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Cells
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Molecular Mechanisms and Biological Roles of Alternative Autophagy
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Molecular Mechanisms and Biological Roles of Alternative Autophagy

A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Autophagy".

Deadline for manuscript submissions: closed (31 July 2024) | Viewed by 5438

Special Issue Editor

Prof. Dr. Shigeomi Shimizu

E-Mail Website
Guest Editor
Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo-ku, Tokyo 113-8510, Japan
Interests: alternative autophagy; cell death; Golgi function; mitochondria; neurodegenerative diseases

Special Issue Information

Dear  Colleagues,

Over the past two decades, studies on canonical autophagy have expanded from molecular mechanisms to human diseases. However, recent studies have revealed the existence of another type of autophagy mechanism, namely alternative autophagy or Golgi-membrane-associated degradation (GOMED). Alternative autophagy is different from canonical autophagy in terms of the molecules involved, membrane sources, and substrates degraded. Therefore, alternative autophagy is a different proteolysis mechanism from canonical autophagy, and importantly, it is shown to be involved in a wide variety of physiological events.

This Special Issue will focus on molecular mechanisms of alternative autophagy, how to monitor alternative autophagy, which molecules are degraded, the physiological roles, and related human diseases.

Prof. Dr. Shigeomi Shimizu
Guest Editor

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Keywords

  • alternative autophagy
  • GOMED
  • Golgi
  • proteolysis

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25 pages, 13938 KiB  
Article
Cisplatin and Starvation Differently Sensitize Autophagy in Renal Carcinoma: A Potential Therapeutic Pathway to Target Variegated Drugs Resistant Cancerous Cells
by Ankita Dutta, Subarna Thakur, Debasish Kumar Dey and Anoop Kumar
Cells 2024, 13(6), 471; https://doi.org/10.3390/cells13060471 - 7 Mar 2024
Cited by 2 | Viewed by 1477
Abstract
Cisplatin, a powerful chemotherapy medication, has long been a cornerstone in the fight against cancer due to chemotherapeutic failure. The mechanism of cisplatin resistance/failure is a multifaceted and complex issue that consists mainly of apoptosis inhibition through autophagy sensitization. Currently, researchers are exploring [...] Read more.
Cisplatin, a powerful chemotherapy medication, has long been a cornerstone in the fight against cancer due to chemotherapeutic failure. The mechanism of cisplatin resistance/failure is a multifaceted and complex issue that consists mainly of apoptosis inhibition through autophagy sensitization. Currently, researchers are exploring ways to regulate autophagy in order to tip the balance in favor of effective chemotherapy. Based on this notion, the current study primarily identifies the differentially expressed genes (DEGs) in cisplatin-treated autophagic ACHN cells through the Illumina Hi-seq platform. A protein–protein interaction network was constructed using the STRING database and KEGG. GO classifiers were implicated to identify genes and their participating biological pathways. ClueGO, David, and MCODE detected ontological enrichment and sub-networking. The network topology was further examined using 12 different algorithms to identify top-ranked hub genes through the Cytoscape plugin Cytohubba to identify potential targets, which established profound drug efficacy under an autophagic environment. Considerable upregulation of genes related to autophagy and apoptosis suggests that autophagy boosts cisplatin efficacy in malignant ACHN cells with minimal harm to normal HEK-293 growth. Furthermore, the determination of cellular viability and apoptosis by AnnexinV/FITC-PI assay corroborates with in silico data, indicating the reliability of the bioinformatics method followed by qRT-PCR. Altogether, our data provide a clear molecular insight into drug efficacy under starved conditions to improve chemotherapy and will likely prompt more clinical trials on this aspect. Full article
(This article belongs to the Special Issue Molecular Mechanisms and Biological Roles of Alternative Autophagy)
►▼ Show Figures

