LOOKING AT THE CHAPERONE-MEDIATED AUTOPHAGY IN FISH – Fish-and-Chap

In silico analyses: Blast search, Phylogenetic and syntenic analyses

Medaka breeding and treatments:
5/8-month-old male and female CAB medaka (Oryzias latipes), wild type or knockout Lamp2a, were used. Fish were raised at 26°C under a growing photoperiod (12h light / 12h dark) and fed with commercial diet three times a day during breeding. Fish were starved for 16 or 24 hours. For organs sampling, fish were euthanized by immersion in a lethal dose of tricaine (MS-222) at 400 mg/L. All experimental procedures were conducted in strict accordance with the legal frameworks of France and the European Union. They respect the directive 2010/63/EU relating to the protection of animals used for scientific purposes as well as the decree No 2013-118, 1 February 2013 of the French legislation governing the ethical treatment of animals. The protocol was approved by the French National Consultative Ethics Committee under the reference number 10494-2017042809304363.


Gene expression analyses: RT-qPCR

Western blot analysis

Histology and electron microscopy

Medaka fibroblast cell line culture and transfection

Imaging procédures: MRic Photonics platform (Microscopy Rennes imaging center) with DeltaVision Elite high resolution microscope (GE Healthcare)

Statistical analysis: For comparisons of more than two groups, we used one-way ANOVA with a Tukey Post-hoc test (parametric) or Pairwise Wilcoxon signed rank test with Bonferroni p-value adjustment (non-parametric). For comparisons between two groups, we used Student’s t test (parametric) or Kruskal-Wallis rank sum test (non-parametric) and Wilcoxon signed rank test (non-parametric).

We demonstrated that LAMP2 emerged from the second round of whole genome duplication that occurred concomitantly with the origin of the vertebrate lineage approximately 500 million years ago. The sequence analysis of the complete genomes of several fish species also revealed high conservation of the genomic organization of the LAMP2 gene across vertebrates. Finally, we conducted a fine expression analysis of the newly characterized lamp2a splice variants in different fish species. The results show that they are expressed in different tissues of a large variety of ray-finned fish species, and therefore imply that CMA function might have appeared much earlier during evolution than initially thought.

We next addressed the existence of functional CMA or a CMA-like pathway in fish. For this purpose, we first characterized the developmental and tissue-specific expression pattern of lamp2a mRNAs in the medaka fish model species. The obtained data clearly demonstrated that lamp2a is ubiquitously expressed during early medaka development and in adult tissues. We then transiently transfected medaka fibroblast cells (OLF cell line) with a KFERQ-CMA-targeting motif fused to a photoactivable mCherry1 reporter, developed to specifically track and evaluate CMA activity in mammalian cells. Our results show that upon starvation, this KFERQ-CMA reporter accumulates in characteristic puncta that co-localize within acidic compartments (lysosomes and/or late endosomes) of the cells. Moreover, being totally dependent of lamp2a expression, our sets of experiments strongly support the existence of a functional CMA activity in fish. Finally, we also demonstrate that lamp2a knockout medakas exhibit pronounced alterations in hepatic carbohydrate and lipid metabolisms in a physiological readout that is similar to what has been observed in mice with defective hepatic CMA.

Overall, our results univocally demonstrate that the CMA function is definitively not restricted to mammals and birds.

Outstanding feature:

Our results shed new light on the evolutionary history of LAMP2A and demonstrate that it appeared at the basis of the vertebrate lineage approximately 500 million years ago, challenging the currently accepted view and suggesting that CMA likely appeared much earlier during evolution than initially thought.
In this regards, we also provide evidences for a functional CMA process in the fish Medaka and show that it plays a critical role in the control of the hepatic metabolism, through the degradation of some key enzymes involved in the use and storage of energy resources, as previously demonstrated in mice.
Overall, these data bring significant conceptual changes and new perspectives in the field, notably the promising use of fish as pertinent model organisms to investigate CMA function as much as either considering fundamental aspects or evolutionary perspectives on the autophagic routes within the different phyla.

They already resulted in the publication of a first article in the journal Autophagy and a second paper is currently under review in Molecular Biology and Evolution.

