FellowDr PhD Michal Cagalinec
Project NameMitochondria-endoplasmic reticulum functional interplay in Wolfram Syndrome: emerging role for heart and brain protection
Host organisationBioscience Center SAS
Duration of the project24.03.2015 - 31.12.2018

Abstract
Wolfram syndrome (WS) is a recessive neurological disorder caused by mutation of the Wfs1 gene. The Wfs1 protein is highly expressed in the brain and heart and is embedded in the endoplasmic reticulum (ER) where it modulates Ca2+ levels and ER stress. Additionally, the main symptoms of the WS are consistent with the ones characteristic for mitochondrial diseases. In fact, our preliminary results showed already that silencing of Wfs1 in mouse neurons decreased the mitochondrial fusion frequency and caused mitochondrial fragmentation, demonstrating strong impact of Wfs1 to mitochondrial function in neurons. Although the high expression of Wfs1 in the heart and cardiac symptoms in WS identified recently emphasize the functional importance of Wfs1 in the heart, the most common causes of morbidity in WS are the neurological manifestations. How is it possible that mutations of Wfs1 causing significant perturbations in the brain functions are not so prominent in the heart? One explanation of this tissue specificity is that a mechanism compensating for loss of Wfs1 protein function is present in the heart but not in neurons. Therefore in this project we propose to study if the knockout of the Wfs1 gene in mice leads to disturbance in contractile properties of the heart both at organ and cell level. As Wfs1 impacts mitochondrial dynamics and morphology in neurons, we plan to analyse mitochondrial dynamics and ultrastructure in Wfs1 deficient myocytes. To understand the functional link of Wfs1 to mitochondria in neurons and myocytes we aim to answer if this link is mediated by direct ER-mitochondria interaction and/or by calcium. Mitochondria-ER contact sites will be analysed by electron microscopy. The role of calcium will be elucidated by measuring of myocyte cytoplasmic calcium transients using confocal microscopy and the interaction of Wfs1 with the two most abundant calcium channels in ER - the IP3- and ryanodine receptors will be resolved by methods of molecular biology

Project Summary with Interim Results

In line with the project objectives, in this period we have fixated and obtained transmission electron microscopy images from the left ventricles of Wolframin deficient rats (Wfs1-/-) at the age of 4 months and their appropriate littermates (Wfs1+/+). Here, we have demonstrated, that the membrane system of mitochondria of the Wfs1-/- is not properly organized (Objective 2). Next, we have obtained the anatomical data of the heart of these animals (Objective 6). For the measurement of myocyte contractility and calcium transients (Objective 3 and 4) we have constructed a new setup of perfusion chamber equipped with electrical field stimulation and have adjusted it to fit into the inverted fluorescence microscope. This setup allows to simultaneously acquire isolated cardiac myocyte shortening and calcium transient.

From the administrative tasks the most critical point which was influencing the progress of the Objectives 1 and 5 was moving of the Host Institute to the new address. This included to physically move all the offices and laboratories; to start running all the laboratory devices, involving special installations and re-calibration of the microscopes and other measuring devices. Additional task was to get the permissions for work with animals and with GMO1 in the new laboratories and in animal house from the Slovak Alimentary and Veterinary Administration Agency and Ministry of Environment of the Slovak Republic.

In spite of all the administrative obstacles mentioned above, we have published an article in PLoS Biology, IF = 8.668 and the results were presented on the prestigious international conference in the USA as well as on the local meeting.

During this period I have successfully applied for the local (VEGA) and international (DoRa, Bilateral Exchange Programm) funding. Other applications are in evaluation (APVV) and in preparation (ERC Consolidator). Finally, beyond this project I am actively involved in three additional international currently running projects.