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Meninges: a new perivascular stem cells niche for widespread neurogenesis

Final Report Summary - MENINGES-NET (Meninges: a new perivascular stem cells niche for widespread neurogenesis)

As listed in the original grant agreement, the project objectives included the investigation of gene fate mapping of the meningeal perivascular cells. In this project, I discovered that perivascular meningeal cells migrate via the choroid plexus to the cortex and contribute to postnatal cerebral cortical neurogenesis in vivo. Using multiple lineage tracing approaches, I was able to demonstrate that PDGFRß+ perivascular meningeal cells generate mostly Satb2+ excitatory neurons in cortical layers I-IV.

The goal of my research project was to demonstrate the presence of an additive cell population contributing to postnatal brain neurogenesis. My overarching hypothesis was that endogenous perivascualr meningeal cell could migrate along vessels and contributed to brain cell genesis of neural (neurons and glia) and non-neural (perycites and endothelial) cells. My work began with the investigation of possible meningeal cell migration to the brain parenchyma. Therefore, to explore the hypothesis that perivascular cells in the meninges outside the brain parenchyma are able to migrate to the brain parenchyma, I used a technique to selectively transfect only meningeal and not cortical parenchymal cells, by electroporating a red fluorescence protein (RFP) reporter plasmid into the meninges. In contrast to other gene transfer techniques (i.e. lentiviral vector), plasmid electroporation allows rapid detection of transfected cells within hours, but only for a limited period. Indeed, RFP electroporation in meninges of newborn (P0) wild type mice resulted in specific labeling of meningeal cells 8 hours later, without however labeling cortical cells. I observed a rapid and progressive spreading of labeled cells from the meninges on the surface of the brain, alongside the projections of the meninges within the brain (16-24 hours) to the lateral ventricles (24-48 hours) and thereafter into the cerebral cortex. At 7 and 30 days after electroporation, labeled cells were detected in the upper cerebral cortical layers I-IV and exhibited primarily a neuronal morphology. I quantified the fate of the meningeal-derived cells at postnatal day 30 when neuronal pruning had already occurred. Up to 77.3% of the parenchymal labeled cells in the cortex showed a neuronal morphology and expressed the pan-neuronal marker NeuN, while 12.1% expressed the astrocyte marker GFAP. None expressed the oligodendrocyte precursor marker NG2 or the microglia marker Iba1. Of the NeuN+ cells, 71.9% expressed Satb2, a marker of excitatory neurons that establish callosal projections, while 13.2% expressed the interneuron marker GAD65/67. Thus, our results show that quiescent embryonically derived meningeal cells migrate into the brain parenchyma and give rise to functional cortical neurons in the retrosplenial and visual motor cortex of postnatal mouse brains.

During the final project period, I was able to develop a tissue specific cell population gene fate mapping. I intercrossed a Wnt1-Cre driver line with a Rosa26-lox-stop-lox-YFP reporter line (yielding Wnt1-YFP mice), resulting in permanent labeling of Wnt1-expressing cells and its descendants. In P15 and adult Wnt1-YFP mice, I observed Wnt1-YFP+ cells in the upper cortical layers I to IV. Up to 82.7% of the cortical Wnt1-YFP+ cells in adult mice showed a typical neuronal morphology and expressed the pan-neuronal marker NeuN. Of the NeuN+ cells, 60.8% expressed Satb2, while 12.1% expressed GAD65/67. However, since other cell types in the brain also expressed Wnt1, this experiment did not exclude the possibility that Wnt1-YFP+ cortical neurons originated from other Wnt1-YFP+ cells in non-meningeal brain regions. To gene fate map only meningeal cells, I injected in meninges of different Cre mouse lines a Brainbow 1.0(L) reporter vector encoding for a lox-stop-lox- reporter protein. This Brainbow 1.0(L) reporter is engineered such that it expresses tdTomato before, and CFP or YFP after Cre-mediated excision of the floxed stop cassette. This construct will be than expressed only in the Cre expressing cells. Similar to what I revealed in Wnt1-YFP mice, analysis of Wnt1-Cre and PDGFRb-Cre mice, 1 month after lentiviral transduction with the Brainbow 1.0(L) reporter in the meninges, the presence of substantial numbers of CFP+ and YFP+ cortical cells (70.2 % of all labeled cells) in the upper cortical layers I to IV. Of those, 72.7% showed a typical neuronal morphology and expressed NeuN. Another fraction of the CFP+ and YFP+ cortical cells (13.9%) had a non-neuronal morphology and expressed GFAP. NG2+ and Iba1+ cells were respectively, minimally or not detected amongst the CFP+ and YFP+ cortical cells. Of the CFP+ and YFP+ neurons (NeuN+), up to 66.0% expressed Satb2, while 6.7% expressed GAD65/67. Those results indicate that perivascular (but not vascular endothelial) meningeal cells were a source of newly generated cortical neurons.

