Third party funded individual grant
Start date : 01.01.2016
End date : 31.12.2017
Brain diseases, e.g. multiple sclerosis or encephalitis, but also brain tumors (e.g. malignant glioblastomas) are associated with marked inflammation of the brain parenchyma which results in activation of immune-competent microglia cells in the brain. Microglia are activated by sensing purinergic metabolites in their environment (e.g. ATP, ADP), that result from necrosis of brain cells. The normally dormant microglia (MG) cells possess purinergic receptors, e.g. P2X/P2Y-receptor that bind those metabolites and initiate MG activation via intracellular signaling cascades, but also through receptor-operated Ca2+ influx (ROCE), followed by formation of macropores. MG cells then become motile and phagocytic, attack surrounding cells and sustain inflammation. As a result, edema formation may compromise the blood-brain barrier and increase intracranial pressure. In malignant tumors, this inflammation-induced component of brain edema worsens prognosis, while in primary inflammatory brain disorders, brain function becomes compromised. The elucidation of the exact role of Ca2+ activation and signaling cascades alongside with screening for drugs to prevent MG activation is thus, of paramount interest in neuroscience.
In this project, the scientific goals are:
1. To study the mechanistic roles of various Ca2+ entry pathways (e.g. ROCE, store-operated Ca2+ entry, SOCE) upon purinergic receptor activation and their dependence on extracellular glutamate (excitatory transmitter) by use of high-end multiphoton fluo-4 Ca2+ fluorescence microscopy
2. To dissect the time course of MG Ca2+ activation from macropore formation and motility in isolated 2D MG cultures and co-cultures with neurons
3. To engineer a high-throughput screening platform based on optical multi-well plate readout systems, i.e. ToxFinder, suitable for large compound screenings
4. To screen for compound libraries (e.g. small molecule libraries, algae extracts, marine organic molecules), traditional pathway blockers (e.g. U731- U733, wortmanin) and antibiotics (e.g. minocycline) on their potency to prevent MG activation
To test effective compounds from (4) in a setting of (1) on an animal model of malignant glioblastoma cell lines (rodent RG2, murine GL261 glioma cells) in co-cultures with MG cells (BV2)