Uurimisrühma töö eesmärkideks on arendada välja puromütsiin-märkimisel põhinev rakutüübi spetsiifiline proteoomi analüüsi meetod ning rakendada seda kesknärvisüsteemi rakkude uurimiseks. Lisaks kasutatakse Ribotag meetodit rakutüübi-spetsiifiliseks transkriptoomi analüüsiks. Üheks lähenemiseks interaktsioonide uurimisel on ühe rakutüübi aktiveerimine rakusisese Ca2+ vabastamisega (DREADD kemogeneetiline süsteemi) ning teise rakutüübi proteoomi/transkriptoomi analüüs. Lisaks uurime valgusünteesi regulatsiooni gliiarakkudes. Kompetentsid: neuronite ja gliiarakkude kasvatamine rakukultuuris; rakutüübi spetsiifiline RNA ja valkude analüüs; adeno-assiotsieeritud viirusvektorite (AAV) tootmine ja kasutamine.
The central nervous system tissues are made of a number of different cell types, among which astrocytes are one of the most abundant type. In the CNS tissue cells are highly intermixed, posing a challenge when trying to analyze their transcriptomes and proteomes separately. Owing to the difficulties separating these cells, bulk tissue analysis has been used previously to profile mRNA and protein in tissue, giving averaged readouts across the tissue. In the past decade, cell type specific RNA analysis has seen enormous progress with the advent of single cell RNA sequencing and genetic tools for cell type specific RNA isolation (TRAP, Ribotag). However, cell type specific proteome analysis is lagging behind and widely used, straightforward methods are not available. Our research aim is to develop a cell type-specific proteome analysis method that is based on puromycin labeling, and to apply the method to studying neuron-astrocyte interactions in an in vitro co-culture system. In addition, we shall use the Ribotag method for cell type-specific mRNA analysis. A key strategy we are planning to use for studying intercellular communication is activation of either neurons of astrocytes by triggering intracellular Ca2+ release by using the DREADD chemogenetic system – followed by proteomic and transcriptomic analysis of the other cell type in culture. Our additional research interest is regulation of neurotrophin BDNF in astrocytes and cardiomyocytes. Key competences: cell cultures of neurons, astrocytes and cardiomyocytes; cell type-specific RNA and protein analysis; adeno-associated virus(AAV) vector production and use