Figure 1

Figure 1
<p>Effect of cisplatin in starvation-induced autophagic cell lines. (<b>A</b>) Autophagosomes were detected by CYTO-ID autophagy detection kit in nutrient-deficient normal (HEK-293) and cancer cell (ACHN) lines. Cells were treated in the presence of complete media (CM), PBS (autophagy inducer), and rapamycin (positive control) in both cell lines. (<b>B</b>) Antibody-based indirect ELISA was used to assess autophagy progression in control (cells without treatment) and in PBS-treated (nutrient-deprived for 3 h) cells. The cytosolic protein was extracted from each treatment condition from both cell lines, and quantification of autophagy-related biomarkers was achieved by recording absorbance at 450 nm using SPECTROStar Nano plate reader (BMG Labteck, Germany). (<b>C</b>) Percentage of cell viability after cisplatin treatment in HEK-293 and ACHN cell by MTT assay after 24 h. (<b>D</b>) Trypan blue exclusion assay for precise quantification of viable and non-viable cells in all conditions—control, PBS (without nutrient), control + cisplatin, and PBS + cisplatin (starvation-induced autophagic condition + cisplatin). All data are mean ± SD and are indicative of three separate studies. The significance level was set at <span class="html-italic">p</span> &lt; 0.05 (*: <span class="html-italic">p</span> ≤ 0.05, **: <span class="html-italic">p</span> ≤ 0.01, ***: <span class="html-italic">p</span> ≤ 0.001, ****: <span class="html-italic">p</span> ≤ 0.0001), and the standard deviations of the data were displayed as error bars.</p> Full article ">Figure 1 Cont.
<p>Effect of cisplatin in starvation-induced autophagic cell lines. (<b>A</b>) Autophagosomes were detected by CYTO-ID autophagy detection kit in nutrient-deficient normal (HEK-293) and cancer cell (ACHN) lines. Cells were treated in the presence of complete media (CM), PBS (autophagy inducer), and rapamycin (positive control) in both cell lines. (<b>B</b>) Antibody-based indirect ELISA was used to assess autophagy progression in control (cells without treatment) and in PBS-treated (nutrient-deprived for 3 h) cells. The cytosolic protein was extracted from each treatment condition from both cell lines, and quantification of autophagy-related biomarkers was achieved by recording absorbance at 450 nm using SPECTROStar Nano plate reader (BMG Labteck, Germany). (<b>C</b>) Percentage of cell viability after cisplatin treatment in HEK-293 and ACHN cell by MTT assay after 24 h. (<b>D</b>) Trypan blue exclusion assay for precise quantification of viable and non-viable cells in all conditions—control, PBS (without nutrient), control + cisplatin, and PBS + cisplatin (starvation-induced autophagic condition + cisplatin). All data are mean ± SD and are indicative of three separate studies. The significance level was set at <span class="html-italic">p</span> &lt; 0.05 (*: <span class="html-italic">p</span> ≤ 0.05, **: <span class="html-italic">p</span> ≤ 0.01, ***: <span class="html-italic">p</span> ≤ 0.001, ****: <span class="html-italic">p</span> ≤ 0.0001), and the standard deviations of the data were displayed as error bars.</p> Full article ">Figure 2
<p>(<b>A</b>) Hierarchical clustering of differentially expressed up−regulated and (<b>B</b>) down−regulated genes in autophagic-treated (cisplatin-treated autophagic ACHN) and autophagic non-treated ACHN (control) cell lines. Heat−map was generated by TBtools (<a href="https://github.com/CJ-Chen/TBtools/releases" target="_blank">https://github.com/CJ-Chen/TBtools/releases</a>) with the FPKM (fragments per kilobase of transcript per million mapped reads) value of both samples. (<b>C</b>) Cnet plot of genes regulating major biological processes (BP); (<b>D</b>) Cnet plot of major cellular components involved (CC); (<b>E</b>) Cnet plot of major molecular function (MF)-regulating genes. The plots were generated by SRPLOT (<a href="http://www.bioinformatics.com.cn/srplot" target="_blank">http://www.bioinformatics.com.cn/srplot</a>) based on the results of the enriched KEGG pathway. Size = number of differentially expressed genes in the enriched KEGG pathway; fold change = the fold change difference between cisplatin-treated autophagic ACHNs and non-treated autophagic ACHNs. (<b>F</b>) Bubble plot showing GO results of all three ontologies ((<b>a</b>–<b>c</b>): bubble plot showing significant pathways for up-regulated DEGs in terms of BP, CC, MF respectively. Larger bubbles indicate higher number of genes. The colour of each bubble reflects significance; (<b>d</b>): combined GO results of three different ontologies) with defined enrichment score.</p> Full article ">Figure 2 Cont.
<p>(<b>A</b>) Hierarchical clustering of differentially expressed up−regulated and (<b>B</b>) down−regulated genes in autophagic-treated (cisplatin-treated autophagic ACHN) and autophagic non-treated ACHN (control) cell lines. Heat−map was generated by TBtools (<a href="https://github.com/CJ-Chen/TBtools/releases" target="_blank">https://github.com/CJ-Chen/TBtools/releases</a>) with the FPKM (fragments per kilobase of transcript per million mapped reads) value of both samples. (<b>C</b>) Cnet plot of genes regulating major biological processes (BP); (<b>D</b>) Cnet plot of major cellular components involved (CC); (<b>E</b>) Cnet plot of major molecular function (MF)-regulating genes. The plots were generated by SRPLOT (<a href="http://www.bioinformatics.com.cn/srplot" target="_blank">http://www.bioinformatics.com.cn/srplot</a>) based on the results of the enriched KEGG pathway. Size = number of differentially expressed genes in the enriched KEGG pathway; fold change = the fold change difference between cisplatin-treated autophagic ACHNs and non-treated autophagic ACHNs. (<b>F</b>) Bubble plot showing GO results of all three ontologies ((<b>a</b>–<b>c</b>): bubble plot showing significant pathways for up-regulated DEGs in terms of BP, CC, MF respectively. Larger bubbles indicate higher number of genes. The colour of each bubble reflects significance; (<b>d</b>): combined GO results of three different ontologies) with defined enrichment score.</p> Full article ">Figure 2 Cont.