Future prospect:

Depending on the comments of the reviewers, we will have to carry out new experiments to complete the manuscript. We will also seek to characterize the CMA protein substrates in fish in accordance to the step 4 of the task2 proposed in the project.

Scientific article in peer reviewed international journals:

Lescat L, Herpin A, Mourot B, Véron V, Guiguen Y, Bobe J, Seiliez I. CMA restricted to mammals and birds: myth or reality? Autophagy. 2018; 14(7): 1267-1270. doi: 10.1080/15548627.2018.1460021.

Communication in international conférences:

1. Laury Lescat, Amaury Herpin, Yann Guiguen, Julien Bobe, Iban Seiliez. Lamp2a in the light of evolution. 7th Scientific days on Autophagy, Nov 27-29. 2017, Paris (France) – Flash Talk + Poster
2. Laury Lescat, Brigitte Mourot, Vincent Véron, Karine Dias, Natàlia Riera, Yann Guiguen, Julien Bobe, Amaury Herpin, Iban Seiliez. On the existence of chaperone-mediated autophagy in fish. Keystone meeting on autophagy February 17-21 2019, Santa Fe, NM (USA) - Poster

Communication in national (France) conférences:

1. Laury Lescat, Amaury Herpin, Yann Guiguen, Julien Bobe, Iban Seiliez. Lamp2a in the light of evolution. Deuxième journée de rencontre du Club d'Autophagie Bordelais. 11 Avril 2017. Bordeaux (France) - Oral
2. Laury Lescat, Amaury Herpin, Brigitte Mourot, Vincent Véron, Yann Guiguen, Julien Bobe, Iban Seiliez. Lamp2A à la lumière de l’évolution. Journées d’Animation Scientifique du département PHASE de l’INRA. 4-5 Avril 2018, Rennes (France) - Poster

Chaperone-Mediated Autophagy (CMA) is a major pathway of lysosomal proteolysis recognized as a key player of the control of intermediary metabolism. To date, this cellular function is presumed to be restricted to mammals and birds, due the absence of an identifiable LAMP2A, a limiting and essential protein for CMA, in phylogenetically earlier species. However, we recently identified the existence of lamp2a in several fish species. In this context, we propose to define for the first time whether or not a CMA (or a CMA-like) process is ancestral to mammalian/bird species and determine the physiological relevance of the newly identified lamp2a homolog in fish with respect to that process.
To address this issue, our strategy will be based on two complementary approaches. We will first characterize the repertoire and the expression of genes involved in CMA in a large number of fish species of agronomical, ecological and scientific interest. The growing number of fish species whose genome was completely sequenced as well as our advances in transcriptome analysis allows including in our study a large number of fish species and thus to provide a comprehensive picture of the “genetic structure” of CMA in fish. Secondly, we will decipher the physiological role of the newly identified lamp2a homolog in fish. Recently, we have generated knockout medaka (Oryzias latipes) for the corresponding lamp2a splice variant of the lamp2 gene, by using the genome-editing tool CRISPR-Cas9. We will therefore perform an exhaustive phenotyping of the lamp2a mutant medaka (at both histological, biochemical and molecular levels) in order to determine the metabolic changes induced by the deletion of lamp2a and the possible existence of a CMA activity in medaka.
This project gathers two partners (UMR1419 NuMeA and UR1037 LPGP) with highly complementary expertise and skills in autophagy, fish genomics, gene editing technology and fish metabolism. A major aim of the team members is to put together and mutually benefit from complementary scientific as well as technical expertise of each one of the scientists involved in this project. The present consortium has thus the required complementarity and added value to successfully carry out the present project. The involvement of a PhD student in the present project will also strengthen the cohesion of the consortium.
Overall this project, which does not present any particular risk, will demonstrate the existence of a CMA activity - not yet suspected - in fish. Addressing the role of lamp2a and the possible existence of CMA activity in fish will represent a major breakthrough in our understanding of the mechanisms involved in the control of metabolism in these species and provide the necessary fundamental knowledge to cope with the challenges of aquaculture. Moreover, the comparative functional genomics approach across phylogenetical distant species will provide an entirely new aspect of the autophagy research by exploring the extent to which the CMA network has diverged during vertebrate evolution. Finally, the national and international partnership network of the members involved in this project will guarantee an effective dissemination of the obtained results to both the aquaculture industry and the academic partners.