InteractiveFly: GeneBrief

orion: Biological Overview | References

Phagocytic clearance of degenerating neurons is triggered by 'eat-me' signals exposed on the neuronal surface. The conserved neuronal eat-me signal phosphatidylserine (PS) and the engulfment receptor Draper (Drpr) mediate phagocytosis of degenerating neurons in Drosophila. However, how PS is recognized by Drpr-expressing phagocytes in vivo remains poorly understood. Using multiple models of dendrite degeneration, this study shows that the Drosophila chemokine-like protein orion can bind to PS and is responsible for detecting PS exposure on neurons; it is supplied cell-non-autonomously to coat PS-exposing dendrites and to mediate interactions between PS and Drpr, thus enabling phagocytosis. As a result, the accumulation of orion on neurons and on phagocytes produces opposite outcomes by potentiating and suppressing phagocytosis, respectively. Moreover, the orion dosage is a key determinant of the sensitivity of phagocytes to PS exposed on neurons. Lastly, mutagenesis analyses show that the sequence motifs shared between orion and human immunomodulatory proteins are important for orion function. Thus, these results uncover a missing link in PS-mediated phagocytosis in Drosophila and imply conserved mechanisms of phagocytosis of neurons (Ji, 2023).

Phagocytic clearance of degenerating neurons is triggered by 'eat-me' signals exposed on the neuronal surface. The conserved neuronal eat-me signal phosphatidylserine (PS) and the engulfment receptor Draper (Drpr) mediate phagocytosis of degenerating neurons in Drosophila. However, how PS is recognized by Drpr-expressing phagocytes in vivo remains poorly understood. Using multiple models of dendrite degeneration, this study shows that the Drosophila chemokine-like protein orion can bind to PS and is responsible for detecting PS exposure on neurons; it is supplied cell-non-autonomously to coat PS-exposing dendrites and to mediate interactions between PS and Drpr, thus enabling phagocytosis. As a result, the accumulation of orion on neurons and on phagocytes produces opposite outcomes by potentiating and suppressing phagocytosis, respectively. Moreover, the orion dosage is a key determinant of the sensitivity of phagocytes to PS exposed on neurons. Lastly, mutagenesis analyses show that the sequence motifs shared between orion and human immunomodulatory proteins are important for orion function. Thus, these results uncover a missing link in PS-mediated phagocytosis in Drosophila and imply conserved mechanisms of phagocytosis of neurons (Ji, 2023).

Phagocytosis of apoptotic and degenerative neurons is essential for the development and homeostasis of the nervous system. Abnormal phagocytosis is also associated with neuroinflammation and neurodegenerative diseases. Neuronal debris is recognized and cleared by resident phagocytes of the nervous system through 'eat-me' signals exposed on the neuronal surface. A conserved 'eat-me' signal is phosphatidylserine (PS), a negatively charged phospholipid normally kept in the inner leaflet of the plasma membrane by P4-ATPase flippases. During neurite degeneration and apoptosis, PS is externalized to the outer surface of neuronal membranes. The exposed PS dominantly triggers phagocytosis of neurons. Besides clearing neuronal debris, PS-mediated phagocytosis also drives the degeneration of injured neurites and neurons with certain genetic perturbations. In the central nervous system (CNS), local PS exposure enables microglia-mediated synaptic elimination. Thus, the regulation and recognition of neuronal PS exposure are critical for the development and homeostasis of the nervous system (Ji, 2023).

Drosophila has been an important model organism for studying neuronal phagocytosis. In Drosophila, Draper (Drpr) is the best-known receptor responsible for phagocytosis of neurons. As a homolog of the Caenorhabditis elegans engulfment receptor CED-1 and the mammalian engulfment receptors Jedi-1 and MEGF10, Drpr is involved in many contexts of neuronal phagocytosis, including the clearance of apoptotic neurons during embryonic development, axon and dendrite pruning during neuronal remodeling, injury-induced neurite degeneration, and removal of destabilized boutons at neuromuscular junctions. Despite the well-known importance of Drpr in sculpting the nervous system, how Drpr recognizes degenerating neurons in vivo is still unclear (Ji, 2023).

Recently, the secreted protein orion was discovered as being required for the developmental pruning and clearance of Drosophila mushroom body (MB) axons (Boulanger, 2021). orion shares a CX3C motif with mammalian CX3CL1 (also known as fractalkine), which is required for the elimination of synapses in the mouse barrel cortex. CX3CL1 is known as a chemokine because of its ability to direct migration of leukocytes and microglia. Thus, orion represents the first known chemokine-like molecule in Drosophila and shares conserved functions with CX3CL1 in the remodeling of the nervous system. However, how orion and CX3CL1 function in the phagocytosis of neurons is still unknown (Ji, 2023).