<p>(<b>A</b>) Hierarchical clustering of differentially expressed up−regulated and (<b>B</b>) down−regulated genes in autophagic-treated (cisplatin-treated autophagic ACHN) and autophagic non-treated ACHN (control) cell lines. Heat−map was generated by TBtools (<a href="https://github.com/CJ-Chen/TBtools/releases" target="_blank">https://github.com/CJ-Chen/TBtools/releases</a>) with the FPKM (fragments per kilobase of transcript per million mapped reads) value of both samples. (<b>C</b>) Cnet plot of genes regulating major biological processes (BP); (<b>D</b>) Cnet plot of major cellular components involved (CC); (<b>E</b>) Cnet plot of major molecular function (MF)-regulating genes. The plots were generated by SRPLOT (<a href="http://www.bioinformatics.com.cn/srplot" target="_blank">http://www.bioinformatics.com.cn/srplot</a>) based on the results of the enriched KEGG pathway. Size = number of differentially expressed genes in the enriched KEGG pathway; fold change = the fold change difference between cisplatin-treated autophagic ACHNs and non-treated autophagic ACHNs. (<b>F</b>) Bubble plot showing GO results of all three ontologies ((<b>a</b>–<b>c</b>): bubble plot showing significant pathways for up-regulated DEGs in terms of BP, CC, MF respectively. Larger bubbles indicate higher number of genes. The colour of each bubble reflects significance; (<b>d</b>): combined GO results of three different ontologies) with defined enrichment score.</p> Full article ">Figure 2 Cont.
<p>(<b>A</b>) Hierarchical clustering of differentially expressed up−regulated and (<b>B</b>) down−regulated genes in autophagic-treated (cisplatin-treated autophagic ACHN) and autophagic non-treated ACHN (control) cell lines. Heat−map was generated by TBtools (<a href="https://github.com/CJ-Chen/TBtools/releases" target="_blank">https://github.com/CJ-Chen/TBtools/releases</a>) with the FPKM (fragments per kilobase of transcript per million mapped reads) value of both samples. (<b>C</b>) Cnet plot of genes regulating major biological processes (BP); (<b>D</b>) Cnet plot of major cellular components involved (CC); (<b>E</b>) Cnet plot of major molecular function (MF)-regulating genes. The plots were generated by SRPLOT (<a href="http://www.bioinformatics.com.cn/srplot" target="_blank">http://www.bioinformatics.com.cn/srplot</a>) based on the results of the enriched KEGG pathway. Size = number of differentially expressed genes in the enriched KEGG pathway; fold change = the fold change difference between cisplatin-treated autophagic ACHNs and non-treated autophagic ACHNs. (<b>F</b>) Bubble plot showing GO results of all three ontologies ((<b>a</b>–<b>c</b>): bubble plot showing significant pathways for up-regulated DEGs in terms of BP, CC, MF respectively. Larger bubbles indicate higher number of genes. The colour of each bubble reflects significance; (<b>d</b>): combined GO results of three different ontologies) with defined enrichment score.</p> Full article ">Figure 3
<p>(<b>A</b>) Protein–protein interaction network constructed using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, version 11.5; <a href="https://string-db.org" target="_blank">https://string-db.org</a>) with significantly upregulated DEGs in autophagic ACHNs in response to cisplatin. (<b>B</b>) Pathway network constructed using all the upregulated DEGs (<b>a</b>); Pie chart shows all the significantly up-regulated pathways (<b>b</b>). (<b>C</b>) Constructed pathway network from MCODE Cluster 1 generated using Cytoscape plugin ClueGO (<b>a</b>); pie chart shows the stress associated with overexpressed functional categories of the ClueGo pathway analysis (<b>b</b>). (<b>D</b>) Constructed pathway network from MCODE Cluster 2 generated using Cytoscape plugin ClueGO (<b>a</b>); pie chart shows the autophagy- and apoptosis-associated overexpressed functional categories of ClueGo (<b>b</b>) pathway analysis (** <span class="html-italic">p</span> &lt; 0.001). The significantly enriched pathways are denoted by different colors.</p> Full article ">Figure 3 Cont.
<p>(<b>A</b>) Protein–protein interaction network constructed using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, version 11.5; <a href="https://string-db.org" target="_blank">https://string-db.org</a>) with significantly upregulated DEGs in autophagic ACHNs in response to cisplatin. (<b>B</b>) Pathway network constructed using all the upregulated DEGs (<b>a</b>); Pie chart shows all the significantly up-regulated pathways (<b>b</b>). (<b>C</b>) Constructed pathway network from MCODE Cluster 1 generated using Cytoscape plugin ClueGO (<b>a</b>); pie chart shows the stress associated with overexpressed functional categories of the ClueGo pathway analysis (<b>b</b>). (<b>D</b>) Constructed pathway network from MCODE Cluster 2 generated using Cytoscape plugin ClueGO (<b>a</b>); pie chart shows the autophagy- and apoptosis-associated overexpressed functional categories of ClueGo (<b>b</b>) pathway analysis (** <span class="html-italic">p</span> &lt; 0.001). The significantly enriched pathways are denoted by different colors.</p> Full article ">Figure 3 Cont.
