvation-induced autophagy.

Methods to Determine the Role of Autophagy Proteins in C. elegans Aging.

This chapter describes methods for the analysis of autophagy proteins in C. elegans aging. We discuss the strains to be considered, the methods for the delivery of double-stranded RNA, and the methods to measure autophagy levels, autophagic flux, and degradation by autophagy.

Age-associated and tissue-specific decline in autophagic activity in the nematode C. elegans.

Macroautophagy/autophagy is a cellular recycling process that is required for the extended life span observed in many longevity paradigms, including in the nematode C. elegans. However, little is known regarding the spatiotemporal changes in autophagic activity in such long-lived mutants as well as in wild-type animals during normal aging. In a recent study, we report that autophagic activity decreases with age in several major tissues of wild-type C. elegans, including the intestine, body-wall muscle, pharynx, and nerve-ring neurons. Moreover, long-lived daf-2/insulin-signaling mutants and glp-1/Notch receptor mutants display increased autophagic activity, yet with different time- and tissue-specific differences. Notably, the intestine appears to be a critical tissue in which autophagy contributes to longevity in glp-1, but not in daf-2 mutants. Our findings indicate that autophagic degradation is reduced with age, possibly with distinct kinetics in different tissues, and that long-lived mutants increase autophagy in a tissue-specific manner, resulting in increased life span.

Anti-Parkinsonian effects of ß-amyrin are regulated via LGG-1 involved autophagy pathway in Caenorhabditis elegans.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease that is associated with aging and is characterized as a movement disorder. Currently, there is still no complete therapy for PD. In recent years, the identification and characterization of medicinal plants to cure or treat PD has gained increasing scientific interest. PURPOSE: In this study, we investigated a pentacyclic triterpenoid compound, ß-amyrin, which is found in many medicinal plants for its anti-Parkinsonian effects, using Caenorhabditis elegans (C. elegans) disease models and their underlying mechanisms. METHODS: C. elegans treated or untreated with ß-amyrin were investigated for oxidative stress resistance, neurodegeneration, and α-synuclein aggregation assays. The C. elegans ortholog of Atg8/LC3, LGG-1 that is involved in the autophagy pathway was also evaluated by quantitative RT-PCR and transgenic strain experiments. RESULTS: ß-Amyrin exerted excellent antioxidant activity and reduced intracellular oxygen species in C. elegans. Using the transgenic strain BZ555, ß-amyrin showed a protective effect on dopaminergic neurons reducing cell damage induced by 6-hydroxydopamine (6-OHDA). In addition, ß-amyrin significantly reduced the α-synuclein aggregation in the transgenic strain NL5901. Moreover, ß-amyrin up-regulated LGG-1 mRNA expression and increased the number of localized LGG-1 puncta in the transgenic strain DA2123. CONCLUSION: The results from this study suggest that the anti-Parkinsonian effects of ß-amyrin might be regulated via LGG-1 involved autophagy pathway in C. elegans. Therefore, ß-amyrin may be useful for therapeutic applications or supplements to treat or slow the progression of PD.

Approaches for Studying Autophagy in Caenorhabditis elegans.

Macroautophagy (hereafter referred to as autophagy) is an intracellular degradative process, well conserved among eukaryotes. By engulfing cytoplasmic constituents into the autophagosome for degradation, this process is involved in the maintenance of cellular homeostasis. Autophagy induction triggers the formation of a cup-shaped double membrane structure, the phagophore, which progressively elongates and encloses materials to be removed. This double membrane vesicle, which is called an autophagosome, fuses with lysosome and forms the autolysosome. The inner membrane of the autophagosome, along with engulfed compounds, are degraded by lysosomal enzymes, which enables the recycling of carbohydrates, amino acids, nucleotides, and lipids. In response to various factors, autophagy can be induced for non-selective degradation of bulk cytoplasm. Autophagy is also able to selectively target cargoes and organelles such as mitochondria or peroxisome, functioning as a quality control system. The modification of autophagy flux is involved in developmental processes such as resistance to stress conditions, aging, cell death, and multiple pathologies. So, the use of animal models is essential for understanding these processes in the context of different cell types throughout the entire lifespan. For almost 15 years, the nematode Caenorhabditis elegans has emerged as a powerful model to analyze autophagy in physiological or pathological contexts. This review presents a rapid overview of physiological processes involving autophagy in Caenorhabditis elegans, the different assays used to monitor autophagy, their drawbacks, and specific tools for the analyses of selective autophagy.

