Distinct roles for BAI1 and TIM-4 in the engulfment of dying neurons by microglia.
Mazaheri, F.*, Breus, O.*, Durdu, S., Haas, P., Wittbrodt, J., Gilmour, D. & Peri, F.
Nat Commun. 2014 Jun 5;5:4046. doi: 10.1038/ncomms5046.
* these authors contributed equally.
The removal of dying neurons by microglia has a key role during both development and in several diseases. To date, little is known about the cellular and molecular processes underlying neuronal engulfment in the brain. Here we took a live imaging approach to quantify neuronal cell death progression in embryonic zebrafish brains and studied the response of microglia. We show that microglia engulf dying neurons by extending cellular branches that form phagosomes at their tips. At the molecular level we found that microglia lacking the phosphatidylserine receptors BAI1 and TIM-4, are able to recognize the apoptotic targets but display distinct clearance defects. Indeed, BAI1 controls the formation of phagosomes around dying neurons and cargo transport, whereas TIM-4 is required for phagosome stabilization. Using this single-cell resolution approach we established that it is the combined activity of BAI1 and TIM-4 that allows microglia to remove dying neurons.
Autophosphorylation and Pin1 binding coordinate DNA damage-induced HIPK2 activation and cell death.
Bitomsky, N., Conrad, E., Moritz, C., Polonio-Vallon, T., Sombroek, D., Schultheiss, K., Glas, C., Greiner, V., Herbel, C., Mantovani, F., Del Sal, G., Peri, F. & Hofmann, T.G.
Proc Natl Acad Sci U S A. 2013 Nov 5;110(45):E4203-12. doi:10.1073/pnas.1310001110. Epub 2013 Oct 21.
Excessive genome damage activates the apoptosis response. Protein kinase HIPK2 is a key regulator of DNA damage-induced apoptosis. Here, we deciphered the molecular mechanism of HIPK2 activation and show its relevance for DNA damage-induced apoptosis in cellulo and in vivo. HIPK2 autointeracts and site-specifically autophosphorylates upon DNA damage at Thr880/Ser882. Autophosphorylation regulates HIPK2 activity and mutation of the phosphorylation-acceptor sites deregulates p53 Ser46 phosphorylation and apoptosis in cellulo. Moreover, HIPK2 autophosphorylation is conserved between human and zebrafish and is important for DNA damage-induced apoptosis in vivo. Mechanistically, autophosphorylation creates a binding signal for the phospho-specific isomerase Pin1. Pin1 links HIPK2 activation to its stabilization by inhibiting HIPK2 polyubiquitination and modulating Siah-1-HIPK2 interaction. Concordantly, Pin1 is required for DNA damage-induced HIPK2 stabilization and p53 Ser46 phosphorylation and is essential for induction of apotosis both in cellulo and in zebrafish. Our results identify an evolutionary conserved mechanism regulating DNA damage-induced apoptosis.
Animal models for studying microglia: The first, the popular, and the new.
Sieger, D. & Peri, F.
Glia. 2013 Jan;61(1):3-9. doi: 10.1002/glia.22385. Epub 2012 Sep 17.
Microglia, the resident phagocytes of brain, have been intensively studied since their discovery in the 1920s. There is no doubt that the possibility of culturing microglia in vitro has advanced enormously our understanding of these cells. However, as we know today, that microglia react to even small changes in the brain, it is crucial to also study these cells by preserving as much as possible their natural environment. Nowadays, advances in imaging technologies and transgenic cell labeling methods allow the direct observation of cells at work. These in vivo approaches have already changed our view on microglia by showing that these cells are active even in the healthy adult brain. As today, there is upcoming evidence that microglia can directly influence neuronal activity, understanding their roles and, in particular, their interactions with neurons is of great importance. The aim of this review is to illustrate three animal models that are currently used for microglial research and to discuss their characteristics and advantages by presenting recent achievements in microglial research. In our view the availability of different systems for studying microglia will lead to a more comprehensive understanding of their functions.
Long-range Ca2+ waves transmit brain-damage signals to microglia.
Sieger, D., Moritz, C., Ziegenhals, T., Prykhozhij, S. & Peri, F.
Dev Cell. 2012 Jun 12;22(6):1138-48. doi: 10.1016/j.devcel.2012.04.012. Epub 2012May 24.