<p>(<b>A</b>) Protein–protein interaction network constructed using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, version 11.5; <a href="https://string-db.org" target="_blank">https://string-db.org</a>) with significantly upregulated DEGs in autophagic ACHNs in response to cisplatin. (<b>B</b>) Pathway network constructed using all the upregulated DEGs (<b>a</b>); Pie chart shows all the significantly up-regulated pathways (<b>b</b>). (<b>C</b>) Constructed pathway network from MCODE Cluster 1 generated using Cytoscape plugin ClueGO (<b>a</b>); pie chart shows the stress associated with overexpressed functional categories of the ClueGo pathway analysis (<b>b</b>). (<b>D</b>) Constructed pathway network from MCODE Cluster 2 generated using Cytoscape plugin ClueGO (<b>a</b>); pie chart shows the autophagy- and apoptosis-associated overexpressed functional categories of ClueGo (<b>b</b>) pathway analysis (** <span class="html-italic">p</span> &lt; 0.001). The significantly enriched pathways are denoted by different colors.</p> Full article ">Figure 3 Cont.
<p>(<b>A</b>) Protein–protein interaction network constructed using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins, version 11.5; <a href="https://string-db.org" target="_blank">https://string-db.org</a>) with significantly upregulated DEGs in autophagic ACHNs in response to cisplatin. (<b>B</b>) Pathway network constructed using all the upregulated DEGs (<b>a</b>); Pie chart shows all the significantly up-regulated pathways (<b>b</b>). (<b>C</b>) Constructed pathway network from MCODE Cluster 1 generated using Cytoscape plugin ClueGO (<b>a</b>); pie chart shows the stress associated with overexpressed functional categories of the ClueGo pathway analysis (<b>b</b>). (<b>D</b>) Constructed pathway network from MCODE Cluster 2 generated using Cytoscape plugin ClueGO (<b>a</b>); pie chart shows the autophagy- and apoptosis-associated overexpressed functional categories of ClueGo (<b>b</b>) pathway analysis (** <span class="html-italic">p</span> &lt; 0.001). The significantly enriched pathways are denoted by different colors.</p> Full article ">Figure 4
<p>Sequential workflow of bioinformatics analysis pipeline. Flowchart describing the steps of data processing and subsequent analysis of differentially expressed genes.</p> Full article ">Figure 5
<p>(<b>A</b>) Validation of RNAseq data by measuring the relative expression level of 10 differentially expressed genes in the ACHN cell line in control (autophagic ACHN cells without cisplatin) and treated (cisplatin-treated autophagic ACHN cells) cell lines by qRT-PCR. Values are represented as ±SD of at least three independent experiments. <span class="html-italic">p</span> &lt; 0.05 was considered significant (**: <span class="html-italic">p</span> ≤ 0.01, ***: <span class="html-italic">p</span> ≤ 0.001, ****: <span class="html-italic">p</span> ≤ 0.0001); the standard deviations of the data have been shown in the form of error bars. (<b>B</b>) Cell death was recorded in HEK-293 and ACHN cells through annexin V-FITC/PI assay—HEK-293 and ACHN cells were treated with cisplatin in nutrient-sufficient (control + cisplatin) and nutrient-deficient conditions (PBS + cisplatin). Slides were stained according to the manufacturer’s instructions, and visualization was achieved under the 40× objective of a fluorescence microscope (Magnus MLXi, India). Each image is a representative of at least three independent biological experiments.</p> Full article ">
28 pages, 3658 KiB  
Article
Neuronal atg1 Coordinates Autophagy Induction and Physiological Adaptations to Balance mTORC1 Signalling
by Athanasios Metaxakis, Michail Pavlidis and Nektarios Tavernarakis
Cells 2023, 12(16), 2024; https://doi.org/10.3390/cells12162024 - 8 Aug 2023
Viewed by 1867
Abstract
The mTORC1 nutrient-sensing pathway integrates metabolic and endocrine signals into the brain to evoke physiological responses to food deprivation, such as autophagy. Nevertheless, the impact of neuronal mTORC1 activity on neuronal circuits and organismal metabolism remains obscure. Here, we show that mTORC1 inhibition [...] Read more.
The mTORC1 nutrient-sensing pathway integrates metabolic and endocrine signals into the brain to evoke physiological responses to food deprivation, such as autophagy. Nevertheless, the impact of neuronal mTORC1 activity on neuronal circuits and organismal metabolism remains obscure. Here, we show that mTORC1 inhibition acutely perturbs serotonergic neurotransmission via proteostatic alterations evoked by the autophagy inducer atg1. Neuronal ATG1 alters the intracellular localization of the serotonin transporter, which increases the extracellular serotonin and stimulates the 5HTR7 postsynaptic receptor. 5HTR7 enhances food-searching behaviour and ecdysone-induced catabolism in Drosophila. Along similar lines, the pharmacological inhibition of mTORC1 in zebrafish also stimulates food-searching behaviour via serotonergic activity. These effects occur in parallel with neuronal autophagy induction, irrespective of the autophagic activity and the protein synthesis reduction. In addition, ectopic neuronal atg1 expression enhances catabolism via insulin pathway downregulation, impedes peptidergic secretion, and activates non-cell autonomous cAMP/PKA. The above exert diverse systemic effects on organismal metabolism, development, melanisation, and longevity. We conclude that neuronal atg1 aligns neuronal autophagy induction with distinct physiological modulations, to orchestrate a coordinated physiological response against reduced mTORC1 activity. Full article
(This article belongs to the Special Issue Molecular Mechanisms and Biological Roles of Alternative Autophagy)
►▼ Show Figures

Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Rapamycin treatment alters cognitive and behavioural patterns in flies. (<b>a</b>) Four-day rapamycin treatment decreases the learning ability, LTM formation, and fear-like behaviour, while it increases the exploratory activity and does not alter the STM and MTM formation in 10-day-old <span class="html-italic">W<sup>Dah</sup></span> flies (<span class="html-italic">n</span> = 5). Individual comparisons by two-tailed Mann–Whitney test. (<b>b</b>) Four-day rapamycin treatment increases autophagy in the heads of 10-day-old <span class="html-italic">W<sup>Dah</sup></span> flies. (<b>c</b>) Reduced neuronal <span class="html-italic">atg1</span> expression decreased the LTM and ameliorated the rapamycin effects on behaviour (<span class="html-italic">n</span> = 5). Ten-day-old <span class="html-italic">W<sup>Dah</sup></span> flies were fed with rapamycin for 4 days. For learning delay: F (5, 24) = 10.79, for LTM: F (5, 24) = 12.50, for fear-like behaviour: F (5, 24) = 16.12, for exploratory activity: F (5, 24) = 26.56. One-way ANOVA, individual comparisons by Sidak’s multiple comparisons test. (<b>d</b>) Ellipsoid bodies-specific <span class="html-italic">atg1</span> inhibition ameliorated rapamycin-evoked behavioural patterns (<span class="html-italic">n</span> = 3). Three-day-old flies were fed with rapamycin for 4 days. For the learning delay of <span class="html-italic">GH146</span>, <span class="html-italic">129Y</span>, <span class="html-italic">ok107</span>, <span class="html-italic">c232</span>, and <span class="html-italic">c205</span>, <span class="html-italic">c601</span>: F (3, 8) = 13.56, F (3, 8) = 55.56, F (3, 8) = 53.33, F (3, 8) = 13.83, F (3, 8) = 30.56, and F (3, 8) = 58.00, respectively. For the exploratory activity of <span class="html-italic">GH146</span>, <span class="html-italic">129Y</span>, <span class="html-italic">ok107</span>, <span class="html-italic">c232</span>, <span class="html-italic">c205</span>, and <span class="html-italic">c601</span>: F (3, 8) = 10.77, F (3, 8) = 15.15, F (3, 8) = 27.43, F (3, 8) = 8.175, F (3, 8) = 58.00, and F (3, 8) = 16.26, respectively. One-way ANOVA, individual comparisons by Sidak’s multiple comparisons test. For <span class="html-italic">atg1RNAi</span>: two-tailed Mann–Whitney test. (<b>e</b>) Ellipsoid bodies-specific <span class="html-italic">atg1</span> expression mimics rapamycin-evoked behaviours (<span class="html-italic">n</span> = 5). Three-day-old flies were used. For learning delay: F (4, 20) = 15.98, for LTM: F (4, 20) = 9.589, for fear-like behaviour: F (4, 20) = 11.67, for exploratory activity: F (4, 20) = 25.61. One-way ANOVA with Dunnette’s multiple comparisons test against <span class="html-italic">c232</span>; <span class="html-italic">UAS-atg1</span>. *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, and * <span class="html-italic">p</span> &lt; 0.05. Error bars represent SEM.</p> Full article ">Figure 2
<p>Ellipsoid bodies-specific 5HTR7 activity mediates the effects of neuronal <span class="html-italic">atg1</span> on behaviour and cognition. (<b>a</b>) QRT-PCR analysis of genes expressing serotonin and Dop1R2 receptors in <span class="html-italic">Drosophila</span> heads, normalized to <span class="html-italic">Rpl32</span> expression (<span class="html-italic">n</span> = 3). Ten-day-old <span class="html-italic">W<sup>Dah</sup></span> flies were fed with rapamycin for 4 days. Acute rapamycin treatment upregulated RNA levels of all receptor genes, with a major impact on <span class="html-italic">5htr7</span> expression. (<b>b</b>) Acute rapamycin treatment and low nutrient availability increase 5HTR7 levels in <span class="html-italic">Drosophila</span> head. Ten-day-old <span class="html-italic">W<sup>Dah</sup></span> flies were fed with rapamycin for 4 days or with low-nutrient food for 2 days. (<b>c</b>) <span class="html-italic">5htr7</span> is exclusively expressed in the ellipsoid bodies (dissected brain of 10-day-old <span class="html-italic">5htr7</span>; <span class="html-italic">UAS-sytegfp</span> flies, posterior view) in the brain of <span class="html-italic">Drosophila</span>. (<b>d</b>) <span class="html-italic">5htr7</span> upregulation mimics rapamycin-evoked cognitive and behavioural effects (<span class="html-italic">n</span> = 3). Ten-day-old flies were used. For learning delay: F (2, 6) = 13.30, for LTM: F (2, 6) = 9.091, for fear-like behaviour: F (2, 6) = 10.50, for exploratory activity: F (2, 6) = 12.64. One-way ANOVA with Dunnette’s multiple comparisons test against <span class="html-italic">5htr7</span>; <span class="html-italic">UAS-5htr7</span>. (<b>e</b>) <span class="html-italic">5htr7</span> inhibition ameliorates rapamycin-evoked cognitive and behavioural effects (<span class="html-italic">n</span> = 5). Ten-day-old flies were fed with rapamycin for 4 days. For learning delay: F (5, 24) = 15.76, for LTM: F (5, 24) = 9.059, for fear-like behaviour: F (5, 12) = 26.30, for exploratory activity: F (5, 24) = 55.31. One-way ANOVA, individual comparisons by Sidak’s multiple comparisons test. (<b>f</b>) 5htr7 inhibition ameliorates ellipsoid bodies-specific <span class="html-italic">atg1</span> effects on cognition/behaviour (<span class="html-italic">n</span> = 3). Three-day-old flies were used. For learning delay: F (4, 10) = 7.435, for LTM: F (4, 10) = 10.08, for fear-like behaviour: F (4, 10) = 8.577, for exploratory activity: F (4, 10) = 14.29. One-way ANOVA, individual comparisons by Sidak’s multiple comparisons test. Selected pairs: <span class="html-italic">c232</span>; <span class="html-italic">UAS-atg1</span> vs. <span class="html-italic">c232</span>; <span class="html-italic">UAS-atg1</span>; <span class="html-italic">UAS-5htr7</span>RNAi. (<b>g</b>) Acute feeding (2 days) of 10-day-old flies with a 5HTR7 specific inhibitor (SB269970) blunted <span class="html-italic">atg1</span>-induced learning deficits and enhanced exploratory activity (<span class="html-italic">n</span> = 3). Two-way ANOVA with Tukey’s multiple comparisons test, selected pairs: <span class="html-italic">c232</span>; <span class="html-italic">UAS-atg10</span> nM vs. <span class="html-italic">c232</span>; <span class="html-italic">UAS-atg1</span>50 nM. *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, and * <span class="html-italic">p</span> &lt; 0.05. Error bars represent SEM.</p> Full article ">Figure 3
