Cell Physiology

CELL PHYSIOLOGY aims to understand the mechanisms by which cells interact with each other in health and disease. Eukaryotic cells emerged 1-2 billion years ago and are characterized by subcellular organelles, including SECRETORY VESICLES, where chemical messengers and membrane-bound molecules are stored, playing a key role in cell-to-cell interaction and are the OBJECT of our studies. Neurons communicate rapidly by the propagation of action potentials along axons to nerve terminals, where the merger between the vesicle and the plasma membranes is triggered. This process, REGULATED EXOCYTOSIS, leads to the formation of a fusion pore, through which molecules, stored in the vesicle lumen, may exit into the extracellular space. Not only neurons, practically all eukaryotic cells exhibit exocytosis, although at slower time scale, and we will study several cell types, including pituitary cells and astrocytes, to address questions about: ORGANELLE TRAFFIC AND MEMBRANE FUSION, REGULATION OF CYTOSOLIC SIGNALING, METABOLISM AND MORPHOLOGY, and CELL (PATHO)PHYSIOLOGY IN DISEASE MODELS. We will use METHODS, including super-resolution microscopy, electrophysiology, molecular biology, quantitative FRET-based microscopy, all operational in our labs, to monitor function at cellular, organelle, and single molecule levels by using cell cultures, tissue slices and transgenic animal models (mice, Drosophila) to correlate cell function to pathologic and therapeutic significance. IMPACT and TRANSLATION. As was the case in the past, this strategy will result in publishing results in highly visibile journals (J. Neurosci., JBC, Neuron, Glia, Nature journals), and will generate translational directions. We have established GMP certified labs and based on membrane fusion studies, we developed an advanced cell-based medical immunotherapy product, which was tested in a clinical trial (EudraCT # 2012-005498-29). In the next period we will advance this approach to treat patients with other solid tumors. Based on our discovery of the mechanism of “metabolic excitability”, we will continue to file patents and conduct research to identify a new receptor, mimicking the stimulating action of noradrenaline (NA) in astrocytes, which is impaired in neurodegeneration. NA augments glycogenolysis and lipid metabolism, while it is a potent inhibitor of neuroinflammation, yet the mechanism(s) are poorly understood. The other aspect of translation is the dissemination of our results through education by teaching courses at the University of Ljubljana. FEASIBILITY & IMPLEMENTATION. The proposed research is feasible to be implemented with success. Our team consists of highly motivated and qualified researchers, holds all the necessary infrastructure and operates a quality management system compliant with the ISO:17025 standard, and the Cell & Tissue Establishment regulations, audited externally. During the recent pandemic we have introduced new studies to unravel the neurotropic mechanisms of Covid-19.

The proposed research is significant at several levels (the relevance of the investigated research in this proposal have been acknowledged by the recently awarded Nobel Prizes in 2013 and 2018). First, it is interesting in its own right, it is providing new information about ubiquitous biological processes - exocytosis, vesicle traffic, cytosolic homeostasis of second messengers and metabolites - that at present are not well understood. Second, these are model systems for the study of ‘functional genomics and proteomics’. That is, for most complex biological processes involving the specific interactions of dozens of different genes and proteins, it is not possible to monitor the process in real-time in living organisms. For this, one has to use single cells and multiple techniques, which can be used either individually or simultaneously in combination to address questions related to the properties of functional modules that contribute to the function of cells and organisms as a whole, using also animal models of human diseases. Unitary exocytic events in pituitary cells and astrocytes, which outnumber neurons in the brain, can be studied in this way. Understanding vesicle dynamics prior and after exocytosis can also be studied in single cells such as astrocytes, which are increasingly viewed as essential elements in information processing in the central nervous system in health and disease. The cytosolic events involving second messengers and metabolites that are associated with vesicle traffic are poorly understood and optical techniques in combination with fluorescent molecular probes provide an approach that promises new important directions to be opened in the future. In particular with the advances in super-resolution microscopy, advanced electrophysiology, the use of optical nanosensors and others. All of these methods are introduced into our labs. In combination with tissue slices the function of single cells can be placed in the framework of a cell association and then into animal models of diseases. Finally, it seems reasonable to hope that this work, while targeted to only a few types of diseases (cancer, neurodegeneration, intelligent dissability, virus infection, including SARS-Cov-2) will be useful for discovering new therapeutic processes and targets and thus helping to preserve and promote human health. Anterior pituitary hormones control bodily functions including growth, development, reproduction, and responses to stress. To understand neuroendocrine integration one needs to understand the mechanisms that control exocytosis. Therefore, the proposed research will not only examine fundamental physiological/biophysical aspects of exocytosis, vesicle traffic, cytosolic signaling with messengers and metabolites, but will additionally reveal new aspects of physiological regulation of hormone and neurotransmitter release, vesicle traffic and cytosolic homeostasis in terms of conditions related to cancer, neurodegeneration (Alzheimer’s Disease, Intellectual Dissability), brain trauma, diabetes and neurotropic mechanisms of SARS-CoV-2 action.

Cell Physiology represents an essential discipline for the development of new therapeutic and diagnostic methods in modern molecular »precision-personalized« medicine. Therefore, the strategy to support fundamental and applied cell research represents a strategy to deliver and support a much more rapid development of the whole society, including that in Slovenia. Fundamental research is essential for the education of future experts and also the lay public. The latter needs to be educated in terms of understanding the new developments in cell biology and molecular medicine. For example: the promising new discoveries in stem cell research, subcellular physiology, cell engineering, cell-based therapy, the biology and mechanisms of diseases, including virus infection, the mechanisms of treatment, and others need to be presented to the public. Furthermore, solving problems in the context of universal problems at the highest possible methodological level is increasing the visibility, credibility and competence of the whole society. Expertise in the field of Cell Physiology, which has a tradition of several decades in Slovenia, also in the industry of biologicals, is necessary to support a more rapid development for Slovenia, contributing significance for global efforts with the family of other nations