Monitoring autophagic flux in Caenorhabditis elegans using a p62/SQST-1 reporter

Ploumi, Christina; Sotiriou, Aggeliki; Tavernarakis, Nektarios

doi:10.1016/bs.mcb.2020.10.011

Cited by 3 publications

(7 citation statements)

References 44 publications

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“…The role of MANF-1 in lysosome-mediated signaling and the involvement of HLH-30/TFEB in this process led us to investigate the role of LGG-1/Atg8/LC3 and SQST-1/p62 in regulating autophagy [40, 59–62]. Although an increase in LGG-1 and SQST-1 puncta may be caused by a blockage of autophagy, a reduction in both types of puncta is indicative of an increase in autophagic flux.…”

Section: Discussionmentioning

confidence: 99%

“…The SQST-1::GFP puncta in sqst-1p::SQST-1::GFP animals were examined using a protocol based on published studies [40, 59]. Z stack images were taken at optimal slice intervals determined by the Zeiss Zen2 blue software.…”

Section: Methodsmentioning

confidence: 99%

“…Two additional autophagy genes, lgg-1/LC3 and sqst-1/p62, were examined. LGG-1 is involved in autophagosome formation and SQST-1 is an autophagy receptor that facilitates degradation of ubiquitinated protein cargo (Kumsta et al 2019;Ploumi et al 2021). The results showed that while transcript levels of lgg-1/LC3 and sqst-1/p62 were unaffected in MANF-1 OE worms (Fig.…”

Section: Manf-1 Promotes Autophagy and Lysosome Functionmentioning

confidence: 99%

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Neurotrophic factor MANF regulates autophagy and lysosome function to promote proteostasis inC. elegans

Taylor

Gupta

2023

Preprint

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The neurotrophic factor, Mesencephalic Astrocyte Derived Neurotrophic Factor (MANF), promotes neuronal health and survival. MANF is conserved in metazoans and necessary for the protection of dopaminergic neurons. The neuroprotective role of MANF involves regulation of the ER unfolded protein response (ER-UPR). Recent work has shown that MANF also functions in many other tissues and affects autophagy genes. Previously, we reported thatC. elegans manf-1mutants show defects including increased ER stress, α-Synuclein aggregation, and age-dependent dopaminergic neurodegeneration. In this study, we have further investigated manf-1’s function and describe its mechanism of action in regulating stress response, protein aggregation and aging. Our results showed that exposure to ER stress-inducing agents causedmanf-1transcription and protein expression to be upregulated. Consistent with the protective role ofmanf-1, mutant animals showed greater sensitivity to ER stress and died prematurely. We generatedmanf-1p::manf-1::mCherry(MANF-1OE) andhsp::manf-1transgenic strains overexpressing MANF and found that these animals had an extended lifespan, reduced protein aggregation, and increased neuronal health. Thein vivoanalysis of MANF-1 expression revealed that the protein was secreted and present in many tissues. Subcellular studies showed that MANF-1 was localized to lysosomes. We also observed that MANF-1OEworms had reduced LGG-1 and SQST-1/p62 levels, and increased expression and nuclear localization of the autophagy transcription factorhlh-30/TFEB. Thus, MANF-1 regulates protein homeostasis through increased lysosome activity and autophagic flux. Collectively, our work provides a novel understanding of MANF-1 function in promoting neuronal health, stress response, proteostasis, and lifespan of animals.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Manf-1 Promotes Autophagy and Lysosome Functionmentioning

confidence: 99%

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Neurotrophic factor MANF regulates autophagy and lysosome function to promote proteostasis inC. elegans

Taylor

Gupta

2023

Preprint

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“…Single CISD-1 deficiency also augments autophagosomal number in daf-2(e1370) mutants, albeit to a lesser extent compared to that of double deficient animals. To examine whether autophagosomes accumulate due to a blockade in autophagic flux, we monitored the degradation of the autophagic substrate SQST-1, the nematode ortholog of p62/ sequestosome [37]. The levels of SQST-1::GFP positive puncta remain unaffected in control, cisd-1(RNAi)and ced-9(RNAi)treated worms.…”