<p>Serotonin cell-specific <span class="html-italic">atg1</span> expression mimics rapamycin and enhanced serotonergic signalling-evoked behaviours, while increasing 5HTR7 expression. (<b>a</b>) Serotonin cell-specific constitutive active S6K inhibits rapamycin-induced behavioural and cognitive effects (<span class="html-italic">n</span> = 3). Ten-day-old flies were fed with rapamycin for 4 days. For learning delay: F (5, 12) = 34.63, for LTM: F (5, 12) = 21.70, for fear-like behaviour: F (5, 12) = 8.671, for exploratory activity: F (5, 12) = 20.07. One-way ANOVA, individual comparisons by Sidak’s multiple comparisons test. (<b>b</b>) Serotonin cell-specific <span class="html-italic">atg1</span> expression induces behavioural and cognitive effects similar to rapamycin treatment (<span class="html-italic">n</span> = 3). Three-day-old flies were used. For learning delay: F (2, 6) = 21.00, for LTM: F (2, 6) = 36.33, for fear-like behaviour: F (2, 6) = 17.17, for exploratory activity: F (2, 6) = 13.88. One-way ANOVA with Dunnette’s multiple comparison test against <span class="html-italic">trh</span>; <span class="html-italic">UAS-atg1</span>. (<b>c</b>) <span class="html-italic">Trh</span>; <span class="html-italic">UAS-atg1</span> flies have increased expression of 5HTR7 in the heads. Three-day-old flies were used. (<b>d</b>) Serotonin transporter inhibition induces effects on behaviour and cognition similar to rapamycin treatment (<span class="html-italic">n</span> = 3). Three-day-old flies were used. For learning delay: F (2, 6) = 10.50, for LTM: F (2, 6) = 6.818, for fear-like behaviour: F (2, 6) = 14.60, for exploratory activity: F (2, 6) = 20.17. One-way ANOVA with Dunnette’s multiple comparison test against <span class="html-italic">trh</span>; <span class="html-italic">UAS-sertRNAi</span>. (<b>e</b>) Inhibition of serotonin transporter via RNAi increases 5HTR7 levels in <span class="html-italic">Drosophila</span> heads. Three-day-old flies were used. (<b>f</b>) Paneuronal <span class="html-italic">atg1</span> expression increases autophagy. RNAi inhibition of <span class="html-italic">atg7</span> reduces <span class="html-italic">atg1</span>-induced autophagy in the heads of <span class="html-italic">GSelav</span>; <span class="html-italic">UAS-atg1</span> flies (<span class="html-italic">GSelav</span>; <span class="html-italic">UAS-atg1</span>; <span class="html-italic">UAS-atg7RNAi</span> flies). Three-day-old flies were used. *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, and * <span class="html-italic">p</span> &lt; 0.05. Error bars represent SEM.</p> Full article ">Figure 4
<p>Rapamycin treatment and ellipsoid bodies-specific <span class="html-italic">atg1</span> expression reduce serotonin transporter localization on the plasma membrane, while peptidergic cell-specific <span class="html-italic">atg1</span> expression is lethal. (<b>a</b>) Acute rapamycin treatment (4 days) of 10-day-old <span class="html-italic">W<sup>Dah</sup></span> flies does not decrease SERT levels in <span class="html-italic">Drosophila</span> heads. (<b>b</b>) Acute rapamycin treatment (4 days) of 10-day-old <span class="html-italic">W<sup>Dah</sup></span> flies increases cytosolic levels of SERT, while decreasing its localization on the plasma membrane, at <span class="html-italic">Drosophila</span> heads. Values from imageJ analysis have been normalized to the total protein amount for each fraction and then normalized to the control values (<span class="html-italic">n</span> = 3). (<b>c</b>) Ellipsoid bodies-specific expression of <span class="html-italic">atg1</span> decreases fluorescent expression of a UAS-GFP-SERT fusion protein, while it does not affect the expression of UAS-SYTE-GFP. Ten-day-old flies were used. Posterior view of <span class="html-italic">Drosophila</span> brain. (<b>d</b>) <span class="html-italic">Dilp2</span>; <span class="html-italic">UAS-atg1</span>; <span class="html-italic">UAS-cd8rfp</span> flies (right) are smaller than the controls (left: <span class="html-italic">UAS-atg1</span>; <span class="html-italic">UAS-cd8rfp</span> flies). (<b>e</b>) Ten-day-old <span class="html-italic">dilp2</span>; <span class="html-italic">UAS-atg1</span>; <span class="html-italic">UAS-cd8rfp</span> flies have reduced fecundity (<span class="html-italic">n</span> = 3). F (2, 6) = 24.15. One-way ANOVA with Dunnette’s multiple comparison test against <span class="html-italic">dilp2</span>; <span class="html-italic">UAS-atg1</span>; <span class="html-italic">UAS-cd8rfp</span> flies. (<b>f</b>) <span class="html-italic">Dilp2</span>; <span class="html-italic">UAS-atg1</span>; <span class="html-italic">UAS-cd8rfp</span> mated female flies are long-lived. Median, mean, and maximum lifespans for <span class="html-italic">dilp2</span>; +: 69.5, 68, and 82 days, respectively, for <span class="html-italic">UAS-cd8rfp</span>; +: 69.5, 68, and 80 days respectively, for <span class="html-italic">UAS-atg1</span>: 71, 70, and 78 days, respectively, for <span class="html-italic">dilp2</span>; <span class="html-italic">UAS-atg1</span>; <span class="html-italic">UAS-cd8rfp</span>: 78, 76, and 85 days, respectively. Log-rank test analysis (<span class="html-italic">n</span> = 100). (<b>g</b>) <span class="html-italic">C929</span>; <span class="html-italic">UAS-atg1</span> flies are pharate lethals. They exhibit excessive cuticle sclerotization and extensive melanisation mainly at the pupal head, trachea, and wings imaginal discs. ** <span class="html-italic">p</span> &lt; 0.01. Error bars represent SEM.</p> Full article ">Figure 5
<p>Lowered mTORC1 and neuronal <span class="html-italic">atg1</span> expression increase the brain levels of extracellular serotonin and stimulate serotonergic neurotransmission. (<b>a</b>) Acute rapamycin treatment (4 days) of 10-day-old <span class="html-italic">W<sup>Dah</sup></span> flies does not alter total serotonin levels in <span class="html-italic">Drosophila</span> heads. Head homogenates were left in an EDTA-rich solution (20 mM) for 3 h at room temperature to check the degree of serotonin catabolism. For each biological sample, we used seven heads (<span class="html-italic">n</span> = 3). (<b>b</b>) Acute rapamycin treatment (4 days) of 10-day-old <span class="html-italic">W<sup>Dah</sup></span> flies increases extracellular serotonin in <span class="html-italic">Drosophila</span> brain. Total DNA and RNA levels in the supernatants subjected to ELISA measurements did not differ among the experimental conditions. For each biological sample, 40 brains were used (<span class="html-italic">n</span> = 9). Individual comparisons by one-tailed unpaired <span class="html-italic">t</span> test. (<b>c</b>) Paneuronal expression of <span class="html-italic">atg1</span> with the mifepristone-inducing <span class="html-italic">elavGS-gal4</span> driver, in the absence of mifepristone, increases extracellular levels of serotonin in <span class="html-italic">Drosophila</span> brains. Three-day-old flies were used. Total DNA and RNA levels in the supernatants subjected to ELISA measurements did not differ among the samples. For each biological sample, 40 brains were used (<span class="html-italic">n</span> = 3). F (2, 6) = 2.671. One-way ANOVA with Dunnette’s multiple comparison tests against <span class="html-italic">elavGS</span>; <span class="html-italic">UAS-atg1</span> flies. (<b>d</b>) Expression of <span class="html-italic">atg1</span> in prothoracic gland-innervating R29H01 serotonergic cells in <span class="html-italic">Drosophila</span> larvae decreases the size of pupae and adults, inhibits pupal colorization, and shrinks larval fat body (<span class="html-italic">R29H01</span>; <span class="html-italic">UAS-atg1</span> larvae, pupae and flies are indicated with arrows. <span class="html-italic">UAS-atg1</span> larvae, pupae and flies were used as controls). These phenotypes are reminiscent of flies with enhanced ecdysone signalling. * <span class="html-italic">p</span> &lt; 0.05. Error bars represent SEM.</p> Full article ">Figure 6
