Master thesis project at Translational Neurology, Department of Clinical Sciences, Lund University
Description
The neurovascular unit (NVU) contains endothelial cells joined by tight junctions that shield the brain from the blood via the blood brain barrier (BBB). Pericytes are perivascular cells that line the entire microvasculature. They are required to support the endothelial cells and to maintain the BBB together with endothelial cells and astrocytes.
Upon brain hypoxia, pericytes are one of the first responders at the blood/brain interface. They undergo activation, morphological changes and express a protein called Regulator of G-protein Signaling 5 (RGS5) (1, 2). RGS5 expression is followed by pericyte detachment from the microvessels, leaving the capillaries leaky (1, 3). Interestingly, loss of RGS5 in pericytes in RGS5-KO mice prevents this detachment and reduces BBB leakage suggesting that RGS5 has an important role in the process of BBB breakdown (3, 4). However, changes in cell signalling in response to hypoxia that are mediated by RGS5 stroke.
For investigation of RGS5, we hypothesize that silencing RGS5 expression using siRNA technology (5) leads to secretome changes that effect cells at the BBB. Human brain vascular pericytes treated with either scrambled or RGS5 targeting siRNA, will be co-cultured in a spheroid model of the BBB together with human brain endothelial cells and human astrocytes. We have established a method allowing to study the cell-specific secretome of pericytes in vitro-using Turbo-ID, a mutant non-toxic biotin ligase (6). Secreted proteins will be purified and analysed using LC-MS/MS and Olink.
Methods to be used
Cell culture primary/immortalised cell lines (as previously established in our lab)
3D-culture (as previously established in our lab)
Cell culture in hypoxic conditions
Lentiviral production/transduction (as previously established in our lab)
Selection of lentiviral transduced cell lines for stable expression of transgenes (as previously established in our lab
siRNA silencing methods (5)
Protein size elusion chromatography (commonly used)
Western blot (as previously established in our lab)
Affinity purification of proteins with tags (commonly used)
LC-MS/MS analysis (as previously established in our lab)
Bioinformatics (as previously established in our lab)
Contact: Gesine.paul-visse@med.lu.se
- Ozen I, Deierborg T, Miharada K, Padel T, Englund E, Genove G, et al. Brain pericytes acquire a microglial phenotype after stroke. Acta Neuropathol. 2014;128(3):381-96.
- Berger M, Bergers G, Arnold B, Hammerling GJ, and Ganss R. Regulator of G-protein signaling-5 induction in pericytes coincides with active vessel remodeling during neovascularization. Blood. 2005;105(3):1094-101.
- Ozen I, Roth M, Barbariga M, Gaceb A, Deierborg T, Genove G, et al. Loss of Regulator of G-Protein Signaling 5 Leads to Neurovascular Protection in Stroke. Stroke. 2018;49(9):2182-90.
- Roth M, Gaceb A, Enstrom A, Padel T, Genove G, Ozen I, et al. Regulator of G-protein signaling 5 regulates the shift from perivascular to parenchymal pericytes in the chronic phase after stroke. FASEB J. 2019:fj201900153R.
- Enstrom A, Carlsson R, Ozen I, and Paul G. RGS5: a novel role as a hypoxia-responsive protein that suppresses chemokinetic and chemotactic migration in brain pericytes. Biol Open. 2022;11(10).
- Branon TC, Bosch JA, Sanchez AD, Udeshi ND, Svinkina T, Carr SA, et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol. 2018;36(9):880-7.