טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHilu Dadia Reut
SubjectMolecular Mechanisms in Glial Phagocytosis of Apoptotic
Neurons During Drosophila CNS Development
DepartmentDepartment of Medicine
Supervisors Professor Adi Salzberg
Dr. Estee Kurant
Full Thesis textFull thesis text - English Version


Abstract

Development of the central nervous system (CNS) involves elimination of superfluous neurons through apoptosis and subsequent phagocytosis. The removal of dying cells by phagocytes is critical for organism development and tissue maintenance in the adult. Defects in apoptotic cell clearance can lead to inflammation and autoimmune diseases. Phagocytes must distinguish between living and dying cells with high level of specificity, which is achieved through function of phagocytic receptors that recognize ‘eat me’ signals exposed on apoptotic cells. In mammals, phagocytic receptors are highly redundant, making it difficult to study their function in vivo. Since phagocytosis is an evolutionarily conserved process, we use the Drosophila melanogaster model to uncover its molecular and cellular mechanisms.

In Drosophila, developmental apoptosis in the CNS occurs mainly during three stages: embryogenesis, metamorphosis and emerging adult. Two transmembrane glial phagocytic receptors, Six-Microns-Under (SIMU, homolog of the mammalian Stabilin-2) and Draper (Drpr, homolog of the mammalian MEGF10 and Jedi), mediate glial phagocytosis of apoptotic neurons during embryogenesis. However, less is known about the removal of apoptotic neurons during later stages of development and of additional phagocytic receptors involved in glial phagocytosis during embryogenesis.

Here we show that during metamorphosis, Drpr plays a critical role in apoptotic cell clearance by glia, whereas SIMU, which is mostly expressed in pupal macrophages, is not involved in glial phagocytosis. We found that Drpr activates Drosophila c-Jun N-terminal kinase (dJNK) signaling predominantly in ensheathing glia and astrocytes, where it is required for efficient removal of apoptotic neurons. Our data suggest that besides the dJNK pathway, Drpr also triggers an additional signaling pathway capable of removing apoptotic neurons in the pupal brain. Collectively, this study reveals that SIMU is not involved in glial phagocytosis of apoptotic neurons during metamorphosis and highlights the novel role of dJNK signaling in developmental apoptotic cell clearance downstream of Drpr.

Furthermore, we discovered a new phagocytic receptor required for glial phagocytosis during embryogenesis. We found that Santa Maria, a CD36 homologue, which is specifically expressed in embryonic glia, plays a major role in removal of apoptotic neurons during CNS development. Our data demonstrate that Santa Maria, like SIMU, is involved in the recognition and engulfment steps of glial phagocytosis. We show that Santa Maria and SIMU physically interact, thus, acting together as a complex to execute the phagocytosis process. Moreover, we discovered that in the absence of all three phagocytic receptors SIMU, Drpr and Santa Maria, phagocytosis of apoptotic neurons is abrogated causing lethality that illuminates the vital role of apoptotic cell clearance in the developing CNS. Interestingly, we revealed that Croquemort (Crq), Santa Maria's closest homologue expressed in macrophages could replace its function in glial cells supporting the notion that phagocytic receptors expressed in specific phagocytic cell populations act similarly.