Although it has long been recognized that there is extensive cell death in the developing peripheral nervous system, the mechanisms responsible for disposing of the cellular “waste” have remained an open question. Our findings demonstrate that SGC precursors in the DRG are the primary cell type responsible for clearing neuronal corpses generated during the period of naturally occurring cell death. In addition, we identified Jedi-1, a novel engulfment receptor, and MEGF10 as two CED-1 homologs expressed in the glial cells and involved in phagocytosing neuronal corpses. Hence, these results reveal the cellular and molecular basis for clearing the neuronal waste generated during the development of sensory ganglia.
Macrophages carry out the removal of cellular debris in many tissues during development or after injury and these cells are known to increase in the DRG after injury to the sciatic nerve35
. However, we found few macrophages in the developing DRG, even in animals with unusually high numbers of apoptotic neurons (nt3−/−
mice). The phagocytic ability of glial cells in the developing nervous system has been known for some time, although the significance has largely been unrecognized. Axonal fragment ingestion by Schwann cells during Wallerian degeneration after injury was first described some 40 years ago 8
. However, macrophages subsequently invade the nerve and clear most of the debris, thereby overshadowing the contribution of the Schwann cells 36
. A more recent study demonstrated that Schwann cells play an important phagocytic role in synapse elimination during the development of the neuromuscular junction 37
. Early EM studies of the developing chick embryo suggested the presence of degenerated axons and apoptotic neurons in astrocytes, satellite glial cells and Schwann cells7, 8, 38
; however, the identity of the engulfing cells was not confirmed due to the absence of immunological markers. Furthermore, in some cases, contradictory observations were reported, suggesting that macrophages were clearing the debris 8
. Our findings demonstrate that SGCs, not macrophages, are the primary phagocytes responsible for clearing the dead neurons generated during the normal development of the DRG.
The physiological roles of SGCs are not well understood. They are found in sensory, sympathetic and parasympathetic ganglia, where they cluster around each neuron and regulate the extracellular environment, taking up neurotransmitters similar to astrocytes 39
. SGCs also produce numerous neuroactive agents such as neurotrophins and bradykinin, although the functions of these in the ganglia are not clear 39
. Following injury the SGCs undergo a morphological change, begin to proliferate and release many of these factors, leading some to suggest that they are involved in neuropathic pain 39, 40
. Their phagocytic ability characterized in this study provides a rare glimpse into an important function of SGCs. Whether the glial cells remain the primary cell type responsible for phagocytosing dead neurons in more mature animals, for example, after axotomy or other inducers of neurodegeneration, remains to be determined.
The molecular mechanisms involved in apoptotic neuron clearance in the developing mammalian PNS were previously unknown. In C. elegans
, CED-1 was identified as a receptor required for engulfment of apoptotic cells 14
. Draper, the Drosophila
homolog of CED-1, was shown to mediate dead neuron removal during development13
and in eliminating degenerating axons during metamorphosis and after injury 15, 16, 33
. We identified MEGF10 and Jedi-1 as possible homologs based on their predicted structural organization and their expression pattern. MEGF10 was previously suggested to be an engulfment receptor 19
, but little is known about Jedi-1. It was shown to be phosphorylated upon platelet activation, although its role in this process was not determined, and its over expression in hematopoietic progenitors reduced the number of cells that committed to a myeloid lineage, suggesting a role in differentiation of these cells. Here, we demonstrate that Jedi-1 functions as a phagocytic receptor, involved in clearing dead sensory neurons.
Interestingly, both Jedi-1 and MEGF10 promoted the engulfment of neuronal corpses by SGCs ( and ); however, over expression or knock-down of both proteins was no more effective than altering the expression of either alone. We, therefore, suggest that these receptors may converge on a common pathway or even form a complex. However, they also exerted somewhat different actions when over expressed in glial cells, suggesting there are some non-overlapping functions. Almost all the cells transfected with MEGF10 had engulfed more than one apoptotic nuclei (90.6 % ± 1.3) and most had numerous apoptotic nuclear remnants in lysosomes. This could be due to an increase in the number of dead neurons taken up by a single cell or an increase in the number of lysosomes digesting the engulfed material. Increased vacuole formation has been described in HEK293 cells over expressing MEGF10, even when no apoptotic cells were added 34
. On the other hand, most of the Jedi-1-over expressing glial cells appeared slimmer with longer processes () and contained only one or two visible vacuoles with ingested nuclei, unlike those over expressing MEGF10 (). It is possible that Jedi has the ability to promote both engulfment and degradation of apoptotic cells, like CED-1 41
; whereas mEGF10 only carries the engulfment activity. Of course, we cannot rule out the possibility that over expression of either protein indirectly increases engulfment, for example, by increasing process formation. In any case, these differences suggest that, upon activation by the “eat-me” signals, MEGF10 and Jedi-1 trigger, at least partially, non-overlapping molecular pathways for engulfment.
Such a need for multiple receptors is typical of the phagocytic process. Multiple ligands and receptors are implicated in the recognition and uptake of apoptotic cells by “professional” phagocytes like macrophages 12, 42
. Just for phosphatidylserine, the most well studied “eat-me” cue, there are at least four transmembrane receptors, PSR, Tim4, BAI1, and Stabilin-2, that have been shown to bind this phospholipid and transduce an engulfment signal 9
. Even in C. elegans
, there are two partially redundant pathways that mediate cell corpse removal, with ced-1
genes functioning in one pathway and ced-2
genes acting in the other 43
. Recently, a second Drosophila receptor, SIMU was identified and suggested to function as the recognition receptor 44
with Draper acting primarily in the engulfment and degradation process through recruitment of the src family kinases 45
Programmed cell death and process elimination are essential for the development and maintenance of functional nervous systems. Consequently, considerable effort has been made to understand the molecular mechanisms involved in these events; however, less attention has been given to the resulting byproducts. During such regressive processes, large amounts of degenerated and excess cellular debris are generated that need to be efficiently eliminated. Several reports have provided convincing evidence for a link between defective clearance of apoptotic cells and the development of autoimmunity 3, 46, 47
. Hence, it is likely that defects in neural waste clearance during development predisposes an organism to autoimmune attack on the nervous system later in life, although this has yet to be demonstrated.