My findings are relevant for multiple reasons: i) they indicate that quiescent embryonically-derived neural progenitors may contribute to retrosplenial and visual motor cortex postnatal brain neurogenesis; ii) they broaden the concept of brain plasticity; and iii) they challenge the dogma that neural precursor reside only in the parenchyma, highlighting the importance of perivascular tissue as a reservoir of neurogenic cells.

The results from this work are under revision in the journal Cell Stem Cell. It has also facilitated the training of several junior and senior laboratory members, as well as national and international collaborations. Further, as part of this research proposal, we have generated several transgenic mouse lines to gene fate map meningeal cell and neural stem cell and pericytes. Moreover, we have set up protocols for the isolation and culture of different types of cells that are now used to perform metabolic studies in several projects of the lab.

Finally, the research focus of this project has been an instrumental part to generate key publications in the Laboratory on both metabolism and neurovascular link, including papers published in Cell (1), Cell Cycle (2), Cell Metabolism (3, 4), EMBO J (5) and Cell Reports (6), or in revision in Cell Stem Cell (7).

Overall, this proposal has been a great success.

1. De Bock K, Georgiadou M, Schoors S, Ghesquière B, Cauwenberghs S, Cantelmo AR, Kuchnio A, Wong BW, Quaegebeur A, Eelen G, Phng L-K, Betz I, Tembuyser B, Brepoels K, Welti J, Geudens I, Segura I, Cruys B, Bifari F, Decimo I, Blanco R, Diaz-Moralli S, Wyns S, Vangindertael J, Rocha S, Collins R, Munck S, Corhout N, Daelemans D, Imamura H, Devlieger R, Rider M, Van Veldhoven PP, Schuit F, Bartrons R, Hofkens J, Fraisl P, Cascante M, Telang S, DeBerardinis RJ, Schoonjans L, Vinckier S, Chesney J, Gerhardt H, Dewerchin M & Carmeliet P. Role of PFKFB3-driven glycolysis in vessel sprouting. Cell 2013 154, 651-663. (IF = 34.8)
2. Schoors S, Cantelmo AR, Georgiadou M, Stapor P, Wang X, Quaegebeur A, Cauwenberghs S, Wong BW, Bifari F, Decimo I, Schoonjans L, De Bock K, Dewerchin M, Carmeliet P. Incomplete and transitory decrease of glycolysis: a new paradigm for anti-angiogenic therapy? Cell Cycle. 2014 Jan 1;13(1):16-22. (IF = 5.3)
3. Schoors S, De Bock K, Cantelmo AR, Georgiadou M, Ghesquière B, Cauwenberghs S, Kuchnio A, Wong BW, Quaegebeur A, Goveia J, Bifari F, Wang X, Blanco R, Tembuyser B, Cornelissen I, Bouché A, Vinckier S, Diaz-Moralli S, Gerhardt H, Telang S, Cascante M, Chesney J, Dewerchin M, Carmeliet P. Partial and transient reduction of glycolysis by PFKFB3 blockade reduces pathological angiogenesis. Cell Metab. 2014 Jan 7;19(1):37-48. (IF = 13.7)
4. Quaegebeur A, Segura I, Schmieder R, Verdegem D, Decimo I, Bifari F, Dresselaers T, Eelen G, Ghosh D, Davidson SM, Schoors S, Broekaert D, Cruys B, Govaerts K, De Legher C, Bouché A, Schoonjans L, Ramer MS, Hung G, Bossaert G, Cleveland DW, Himmelreich U, Voets T, Lemmens R, Bennett CF, Robberecht W, De Bock K, Dewerchin M, Ghesquière B, Fendt SM, Carmeliet P. Deletion or inhibition of the oxygen sensor PHD1 protects against ischemic stroke via reprogramming of neuronal metabolism. Cell Metab. 2016 Feb 9;23(2):280-91. (IF = 16.7).
5. Lange C, Turrero Garcia M, Decimo I, Bifari F, Eelen G, Quaegebeur A, Boon R, Zhao H, Boeckx B, Chang J, Wu C, Le Noble F, Lambrechts D, Dewerchin M, Kuo CJ, Huttner WB, Carmeliet P. Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis. EMBO J. 2016 Feb 8. (IF=13.012).
6. Segura I, Lange C, Knevels E, Moskalyuk A, Pulizzi R, Eelen G, Chaze T, Tudor C, Boulegue C, Holt M, Daelemans D, Matondo M, Ghesquière B, Giugliano M, Ruiz de Almodovar C, Dewerchin M, Carmeliet P. The Oxygen Sensor PHD2 Controls Dendritic Spines and Synapses via Modification of Filamin A. Cell Rep. 2016 Mar 22;14(11):2653-67. (IF=8.358).
7. Bifari F, Decimo I, Pina A, Lange C, et al, Carmeliet P. Meningeal cells contribute to postnatal cortical neurogenesis. Cell Stem Cell 2016 (in revision)