Modulating autophagy to treat CV disease

Brought to you by the Fondation Leducq Transatlantic Network of Excellence

Research Exchange visit of Dr. Julio Madrigal-Matute from Dr. Cuervo‘s group in NY, USA to the lab of Dr. Kroemer in Paris, France.

The purpose of this Leducq fellow exchange was to perform experiments related to the project “CMA and calorie mimetics in the protection against cardiovascular disease-associated metabolic dysregulation” funded within the Leducq Young Investigator grant application. Some of the experiments proposed in the project required  expertise and technical skills well established in the Kroemer lab. Therefore, it was essential for the successful outcome of the project the visit of Dr. Madrigal-Matute to the the Kroemer lab to complete part of this project and acquire expertise in metabolomic analysis.

Metabolomic analysis: Previous data from the Cuervo lab has shown that Chaperone-mediated autophagy (CMA) regulates glucose and lipid metabolism. Key metabolic enzymes are degraded through CMA and therefore we proposed that decline of CMA with aging or high dietary lipids is involved in the development of metabolic dysfunction. The role of CMA in the response to high carbohydrate challenges, typical from the western countries, is yet to be explored. Interestingly, Dr. Madrigal-Matute’s preliminary data suggest that CMA might have an essential role in the protection against the metabolic syndrome resulting from a chronic consumption of elevated levels of carbohydrates. In this context, the project was designed to  investigate in detail the metabolic dysregulation occurring in mice that lack CMA in response to a high carbohydrate diet. To that end, we performed a state-of-the-art metabolomic approach of samples from CMA-deficient mice (LAMP-2KO) and wild type (WT) littermates that have been fed for 8 weeks with either chow or high carbohydrate diet. These mice showed a marked metabolic dysfunction and tissue samples and serum have been already collected and stored. The expertise from Kroemer lab was key for the correct performance of the analysis and interpretation of the data. Training of Dr. Madrigal-Matute in these procedures was extremely beneficial for his currently ongoing project as well as his future independent research program.

The results obtained from this project were presented in the Leducq Meeting in Croatia, 2017 and will be incorporated as part of a manuscript in preparation describing the role of CMA in modulation of carbohydrate metabolism.

Modulation of CMA activity by acetylase and deacetylases: Calorie restriction (CR) in mice promotes constitutive CMA activation (Kaushik et al. unpublished) suggesting that upregulation of this type of autophagy could be one of the many factors behind the beneficial effects of CR in life- and healthspan extension. Caloric restriction mimetics (CRMs) are drugs that reproduce the biochemical effects of nutrient deprivation as they are able to reduce the levels of lysine acetylation of the cellular proteome. As part of the collaborative efforts between the Kroemer and Cuervo labs and under the Leducq Young Investigator Award, we have investigated the effect of CRMs on CMA and found that prototypical CRMs including spermidine, hydroxycitrate and sodium salicylate activate CMA in fibroblasts (3T3-NIH cells). To identify and characterize at the molecular level the acetyltransferases/deacetylases that were responisible for the CMA inducing effect of CRMs, we performed siRNA screening with  libraries of acetylases/deacetylases available from the Kroemer lab and 3T3-NIH cells with the KFERQ-Dendra reporter from Cuervo lab which allows to measure CMA activity and we have already adapted to high content microscopy analysis. The expertise in the Kroemer lab using siRNA screen libraries of acetylases/deacetylases and the availability of the BD Attovision Imaging System combined with Dr. Madrigal-Matute’s extensive knowledge on the usage of the KFERQ-Dendra reporter was essential for the succesful development of this project. These studies have led to the identification of two acetylation modifying enzymes that when knock-down from the cell result in reproducible changes on CMA. Dr. Madrigal-Matue will continue the characterization of these enzymes and we anticipate a manucript with this findings will be submitted by the end of this year.

The results obtained from this project were presented in the Leducq Meeting in NY, 2018.

 

 

Fellow Exchange of Salwa Sebti to the lab of Dr. Junichi Sadoshima at Rutgers New Jersey Medical School in Newark, New Jersey, USA

My postdoctoral research is focused on investigating the molecular mechanisms of mitophagy and the role of mitophagy in cardiovascular and other diseases.