<p>Activation of the 5HTR7 receptor mediates rapamycin-induced dephosphorylation of NMDAR2 receptor and causes NMDA signalling inhibition-like effects in flies. (<b>a</b>) Four-day rapamycin treatment of 10-day-old flies reduces NMDA receptor 2 phosphorylation in Tyr1472 in <span class="html-italic">Drosophila</span> heads. (<b>b</b>) Inhibition of the <span class="html-italic">nmdar2</span> receptor gene mimics rapamycin-induced cognitive and behavioural effects (<span class="html-italic">n</span> = 3). Ten-day-old flies were used. For learning delay: F (2, 6) = 34.40, for LTM: F (2, 6) = 13.00, for fear-like behaviour: F (2, 6) = 11.40, for exploratory activity: F (2, 6) = 18.38. One-way ANOVA with Dunnette’s multiple comparisons test against <span class="html-italic">nmdar2</span>; <span class="html-italic">UAS-nmdar2RNAi</span>. (<b>c</b>) <span class="html-italic">5htr7</span> expression in NMDAR2-expressing cells mimics the effects of rapamycin on behaviour and cognition (<span class="html-italic">n</span> = 5). Ten-day-old flies were used. For learning delay: F (2, 6) = 13.87, for LTM: F (2, 6) = 9.000, for fear-like behaviour: F (2, 6) = 26.60, for exploratory activity: F (2, 6) = 52.33. One-way ANOVA with Dunnette’s multiple comparisons test against <span class="html-italic">nmdar2</span>; <span class="html-italic">UAS-5htr7</span>. (<b>d</b>) <span class="html-italic">5htr7</span> expression in NMDAR2-expressing cells enhances the lifespan in female mated flies. Median, mean, and maximum lifespans for <span class="html-italic">nmdar2</span>; +: 67, 69.1, and 85 days, respectively, for <span class="html-italic">UAS-5htr7</span>; +: 71.5, 71.4, and 89 days, respectively, for <span class="html-italic">nmdar2</span>; <span class="html-italic">UAS-5htr7</span>: 84, 81.5, and 93 days, respectively. Log-rank test analysis (<span class="html-italic">n</span> = 100). (<b>e</b>) RNAi inhibition of <span class="html-italic">5htr7</span> at NMDAR2-expressing cells blocks rapamycin-induced NMDAR2 dephosphorylation in <span class="html-italic">Drosophila</span> heads. Ten-day-old flies were fed with rapamycin for 4 days. *** <span class="html-italic">p</span> &lt; 0.001, ** <span class="html-italic">p</span> &lt; 0.01, and * <span class="html-italic">p</span> &lt; 0.05. Error bars represent SEM.</p> Full article ">Figure 7
<p>Rapamycin treatment induces cAMP/PKA signalling non-cell autonomously. (<b>a</b>) Rapamycin treatment-evoked behaviours are ameliorated by RNAi inhibition of Rutabaga adenylyl cyclase in NMDAR2-expressing cells (<span class="html-italic">n</span> = 3). For learning delay: F (5, 12) = 17.48, for LTM: F (5, 12) = 7.965, for fear-like behaviour: F (5, 12) = 28.43, for exploratory activity: F (5, 12) = 29.36. One-way ANOVA, individual comparisons by Sidak’s multiple comparisons test. (<b>b</b>) RNAi expression of the gene coding for the catalytic subunit of PKA (<span class="html-italic">pkac1</span>) ameliorates peptidergic cell-specific <span class="html-italic">atg1</span>-induced melanisation, cuticle hypersclerotization, and developmental death. (<b>c</b>) Starvation, rapamycin feeding, and neuronal <span class="html-italic">atg1</span> expression cause epidermal melanisation in larvae. (<b>d</b>) Paneuronal <span class="html-italic">atg1</span> and rapamycin treatment induce epidermal cAMP. Rapamycin treatment induces cAMP-related melanisation in scabrous-expressing neuroblasts, in a non-cell autonomous way. Larvae were screened for CFP fluorescence and epidermal melanisation under a ZEISS/Axioskop 2 Plus microscope, with a DAPI filter. Then, samples were further analysed with confocal microscopy. *** <span class="html-italic">p</span> &lt; 0.001 and ** <span class="html-italic">p</span> &lt; 0.01. Error bars represent SEM.</p> Full article ">Figure 8
<p>Rapamycin treatment alters behaviour and cognition in zebrafish via serotonergic signalling. (<b>a</b>) Rapamycin injection delays learning and inhibits long-term memory (<span class="html-italic">n</span> = 8). Two-tailed Mann–Whitney test. (<b>b</b>) Prolonged training abrogates rapamycin-induced LTM impairment. Two-tailed Mann–Whitney test (<span class="html-italic">n</span> = 7). (<b>c</b>) Rapamycin-injected zebrafish spent more time in the top area of a water tank (novel tank test) (<span class="html-italic">n</span> = 10). Two-tailed Mann–Whitney test. (<b>d</b>) Rapamycin injection increases HTR1B expression in the brain of zebrafish, as well as RNA levels of <span class="html-italic">htr1b</span> (<span class="html-italic">n</span> = 3). (<b>e</b>) GR55562 mild treatment of rapamycin-injected zebrafish abrogates learning defects, and galantamine hydrobromide enhances rapamycin-induced learning defects, while Prozac, although it worsened learning ability compared to controls (<span class="html-italic">p</span> &lt; 0.05), did not significantly further enhance rapamycin-induced learning defects. Day 6 of training protocol (<span class="html-italic">n</span> = 9). Two-tailed Mann–Whitney test. (<b>f</b>) GR55562 mild treatment abrogates altered the swimming behaviour of rapamycin-injected zebrafish (<span class="html-italic">n</span> = 9). F (3, 26) = 3.734. One-way ANOVA with Dunnette’s multiple comparison tests against untreated zebrafish. (<b>g</b>) Rapamycin injection decreased pTyr1472 phosphorylation of NR2B in the forebrain, while it increased HTR1B in the midbrain and forebrain. Mild treatment of rapamycin-injected zebrafish with GR55562 reduced rapamycin-induced pTyr1472 dephosphorylation of NR2B in whole brain extracts. F (2, 12) = 4.491. One-way ANOVA with Dunnette’s multiple comparison test against untreated zebrafish (<span class="html-italic">n</span> = 5). *** <span class="html-italic">p</span> &lt; 0.001 and * <span class="html-italic">p</span> &lt; 0.05. Error bars represent SEM.</p> Full article ">Figure 9
<p>Acute mTORC1 inhibition enhances ATG1 at serotonergic cells, which inhibits serotonin transporter (SERT) activity and results in increased extracellular serotonin and expression of postsynaptic 5HTR7. The latter enhances cAMP/PKA signalling: (a) at NMDA2 receptor expressing cells in the brain, where it inhibits NMDA signalling and induces behavioural/cognitive modulations, and (b) at prothoracic gland cells, where it stimulates ecdysone (20E) signalling and systemic catabolism. Both effects enhance longevity and physiological adaptations to nutrient deprivation.</p> Full article ">