Section: Mitochondrial Massmentioning

confidence: 99%

“…We also used the MAH14 strain as an additional control for induced autophagy and similarly measured the number of GFP::LGG-1 positive puncta in hypodermal seam cells. The sequestration of autophagic substrates as an indicator for autophagic flux, was monitored by using the SQST-1::GFP reporter strain (HZ589), as previously described [37]. Approximately 20-30 day-1 adult animals (second generation progeny grown on RNAi), were collected, anaesthetized with 20 mM tetramisole and mounted on agarose pads for microscopic observation.…”

Section: Autophagy Measurementsmentioning

confidence: 99%

Coupling of autophagy and the mitochondrial intrinsic apoptosis pathway modulates proteostasis and ageing in Caenorhabditis elegans

2023

Self Cite

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Mitochondria preserve metabolic homeostasis and integrate stress signals, to trigger cytoprotective, or cell death pathways. Mitochondrial homeostasis and function decline with age. The mechanisms underlying the deterioration of mitochondrial homeostasis during ageing, or in age-associated pathologies, remain unclear. Here, we show that CISD-1, a mitochondrial iron-sulfur cluster binding protein, implicated in the pathogenesis of Wolfram neurodegenerative syndrome type 2, modulates longevity in the nematode Caenorhabditis elegans by engaging autophagy and the mitochondrial intrinsic apoptosis pathway. The anti-apoptotic protein CED-9 is the downstream effector that mediates CISD-1-dependent effects on proteostasis, neuronal integrity and lifespan. Moreover, intracellular iron abundance is critical for CISD-1 function, since mild iron supplementation is sufficient to decelerate ageing and partly ameliorate the disturbed mitochondrial bioenergetics and proteostasis of CISD-1 deficient animals. Our findings reveal that CISD-1 serves as a mechanistic link between autophagy and the apoptotic pathway in mitochondria to differentially modulate organismal proteostasis and ageing, and suggest novel approaches which could facilitate the treatment of Wolfram Syndrome or related diseases.

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Salsolinol as an RNA m6A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy

Wang,

Ran,

et al. 2024

Neural Regeneration Research

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JOURNAL/nrgr/04.03/01300535-202503000-00032/figure1/v/2024-06-17T092413Z/r/image-tiff Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, Sal) is a catechol isoquinoline that causes neurotoxicity and shares structural similarity with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an environmental toxin that causes Parkinson’s disease. However, the mechanism by which Sal mediates dopaminergic neuronal death remains unclear. In this study, we found that Sal significantly enhanced the global level of N6-methyladenosine (m6A) RNA methylation in PC12 cells, mainly by inducing the downregulation of the expression of m6A demethylases fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5). RNA sequencing analysis showed that Sal downregulated the Hippo signaling pathway. The m6A reader YTH domain-containing family protein 2 (YTHDF2) promoted the degradation of m6A-containing Yes-associated protein 1 (YAP1) mRNA, which is a downstream key effector in the Hippo signaling pathway. Additionally, downregulation of YAP1 promoted autophagy, indicating that the mutual regulation between YAP1 and autophagy can lead to neurotoxicity. These findings reveal the role of Sal on m6A RNA methylation and suggest that Sal may act as an RNA methylation inducer mediating dopaminergic neuronal death through YAP1 and autophagy. Our results provide greater insights into the neurotoxic effects of catechol isoquinolines compared with other studies and may be a reference for assessing the involvement of RNA methylation in the pathogenesis of Parkinson’s disease.

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Monitoring autophagic flux in Caenorhabditis elegans using a p62/SQST-1 reporter

Cited by 3 publications

References 44 publications

Neurotrophic factor MANF regulates autophagy and lysosome function to promote proteostasis inC. elegans

Neurotrophic factor MANF regulates autophagy and lysosome function to promote proteostasis inC. elegans

Coupling of autophagy and the mitochondrial intrinsic apoptosis pathway modulates proteostasis and ageing in Caenorhabditis elegans

Salsolinol as an RNA m6A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy

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