Dr. Sadoshima’s laboratory has significant expertise in precisely quantifying the degradation of mitochondria by autophagy (mitophagy), in mouse hearts using the fluorescent marker, mito-Keima.

I therefore visited the laboratory of Dr. Sadoshima in March 2017 to learn this important technique to measure basal mitophagy levels in the heart and other tissues. The Postdoctoral Research Fellow, Dr.Toshiro Saito, accompanied me throughout my journey at Rutgers University; he taught me all procedures and how to use the platform for mitophagy analyses and introduced me to his colleagues with expertise in different steps of mitophagy analyses. More particularly, he showed me how to quantify the ratio of mito-Keima fluorescence in vivo and determine mitophagy levels in cardiac tissues.

My visit to Dr Sadoshima’s laboratory was really instructive and facilitates the success of my postdoctoral research project by providing me expertise in an essential tool for measuring mitophagy in vivo. I am indeed very grateful for this wonderful training experience and thank Dr. Sadoshima for organizing and hosting my stay in his lab.

 

Fellow exchange of Toshiro Saito to the lab of Dr. Luca Scorrano at Venetian Institute for Molecular Medicine Room Padova, Italy

Based upon my clinical experience as a cardiologist, I started the basic research of cardiovascular medicine in 2007 in Japan. Since then, I have expanded my field of interest from vascular biology to heart disease and mitochondrial biology. At present, I am engaged in the research of mitophagy/autophagy as a postdoctoral fellow in the laboratory of Prof. Sadoshima at Rutgers New Jersey Medical School in the USA.

Thanks to his guidance, I have successfully developed insight into the molecular basis of mitophagy/autophagy in the ischemic heart. In the near future, I would like to go back Japan, advance this field with my PhD students, and expand my interest to vascular biology and stem cell research. For these reasons, the mitochondrial biology in other organs is of great interest to me.   

In November 2017, I visited the laboratory of Prof. Scorrano at the Venetian Institute for Molecular Medicine in Italy. Mitochondrial dynamics, especially fusion/fission mechanism, were intensively investigated there in various organs. His kind fellows welcomed me and introduced their fine projects, which clarify how mitochondrial events govern the cellular homeostasis. The stem cell research regarding the hair follicle still remains vividly in my mind since the phenotype of knockout mice was obvious from their appearance. Although I am not familiar with cancer biology, their projects which connect the resistance to chemo-therapy with mitochondrial function has attracted me a lot. Apart from the cardiomyocytes, these cells possess the frequent turn-over of mitochondria. They may be better subjects than cardiomyocytes for the drug screening to target mitochondrial dynamics.

Despite the short stay, this great experience inspired me and gave precious guidance for my future direction. I greatly appreciate Profs. Sadoshima and Scorrano for this opportunity.

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Fellow exchange of Lorenza Tsansizi to the lab of Dr. Guido Kroemer at the Centre De Recherche Les Cordelier, Paris

In the laboratory of Professor Guido Kroemer groundbreaking research in cancer and metabolism with state-of-the-art facilities and techniques is performed. In December 2017 I spent two weeks in Centre De Recherche Les Cordelier in Paris, in order to perform metabolomic experiments on the project of my PhD thesis, which is part of our Leducq network on “Modulation of autophagy to treat cardiovascular disease”. More precisely, one of the main aims of my PhD project is to study the role of a phosphatase important for the catabolism of Branched Chain Aminoacids in autophagy, and to that end I needed a fluxomics approach. Fluxomics is a recent high-through put approach to determine the metabolic fluxes of a big number of metabolites. Therefore, during my stay, I visited also Prof. Kroemer’s metabolomic platform in Gustave Roussy Comprehensive Cancer Institute in Paris, where experts perform all the activities required for the project, including measurements, analysis and interpretation of results.

My stay was a great experience not only from a professional and experimental point of view, but also from a collaborative one. I am most thankful to Dr. Federico Pietrocola for his help and constant availability when needed. It was of an immense benefit for me to be able, even for such a short period, to work and net-work within this excellent environment with great such great researchers.