Review

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Review
An Overview of Golgi Membrane-Associated Degradation (GOMED) and Its Detection Methods
by Hajime Tajima Sakurai, Satoko Arakawa, Hirofumi Yamaguchi, Satoru Torii, Shinya Honda and Shigeomi Shimizu
Cells 2023, 12(24), 2817; https://doi.org/10.3390/cells12242817 - 11 Dec 2023
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Abstract
Autophagy is a cellular mechanism that utilizes lysosomes to degrade its own components and is performed using Atg5 and other molecules originating from the endoplasmic reticulum membrane. On the other hand, we identified an alternative type of autophagy, namely, Golgi membrane-associated degradation (GOMED), [...] Read more.
Autophagy is a cellular mechanism that utilizes lysosomes to degrade its own components and is performed using Atg5 and other molecules originating from the endoplasmic reticulum membrane. On the other hand, we identified an alternative type of autophagy, namely, Golgi membrane-associated degradation (GOMED), which also utilizes lysosomes to degrade its own components, but does not use Atg5 originating from the Golgi membranes. The GOMED pathway involves Ulk1, Wipi3, Rab9, and other molecules, and plays crucial roles in a wide range of biological phenomena, such as the regulation of insulin secretion and neuronal maintenance. We here describe the overview of GOMED, methods to detect autophagy and GOMED, and to distinguish GOMED from autophagy. Full article
(This article belongs to the Special Issue Molecular Mechanisms and Biological Roles of Alternative Autophagy)
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Figure 1

Figure 1
<p>Comparison of autophagy and Golgi membrane-associated degradation (GOMED). Both autophagy and GOMED proceed in the following order: (1) phagophore membrane formation, (2) membrane elongation, (3) autophagosome(-like) structure formation, and (4) autolysosome(-like) structure formation (fusion with lysosomes). Both pathways start with Ulk1, and are induced via the PI3K complex. Autophagy originates from the ER membrane, and the phagophore membrane is elongated via Wipi1/2. Downstream of Wipi1/2, Atg5 is an essential molecule, and LC3 binds to the phagophore membrane in an Atg5-dependent manner, contributing to autophagosome formation. In GOMED, autophagosome(-like) structure formation proceeds in a Wipi3- and Rab9-dependent manner using the Golgi membrane.</p> Full article ">Figure 2
<p>GOMED induction during DNA damage is mediated by the phosphorylation of Ulk1 serine<sup>746</sup> by RIPK3. Schematic diagram of GOMED induction by DNA damage. DNA damage induces the p53-dependent upregulation of PPM1D and RIPK3; PPM1D induces dephosphorylation of the 637th serine residue of Ulk1, which results in the induction of both autophagy and GOMED. Subsequent phosphorylation of the 746th serine residue of Ulk1 activates GOMED, whereas the absence of this phosphorylation leads to canonical autophagy.</p> Full article ">Figure 3
<p>Phenotype of Wipi3-deficient mice. (<b>A</b>) Mice lacking Wipi3 show loss of limb-clasping reflex. (<b>B</b>) Mice lacking Wipi3 had degenerative loss of cerebellar Purkinje cells (calbindin staining). (<b>C</b>) Purkinje cells in mice lacking Wipi3 showed iron deposition and ceruloplasmin accumulation. (Modified from [<a href="#B26-cells-12-02817" class="html-bibr">26</a>]).</p> Full article ">Figure 4
<p>Visualization of autolysosome(-like) structures by mRFP-EGFP. (<b>top</b>) Schematic diagram of the method using mRFP-EGFP. mRFP-EGFP can localize on phagophore(-like) structures and autophagosome(-like) structures. In autolysosome(-like) structures, only EGFP is quenched and degraded in an acidic environment. (<b>bottom</b>) GOMED is induced in mRFP-GFP-expressing Atg5<sup>KO</sup> MEFs. mRFP-EGFP can be detected as red only in autolyssome(-like) structures, because only EGFP is quenched and degraded. The right panel is an enlarged image of the boxed area in the left panel. White arrowheads indicate GOMED structures. (Modified from [<a href="#B7-cells-12-02817" class="html-bibr">7</a>]).</p> Full article ">Figure 5
<p>Detection of autophagy and GOMED dynamics by FLAD. Schematic model of FLAD. When GOMED is induced after the expression of EGFP in the cytoplasm, GFP is not confined within the phagophore(-like) structures, but once autophagosome(-like) structures are formed, EGFP is confined and sequestered from the surroundings. When a portion of the cell (ROI) is photobleached with a 488 nm laser, EGFP in the ROI is quenched. By continuous photobleaching, EGFP fluorescence outside the ROI is also reduced owing to free diffusion of the EGFP molecules. On the other hand, EGFP sequestered in autophagosome(-like) structures cannot diffuse, and is detected as puncta that retain their fluorescence intensity.</p> Full article ">Figure 6
<p>Visualization of GOMED-associated structures by DAPGreen, DAPRed, and DALGreen. Illustration showing the steps of GOMED that are visualized by each probe (bottom left images). When Atg5<sup>KO</sup> MEFs were stained with DAPGreen/DAPRed and GOMED was induced, early GOMED structures were labeled with DAPGreen alone, and later structures were costained with DAPGreen/DAPRed.</p> Full article ">
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