The recycling endosome protein RAB-10 promotes autophagic flux and localization of the transmembrane protein ATG-9.

Macroautophagy/autophagy involves the formation of an autophagosome, a double-membrane vesicle that delivers sequestered cytoplasmic cargo to lysosomes for degradation and recycling. Closely related, endocytosis mediates the sorting and transport of cargo throughout the cell, and both processes are important for cellular homeostasis. However, how endocytic proteins functionally intersect with autophagy is not clear. Mutations in the DAF-2/insulin-like IGF-1 (INSR) receptor at the permissive temperature result in a small increase in GFP::LGG-1 foci, i.e. autophagosomes, but a large increase at the nonpermissive temperature, allowing us to control the level of autophagy. In a RNAi screen for endocytic genes that alter the expression of GFP::LGG-1 in daf-2 mutants, we identified RAB-10, a small GTPase that regulates basolateral endocytosis. Loss of rab-10 in daf-2 mutants results in more GFP::LGG-1-positive foci at the permissive, but less GFP::LGG-1 or SQST-1::GFP foci at the nonpermissive temperature. As previously reported, loss of rab-10 alone resulted in an increase of GFP:LGG-1 foci. Exposure of rab-10 mutant animals to chloroquine, a known inhibitor of autophagic flux, failed to increase the number of GFP::LGG-1 foci. Moreover, colocalization between LMP-1::tagRFP and GFP::LGG-1 (the lysosome and autophagosome reporters) was decreased in daf-2; rab-10 dauers at the nonpermissive temperature. Intriguingly, RAB-10 was required to maintain the normal size of GFP::ATG-9-positive structures in daf-2 mutants at both the permissive and nonpermissive temperature. Finally, we found that RAB-10 GTPase cycling was required to control the size of GFP::ATG-9 foci. Collectively, our data support a model where rab-10 controls autophagic flux by regulating autophagosome formation and maturation.

Small differences make a big impact: Structural insights into the differential function of the 2 Atg8 homologs in C. elegans.

The 2 C. elegans homologs of Atg8, LGG-1 and LGG-2, show differential function in the degradation of protein aggregates during embryogenesis. LGG-1 is essential for the degradation of various protein aggregates, while LGG-2 has cargo-specific and developmental stage-specific roles. LGG-1 and LGG-2 differentially interact with autophagy substrates and ATG proteins. LGG-1 and LGG-2 possess 2 hydrophobic pockets, the W-site and the L-site, which recognize the LIR motif in Atg8-binding proteins. The plasticity of the W-site and the size and shape of the L-site differ between LGG-1 and LGG-2, thus determining their preferences for distinct LIR motifs. The N-terminal tails of LGG-1 and LGG-2 adopt unique closed and open conformations, respectively, which may result in distinct membrane tethering and fusion activities. LGG-1 and LGG-2 have different affinities for ATG-7 and ATG-3, and lipidation of LGG-2 is regulated by levels of lipidated LGG-1. Taken together, the structural differences between LGG-1 and LGG-2 provide insights into their differential functions in the aggrephagy pathway.

Guidelines for monitoring autophagy in Caenorhabditis elegans.

The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.

Need an ESCRT for autophagosomal maturation?

Several reports in fly, nematode and mammalian cells have revealed that the inactivation of endosomal sorting complexes required for transport (ESCRT) blocks the endosomal maturation but also leads to the increased number of autophagosomal structures. In this review we compare these data and conclude that the way ESCRT mutations affect the relationships between autophagosomes and endosomes cannot be generalized but depends on the studied species. We propose that the effect of ESCRT mutations on autophagy is directly dependent of the level of interaction between autophagosomes and endosomes. In particular, the formation of amphisomes during autophagosomal maturation could be the key point to explain the differences observed between species. These observations highlight the importance of multiple model organisms to decipher the complexity of relationships between such dynamic vesicles.