Periodic Reporting for period 1 - aMUST (Alteration of Mfn2 protein and Endoplasmic Reticulum to Mitochondria calciUm transfer in STress)
Reporting period: 2020-05-01 to 2022-04-30
Chronic stress has been described as a risk factor for anxiety and depression, currently affecting 25% of the population and increasing the economic burden on society (representing more than €170 billion per year in EU). In this context, this MSC action aimed to decipher the neurobiological mechanisms underlying stress response under adaptative (acute) or pathological (chronic threats) conditions. Understanding the molecular differences between these responses is crucial to address the impaired mechanism that occurs during chronic stress and reduce the impact of stress on psychopathologies. As energy demand could be involved in this process, this project has been focused on the study of mitochondria and mitochondrial-endoplasmic reticulum contact sites, as they can modulate several functions within the cell, including those that may be key to providing energy to the cell. With this porpoise the researcher had two objectives: to determine the impact of stress on mitochondrial function through Mfn2 expression, Mitochondrial Associated Membranes (MAMs) structure and function, and to test the implication of Mfn2 and MAMs in stress through a behavioural readout. This project allowed the researcher to use in vivo models and employ gold standard techniques to unveil this crucial biological question, as the use of behavioural tests to measure the impact of stress on social dominance, in vivo calcium imaging and techniques that allowed the identification of different cell types in this process. In the course of the action, the investigator was able to conclude that corticosterone correlated with the adaptative situational dominance response observed in the face of acute stress and that the effect changed as a function of the duration of stress. A different contribution of corticosterone was also observed in mitochondrial oxidative respiration in mPFC and NAc, two regions involved in stress, and depending on whether the stress was acute or chronic. Finally, corticosterone could modify MAMs in a cell-specific. The conclusions of the action shed light on the regions affected upon adaptative or pathologic stress and open new avenues to interfere with the corticosterone pathway to treat associated psychopathologies.
During this period, the researcher was able to perform the six work packages (WP) included in the two objectives described above. In the first WP, she was able to perform behavioural tests under acute and chronic stress. She could also measure mitochondrial calcium dynamics ex vivo, in vitro and in vivo (2nd WP), after corticosterone treatment and acute stress. The principal investigator could also measure ATP, mitochondrial respiration and ROS production by high-resolution respirometry. MAMs were also measured from acutely stressed animals. Protein expression of several MAMs-localized proteins involved in calcium transfer was also evaluated by qPCR in chronic stress samples. During the completion of this WP, the investigator received trainings related to dissection methods, OROBOROS, confocal trainings and other techniques necessary to complete the experiments included in the appendix 1 of the project. The third WP of the project included the set up and proteomics of MAMs in a region and cell-specific manner. During the period of the action the main investigator was able to design and produce the constructs and test them in vitro and in vivo, optimizing the amount of biotinylated proteins detected in the NAc. Further experiments are in progress. For the WP 4, the investigator worked with the Mfn2 flox/flox line to knocked out the Mfn2 specifically in D1 neurons and test the impact in social dominance after the acute stress. The main results obtained during the development of this project were that the correlation of corticosterone with mitochondrial respiration in NAc was lost under acute stress and inversely correlated in the mPFC under chronic stress. Acute stress produced metabolic stress in NAc, affecting ATP production that correlated with animal performance in the tube test. These corticosterone-mediated effects also differently affected MAMs in the two types of medium spiny neurons in the NAc. These results were reported and disseminated in several congresses where the investigator presented her work as a poster. She also participated in several dissemination activities. The researcher will publish the main results after the end of all experimentation duly acknowledging the EU and specifying the number of the action. In case the experimenter finds other results that can be exploited in another way, they will be exploited by applying for a patent with the help of the EPFL Technology Transfer Office, as specified in the grant agreement of the action. During the duration of the action, the researcher already worked on her professional development plan, participating in mentorship programs. The researcher applied earlier this year for the Serra Hunter program to be eligible for a tenure-track position in her home country. In parallel, she is training, collaborating, networking and pursuing other options for positions in industry in Switzerland. In addition, other competencies that the researcher developed during the course of the action were the opportunity to supervise two bachelor students and training in license writing. After writing and being interviewed she obtained a Robert Gnehm grant to hire a technician for her project; concurrently, she also obtained a FENS/IBRO society travel grant. She collaborated on two scientific publications with laboratory and external colleagues during the period of the action. The researcher attended several courses on technical data management and transfer to industry and during the period of the action and currently, she is attending French courses to improve her communication skills and job opportunities.
To our knowledge, the aMUST action made it possible for first time to measure calcium dynamics in the mitochondria in freely moving live animals. This opens the avenue to measure calcium in different cell organelles and to study its contribution in certain circuits under more physiological conditions than in complementary techniques such as in vitro cultures or ex vivo slices. Some of the cute-edge techniques described in the aMUST action also allow us to isolate the function of inter-organelle contacts under physiological conditions, making it more comprehensible which function and proteins are involved in these structures. In addition, the expected results of this study on the effects of corticosterone on mitochondria and brain energetics, in acute and chronic stress in different brain regions and in a cell-specific way may also allow discerning the effects of stress in a more targeted manner. Understanding the details of the molecular pathways affected in complex behavior, although a very challenging process, can make a difference in drug discovery with reduced targets and fewer side effects. This is why conducting detailed studies such as this one can have a broad impact on society, allowing the development of more specific drugs and more translational studies to human diseases. This, together with the dissemination activities to non-specialized public included in the MSC action, could not only reduce the economic burden that psychopathologies represent in the population but also make society more aware of the importance of contributing to the generation of knowledge to promote mental health.