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Perivascular meningeal stem cells: a new player in the Neurovascular Unit.
Characterization, modulation and therapeutic potential of perivascular meningeal stem cell activation in neurological disord

Final Report Summary - STEM-ACTIVATION (Perivascular meningeal stem cells: a new player in the Neurovascular Unit.<br/> Characterization, modulation and therapeutic potential of perivascular meningeal stem cell activation in neurological disord)

The Neurovascular Unit (NVU) consists of cerebral endothelial cells that interact physically and chemically with pericytes, astrocytes and microglial cells connected through the interactions with the basal lamina. The NVU create a functional barrier that regulates the movements of molecules into and out of the brain (1). In the last two decades, the brain vasculature has increasingly entered the center stage as a key player that actively influences and directs brain development, homeostasis, and disease (2). Recently several groups have identified perivascular cell population, including meningeal cells, that shows neural differentiation properties and demonstrate a role in the injury-induced central nervous system reaction (3, 4).

The overall hypothesis of this project was that the modulation of perivascular meningeal stem cell activation and differentiation (i.e. through finding of candidate metabolic key modulators) could yield novel therapeutics in neurological disorders. We evaluated the metabolic profile of the meningeal stem perivascular cells (MeSP) and the in vitro role of candidate metabolic key modulators (i.e. PFKFB3) in MeSP activation/differentiation.

We were able to show a clear shift from glycolytic to oxidative metabolism in growing vs differentiated cells. Neuronal differentiation of MeSP was, as expected, characterized by increased glutamine oxidation, while pericyte differentiation was associated with an increase in fatty acid oxidation. We also demonstrated that the enzyme PFKFB3 is an important regulator of glycolysis in MeSP cells, and that glycolysis is necessary to activate (e.g. increase in proliferation and migration) perivascular meningeal stem cells and to modulate their differentiation.

In addition to the originally proposed experiments investigating MeSP activation, their metabolic profile and the effect of their metabolism modulation in pathological conditions, we have added experimental investigations to determine the MeSP identity, their possible contribution to neurogenesis and their developmental origin. In order to select the best transgenic mice to trace MeSPs in vivo, we screened different CRE (PDGFrß, Wnt1) and CRE inducible (SOX10, Nestin, Glast, PDGFralpha) reporter lines intercrossed with Rosa26-lox-stop-lox-YFP mice. During this screening we found particularly interesting the PDGFrß-Cre:YFP line. The PDGFRß-Cre:YFP mouse lines showed high recombination in the perivascular cells of either the meninges and the brain as expected, however YFP expression is also present in the choroid plexi and in brain parenchyma neurons. Those findings suggest a possible contribution of MesP to neurogenesis in vivo. The in vivo identity and fate of MesP is still largely unknown and the hypothesis of the presence of neurogenic cells in meninges challenge the dogma that only parenchymal cells can contribute to neurogenesis, highlighting the importance of vascular cells as a reservoir of neurogenic cells. Given the potential breakthrough of this hypothesis we focused the project into more detail on the contribution of MeSP to neurogenesis in vivo.

We found 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, we demonstrate that PDGFRß+ perivascular meningeal cells generate mostly Satb2+ excitatory neurons in cortical layers I-IV. Lineage tracing experiments further suggest that these neurogenic meningeal cells likely have a neural crest origin. Thus, aside from ventricular radial glial cells that are well known to give rise to cerebral cortical neurons, a distinct pool of meningeal cells also contributes to cerebral cortical neurogenesis.

The results from this work are under revision in the journal Science (5). 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 human pericytes 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 two papers published in Cell (6), and Cell Cycle (7), and 3 papers under revision or in preparation (8-10).

Overall, this proposal has been a great success, where the recruited researcher has been able to not only contribute to ongoing research efforts to demonstrate the therapeutic efficacy of targeting metabolism in in perivascular cells, but also develop a novel research direction around the potential role of the vasculature as reservoir of neurogenic cells.

As perivascular cells and pericytes play crucial roles in both health and a number of neurodegenerative diseases (such as glioma, stroke, Alzheimer etc.), the future socio-economic impact of this work is great.


References
1. Hermann et al. the Abluminal endothelial membrane in neuorvascular remodeling in health and disease. Cell singalling 2012,
2. A. Quaegebeur, C. Lange, P. Carmeliet, The neurovascular link in health and disease: molecular mechanisms and therapeutic implications. Neuron 71, 406 (Aug 11, 2011).
3. Decimo et al., Nestin- and doublecortin-positive cells reside in adult spinal cord meninges and participate in injury-induced parenchymal reaction. Stem Cells 29, 2062 (Dec, 2011).
4. G. Paul et al., The adult human brain harbors multipotent perivascular mesenchymal stem cells. PLoS One 7, e35577 (2012).
5. Decimo, I. et al., Perivascular meningeal cells contribute to cortical neurogenesis in the mammalian brain. Science under revision
6. De Bock, K. et al. Role of PFKFB3-driven glycolysis in vessel sprouting. Cell 154, 651-663, doi:10.1016/j.cell.2013.06.037 (2013)
7. Schoors S et al. A new paradigm for anti-angiogenic therapy? Cell Cycle. 2013 Dec 13;13(1). [Impact factor – 5.243]
8. Quaegebeur A. Decimo I., et al., Deletion or inhibition of the oxygen sensor phd1 protects against ischemic stroke via reprogramming of neuronal metabolism. Cell Metabolism under revision
9. Lange, C. et al. Relief of physiological hypoxia by formation of niche blood vessels promotes neural stem cell differentiation during cerebral cortex development. Developmental Cell submitted
10. Cantelmo A.R. et al. Partial reduction of glycolysis by pfkfb3-blockade induces tumor vessel normalization and impairs metastasis. Cancer Cell in preparation.