Francesca Peri

Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction

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.

Live Imaging of Neuronal Degradation by Microglia Reveals a Role for v0-ATPase a1 in Phagosomal Fusion In Vivo

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.

Long-Range Ca2+ Waves Transmit Brain-Damage Signals to Microglia

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.

Local Gurken signaling and dynamic MAPK activation during Drosophila oogenesis.

During Drosophila melanogaster oogenesis Gurken, a TGF-alpha like protein localized close to the oocyte nucleus, activates the MAPK cascade via the Drosophila EGF receptor (DER). Activation of this pathway induces different cell fates in the overlying follicular epithelium, specifying the two dorsolaterally positioned respiratory appendages and the dorsalmost cells separating them. Signal-associated internalization of Gurken protein into follicle cells demonstrates that the Gurken signal is spatially restricted and of constant intensity during mid-oogenesis. At the same time MAPK activation evolves in a spatially and temporally dynamic way and resolves into a complex pattern that presages the position of the appendages. Therefore, different dorsal follicle cell fates are not determined by a Gurken morphogen gradient. Instead they are specified by secondary signal amplification and refinement processes that integrate the Gurken signal with positive and negative feedback mechanisms generated by target genes of the DER pathway.

Microglia in the developing brain: from immunity to behaviour.

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.

Microglia: Multitasking Specialists of the Brain

Microglia are macrophages that colonize the brain during development to establish a resident population of professional phagocytes that protect against invading pathogens and contribute to brain development and homeostasis. As such, these cells sit at the interface between immunology and neurobiology. In addition to their key roles in brain physiology, microglia offer a great opportunity to address central questions in biology relating to how migrating cells find their positions in the embryo, adopt a behavior that is appropriate for that position, and interact with their local environment. We aim, in this review, to survey key recent advances in microglial research.

Distinct roles for BAI1 and TIM-4 in the engulfment of dying neurons by microglia.

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.

Animal models for studying microglia: The first, the popular, and the new

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.

Autophosphorylation and Pin1 binding coordinate DNA damage-induced HIPK2 activation and cell death

Significance Activation of the cell death (apoptosis) program is a major principle of DNA-damaging cancer treatments including ionizing radiation and chemotherapeutic drug treatment. The protein kinase HIPK2 plays a key role in radiosensitivity and chemosensitivity. Here, we found that HIPK2 autointeracts and autophosphorylates after DNA damage. HIPK2 autophosphorylation is conserved in evolution and regulates its apoptosis-inducing activity by facilitating binding of the isomerase Pin1. Pin1 couples HIPK2 activation to its stabilization and is essential for DNA damage-induced apoptosis in cancer cells and in zebrafish. Our findings identify a mechanism linking HIPK2 activation to its stabilization and highlight a conserved function of HIPK2 and Pin1 in the DNA damage-induced apoptosis response. 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.

Developmental Apoptosis Mediates Entry and Positioning of Microglia in the Zebrafish Brain

In the brain, neurons that fail to assemble into functional circuits are eliminated. Their clearance depends on microglia, immune cells that colonize the CNS during embryogenesis. Despite the importance of these cells in development and disease, the mechanisms that target and position microglia within the brain are unclear. Here we show that, in zebrafish, attraction of microglia into the brain exploits differences in developmental neuronal apoptosis and that these provide a mechanism for microglial distribution. Reducing neuronal cell death results in fewer microglia, whereas increased apoptosis enhances brain colonization, resulting in more microglia at later stages. Interestingly, attraction into the brain depends on nucleotide signaling, the same signaling system used to guide microglia toward brain injuries. Finally, this work uncovers a cell-non-autonomous role for developmental apoptosis. Classically considered a wasteful process, programmed cell death is exploited here to configure the immune-neuronal interface of the brain.