 
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Research exchange of Dr. Nina Kaludercic with the labs of Dr. Ana Maria Cuervo and Dr. Richard Kitsis at the Albert Einstein College of Medicine, New York

In September 2016, Dr. Julio Madrigal-Matute (from the Cuervo Lab) and I were awarded the Junior Investigator Award for the project entitled “A role for chaperone-mediated autophagy (CMA) in monoamine oxidases degradation - potential relevance for heart disease”. As part of this collaboration, in April and May 2017 I spent six weeks in the Cuervo Lab where I learned some of the techniques required to perform the experiments outlined in our project and used the tools developed in the Cuervo Lab for the investigation of CMA. During my time in the Cuervo Lab, I extensively interacted with Julio and other members of the lab and learned in detail about the techniques employed to investigate not only CMA, but also autophagy in general. The time spent in Ana Maria’s Lab was very fruitful: with Julio’s help, we generated a set of preliminary data that lays the groundwork for our project. These results will be presented at the biannual meeting of our Leducq network in September 2017 in Cavtat, Croatia.

During my time at Einstein, I also spent two additional weeks in the Kitsis Lab where I extensively interacted with Dr. Yun Chen, the Leducq Fellow in charge of the project focused on CMA and mitochondria. Combining Yun’s and my expertise, we developed and set up assays for the study of mitochondrial function in organelles isolated from mouse heart. Moreover, in a joint effort with Julio from the Cuervo Lab, the three of us made significant progress in setting up the method for isolating CMA active and CMA inactive lysosomes from the mouse heart.

Overall, the time I spent at Einstein enriched me scientifically and personally. I learned about multiple topics and techniques and had the opportunity to interact with amazing scientists working in autophagy and cardiovascular fields. This short exchange helped in starting the project and I hope that further exchanges will be possible in order to speed-up our training, data acquisition and project development.

 (From left to right) Drs. Madrigal-Matute, Kaludercic, and Chen at the Albert Einstein College of Medicine in NYC

(From left to right) Drs. Madrigal-Matute, Kaludercic, and Chen at the Albert Einstein College of Medicine in NYC






Research Exchange to the Cuervo Lab in New York by Bianca Sander

I have just started my PhD in May 2016 during which I will investigate the importance of chaperone-mediated autophagy (CMA) in atherosclerosis. The cooperation of my supervisor Dr. Judith Sluimer with Prof. Ana Maria Cuervo gave me the opportunity to learn the most important techniques for working with CMA. Having the chance for a work exchange in a lab with such a great expertise so early in my PhD was really great. When I arrived I was warmly welcomed by Ana Maria, Julio and all the other lab members so that I felt like part of the team from the first day onwards. During the six weeks in the Cuervo Lab I benefitted immensely from Ana Maria’s and Julio’s extensive knowledge of CMA techniques. Likewise, the Cuervo lab benefitted from the expertise of the Sluimer lab in atherosclerosis with hands-on help with sacrifice, sectioning and quantification of atherosclerosis in mice with gain- or loss-of-function of CMA. 

I was able to perform first analyses of the levels of CMA-related proteins in atherosclerosis as well as to establish the best working conditions for determining the CMA activity in macrophages with a fluorescent reporter. Consequently, I am able to set-up techniques in my home lab and received insight and inspiration for follow-up experiments. In the future, a second research visit may be required as 6 weeks were not fully sufficient to acquire all methodology and decisive data. However, this research visit was crucial to align techniques for CMA and atherosclerosis, and firmly established ground for an active and fruitful collaboration. This exchange also opened other opportunities, like attending the “2016 Dr. Paul Janssen Award Symposium” to hear presentations of both Dr. Cuervo Ana Maria, and the 2016 Nobel Prize winner Dr. Ohsumi on groundbreaking research in autophagy.

In conclusion, this 6-week exchange added so much to my experience as a scientist since it was an excellent chance for networking in the field of autophagy as well as broadening my repertoire of techniques with novel methods like the fluorescent CMA reporter assay.