Uurimisrühma töö eesmärkideks on arendada välja puromütsiin-märkimisel põhinev rakutüübi spetsiifiline proteoomi analüüsi meetod ning rakendada seda neuronite-astrotsüütide interaktsioonide uurimiseks in vitro segakultuuri katsesüsteemis. Lisaks kasutatakse Ribotag meetodit rakutüübispetsiifiliseks transkriptoomi analüüsiks.Üheks plaanitud lähenemiseks interaktsioonide uurimisel on ühe rakutüübi aktiveerimine rakusisese Ca2+ vabastamisega (selleks plaanitakse rühma töös kasutada DREADD kemogeneetilist süsteemi) ning teise rakutüübi proteoomi/transkriptoomi analüüs. Rühma täiendavaks huviobjektiks on neurotrofiin BDNF regulatsioon ja rollid astrotsüütides ja kardiomüotsüütides. Kompetentsid: neuronite, astrotsüütide, kardiomüotsüütide kasvatamine rakukultuuris; rakutüübi spetsiifiline RNA ja valkude analüüs; adeno-assiotsieeritud viirusvektorite (AAV) tootmine ja kasutamine.
The central nervous system tissues are made ofa number of different cell types, among whichastrocytes are one of the most abundant type.In the CNS tissue cells are highly intermixed,posing a challenge when trying to analyze theirtranscriptomes and proteomes separately. Owing to the difficulties separating these cells,bulk tissue analysis has been used previouslyto profile mRNA and protein in tissue, givingaveraged readouts across the tissue. In the pastdecade, cell type specific RNA analysis has seenenormous progress with the advent of singlecell RNA sequencing and genetic tools for celltype specific RNA isolation (TRAP, Ribotag).However, cell type specific proteome analysis is lagging behind and widely used, straightforwardmethods are not available.Our research aim is to develop a cell type-specific proteome analysis method that is based onpuromycin labeling, and to apply the method tostudying neuron-astrocyte interactions in an invitro co-culture system. In addition, we shall usethe Ribotag method for cell type-specific mRNAanalysis.A key strategy we are planning to use for studying intercellular communication is activation ofeither neurons of astrocytes by triggering intracellular Ca2+ release by using the DREADDchemogenetic system – followed by proteomicand transcriptomic analysis of the other celltype in culture.Our additional research interest is regulationof neurotrophin BDNF in astrocytes and cardiomyocytes.Key competences: cell cultures of neurons, astrocytes and cardiomyocytes; cell type-specific RNAand protein analysis; adeno-associated virus(AAV) vector production and use