Microglia are the resident phagocytes of the brain that are responsible for the clearance of injured neurons, an essential step in subsequent tissue regeneration. How death signals are controlled both in space and time to attract these cells toward the site of injury is a topic of great interest. To this aim, we have used the optically transparent zebrafish larval brain and identified rapidly propagating Ca2+ waves that determine the range of microglial responses to neuronal cell death. We show that while Ca2+-mediated microglial responses require ATP, the spreading of intercellular Ca2+ waves is ATP independent. Finally, we identify glutamate as a potent inducer of Ca2+-transmitted microglial attraction. Thus, this real-time analysis reveals the existence of a mechanism controlling microglial targeted migration to neuronal injuries that is initiated by glutamate and proceeds across the brain in the form of a Ca2+ wave.
Microglia in the developing brain: from immunity to behaviour.
Schlegelmilch, T., Henke, K. & Peri, F.
Curr Opin Neurobiol. 2011 Feb;21(1):5-10. doi: 10.1016/j.conb.2010.08.004.
For decades, microglia, the resident macrophages of the brain, have been recognized mostly for their role in several, if not all, pathologies affecting the brain. However, several studies under physiological conditions demonstrate that microglial function is indispensable also in the healthy brain. Indeed, microglia implement key functions already during development, such as the clearance of the huge amount of neurons that are produced in large excess in the embryo and later die of apoptosis. Beside these classical functions, however, novel roles are emerging that strikingly link microglia with higher order brain functions and show that these cells can ultimately influence behaviour. Therefore a detailed understanding of microglia under physiological conditions may open unprecedented perspectives in the prevention and treatment of neuropsychiatric diseases.
Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction.
Fantin, A., Vieira, J.M., Gestri, G., Denti, L., Schwarz, Q., Prykhozhij, S., Peri, F., Wilson, S.W. & Ruhrberg, C.
Blood. 2010 Aug 5;116(5):829-40. doi: 10.1182/blood-2009-12-257832. Epub 2010 Apr19.
Blood vessel networks expand in a 2-step process that begins with vessel sprouting and is followed by vessel anastomosis. Vessel sprouting is induced by chemotactic gradients of the vascular endothelial growth factor (VEGF), which stimulates tip cell protrusion. Yet it is not known which factors promote the fusion of neighboring tip cells to add new circuits to the existing vessel network. By combining the analysis of mouse mutants defective in macrophage development or VEGF signaling with live imaging in zebrafish, we now show that macrophages promote tip cell fusion downstream of VEGF-mediated tip cell induction. Macrophages therefore play a hitherto unidentified and unexpected role as vascular fusion cells. Moreover, we show that there are striking molecular similarities between the pro-angiogenic tissue macrophages essential for vascular development and those that promote the angiogenic switch in cancer, including the expression of the cell-surface proteins TIE2 and NRP1. Our findings suggest that tissue macrophages are a target for antiangiogenic therapies, but that they could equally well be exploited to stimulate tissue vascularization in ischemic disease.
Breaking ranks: how leukocytes react to developmental cues and tissue injury.
Curr Opin Genet Dev. 2010 Aug;20(4):416-9. Epub 2010 Jun 2.
Leukocytes are arguably the most motile cells in metazoans. Besides their well-described ability to migrate rapidly toward sites of tissue injury, tissue-specific macrophages migrate already during embryogenesis, when they take up residence in a wide range of organs. The recent identification of molecules responsible for the guidance of leukocytes during development and in response to injury has revealed that these modes of migration are under the control of surprisingly different signaling systems. While the developmental migrations are regulated by hard-wired pre-patterns of secreted proteins, the rapid acute response to injury involves signals like hydrogen peroxide or extracellular nucleotides such as ATP. Ongoing work aims to understand how these distinct signals are integrated in the cell to determine different cellular responses.
Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo.
Peri, F. & Nusslein-Volhard, C.
Cell. 2008 May 30;133(5):916-27.
A significant proportion of neurons in the brain undergo programmed cell death. In order to prevent the diffusion of damaging degradation products, dying neurons are quickly digested by microglia. Despite the importance of microglia in several neuronal pathologies, the mechanism underlying their degradation of neurons remains elusive. Here, we exploit a microglial population in the zebrafish to study this process in intact living brains. In vivo imaging reveals that digestion of neurons occurs in compartments arising from the progressive fusion of vesicles. We demonstrate that this fusion is mediated by the v0-ATPase a1 subunit. By applying live pH indicators, we show that the a1 subunit mediates fusion between phagosomes and lysosomes during phagocytosis, a function that is independent of its proton pump activity. As a real-time description of microglial phagocytosis in vivo, this work advances our understanding of microglial-mediated neuronal degeneration, a hallmark of many neuronal diseases.