Grant reports

Snapshots from NAS Conference grant receivers:

Report from Lab Exchange Grantee Florentina Negoita

The role of SIK2 in autophagy and its link to human adipose tissue dysfunction in the development of type 2 diabetes

Host: Pr. Eeva-Liisa Eskelinen (University of Turku, Finland)

Duration of visit: 2 weeks (September 30 – October 11, 2019)

The aim of this project is to characterize the molecular mechanisms underlying the regulation of autophagy by SIK2 in adipocytes. Transmission electron microscopy (TEM) is currently considered to be the gold standard test for the evaluation of autophagy, but identification of autophagic structures is challenging, without proper expertise, and might lead to false conclusions. Thus, in order to learn how to correctly identify and differentiate autophagic structures from other cellular structures and how to quantify the TEM images, a research visit was organized in the lab of Pr Eeva-Liisa Eskelinen, a leading scientist on TEM imaging of autophagic structures. Adipocytes treated with a SIK inhibitor were imaged and images were then analyzed. We found a marked change in the number and volume of autolysosomes, in line with our previous data. With the protocol for TEM sample processing obtained through this collaboration, we are now planning to further explore our findings by establishing the TEM method in our own laboratory, and applying it to 3T3-L1 adipocytes treated with SIK2 siRNA. We will maintain an active collaboration with Pr Eeva-Liisa Eskelinen. In conclusion, the research visit was very successful. I received excellent training in TEM analysis of autophagic structures, and we also generated novel, interesting data, which has opened up a new direction of study in the project.

Florentina Negoita (2nd from the left) with the group of Prof Eeva-Liisa Eskelinen (1st from the left)

Report from Lab exchange grantees Pauline Verlhac Yahyah Aman

One of the aims of the Nordic Autophagy Society (NAS) is to promote excellent research in the autophagy field. To achieve this goal, the NAS offer NAS Lab Exchange Grants (https://nordicautophagy.org/lab-exchange-grants/), giving the opportunity for a research stay in another group in a period from 5 to 90 days. These research exchanges allow the application of methodologies which are not available in the NAS member’s laboratory, and eventually they will facilitate valuable collaborations between research groups. Recently, 2 NAS members, Dr. Pauline Verlhac and Dr. Yahyah Aman, have been awarded with the NAS Lab Exchange Grants, and their corresponding scientific reports can be read below.

– Pauline Verlhac (University Medical Center Groningen, The Netherlands)

WIPIng out Flu: Role of WIPI2 in restricting Influenza A virus

Host: Prof. Jörn Dengjel (University of Fribourg, Switzerland)

Duration of visit: 7 days (June 16th-23rd 2019)

The ultimate goal of the project is to decipher how WIPI2 controls viral replication, using influenza A virus (IAV) as a relevant clinical model. Therefore, we plan to use an unbiased and broad approach to identify potential WIPI2 binding partners involved in the control of IAV infection. To do so, we have used stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS) with proximity-dependent biotin labelling by fusing WIPI2 with APEX2 in order to identify interacting proteins in both infected and non-infected cells. To determine which WIPI2 neighbours are regulated by IAV infection, we combined APEX2 with SILAC-based MS. Neighbours identified thanks to this Lab Exchange will now be tested for their impact on IAV replication by siRNA interference and their interaction with WIPI2 confirmed by IP. The work was realised with the help of Devanarayanan Siva Sankar, who taught the experimental part, and Professor Jörn Dengjel for the statistical data analysis. The exchange allowed me to implement a new and innovative technique into our laboratory as well as to strengthen the collaboration between our two laboratories.

From left to right: Devanarayanan Siva Sankar, Dr Pauline Verlhac and Prof Jörn Dengjel

– Dr. Yahyah Aman (University of Oslo, Norway)

Is mitophagy protective against tau aggregation in Alzheimer’s disease?

Host: Dr. William A. McEwan (University of Cambridge, United Kingdom)

Duration of visit: One month (May 6th to June 7th 2019)

Mitochondrial quality is tightly regulated by mitophagy, in which damaged mitochondria are degraded and recycled. However, the underlying mechanism remains elusive. We wished to investigate how mitophagy inhibits p-tau using a novel in-vitro system. For this purpose, an overseas visit to Dr McEwan’s lab was arranged in order to acquire and apply high-content quantitative assays for measuring cytosolic tau aggregation in situ. This assay not only allows to assess the impact of mitophagy inducers on the development of p-tau aggregates; but also provide a platform to evaluate whether mitophagy can aid in degradation of already established tau aggregates, which further enhances the translational relevance. The assay was used with success during my stay and already provided some interesting results. In collaboration with Dr McEwan’s lab, we will further explore on the underlying molecular mechanisms that contribute to p-tau pathogenesis with the perspective of the mitophagy and its role using this exciting seeded aggregation of tau model system.

The McEwan group visited by Dr. Yahyah Aman in May-June 2019