This study examined orion's function in the phagocytosis of Drosophila class IV dendritic arborization (C4da) neurons, a well-established in vivo model for studying PS-mediated phagocytosis. In vivo evidence is presented that the transmembrane engulfment receptor Drpr relies on orion to sense PS on neurons. orion is secreted by peripheral nonneural tissues and serves as a nonautonomous permissive factor for phagocytosis of neurons. orion binds to PS-exposing neurons and is also enriched on phagocytes overexpressing Drpr. Strikingly, a membrane-tethered orion expressed by neurons dominantly induces phagocytosis even in the absence of PS exposure, while orion accumulation on the surface of phagocytes makes phagocytes blind to PS-exposing neurons. Importantly, the dosage of orion determines the sensitivity of phagocytes to neuronal PS exposure. Lastly, this study established that the motifs orion shares with human chemokines and neutrophil peptides are critical for engulfment of PS-exposing neurons. These findings reveal key mechanisms of PS recognition in Drosophila and imply potentially conserved roles of chemokines in PS-mediated phagocytosis of neurons (Ji, 2023).

By triggering phagocytosis, the recognition of PS exposed on neurons is a critical event during neurodegeneration and clearance. Although several studies have implied the involvement of Drpr in this process in Drosophila, how Drpr mediates PS recognition in vivo is unclear. This study presents several lines of in vivo evidence that strongly indicate that the Drosophila chemokine-like orion is a PS-binding bridging molecule that enables Drpr to respond to neuronal PS exposure. First, orion is required for multiple scenarios of Drpr-dependent phagocytosis of sensory dendrites and functions upstream of Drpr. Second, orion binds to PS-exposing cell surfaces. This study shows that orion binds to neurons and epidermal cells that expose PS as a result of tissue-specific KO of the PS flippase ATP8A. In addition, ATP8A OE, which retains PS in the inner membrane leaflet, eliminates orion binding on epidermal cells and late-larval dendrites, suggesting that this binding is PS-dependent. Importantly, orion outcompetes Annexin V for binding to injured dendrites, suggesting that orion may directly interact with PS in vivo. Third, overexpressed Drpr proteins can trap orion on the cell surface, suggesting that Drpr interacts with orion in vivo. Lastly, when expressed in neurons, membrane-tethered orion bypasses the requirement for PS in inducing Drpr-dependent engulfment, but when expressed in phagocytes, membrane-tethered orion blocks PS-induced engulfment. Based on these observations, it is proposed that orion functions as a bridging molecule between PS and Drpr (Ji, 2023).

Previously, SIMU, a PS-binding transmembrane protein expressed by Drosophila embryonic phagocytes, was proposed to be a bridging molecule. orion and SIMU contribute to phagocytosis through distinct mechanisms. First, SIMU is expressed by phagocytes to allow them to tether apoptotic cells, while orion is secreted from many peripheral tissues and functions as an opsonin to enable phagocytosis. Second, SIMU is a membrane protein that shares homology with Drpr but functions at a different step in apoptotic neuron clearance compared to Drpr. In contrast, as a secreted protein, orion functions at the same step of phagocytosis as Drpr. Therefore, SIMU behaves more like a tethering receptor, while orion is functionally analogous to PS-bridging molecules in other species. Although the analyses on the engulfment of somatosensory dendrites, the ubiquitous roles of PS and Drpr in phagocytosis and the broad expression patterns of orion suggest that orion may be widely involved in PS-mediated phagocytosis in Drosophila. This view is supported by findings that orion deposited in the hemolymph can mediate phagocytosis in distant tissues and that the accumulation of orion on epidermal cells turns these cells into targets of phagocytosis (Ji, 2023).

Although the role of PS exposure in inducing phagocytosis has been well documented, what determines the sensitivity of phagocytes to PS is much less understood. This study discovered that the available level of orion is a determinant of phagocyte sensitivity to PS in Drosophila. Reducing the dosage of functional orion by one half makes phagocytes blind to dendrites with moderate levels of PS exposure, but the reduced orion does not affect the ability of phagocytes to engulf dendrites that display high levels of PS exposure (i.e., injury). Conversely, an extra copy of the orion locus enhances the ability of phagocytes to engulf dendrites that have mild PS exposure (i.e., CDC50 KO). These results suggest that endogenous orion is likely expressed at a finely balanced level to enable the right amount of phagocytosis: Too much orion may cause unintended phagocytosis of stressed cells that display mild PS exposure, while too little orion may interfere with efficient clearance of sick cells or structures that are beyond rescue. Consistent with this idea, endogenous orion is expressed at a low level during larval development, but is dramatically up-regulated during metamorphosis, a time when large-scale tissue remodeling and clearance take place (Ji, 2023).

orion is required for axonal pruning and clearance of MB γ Kenyon neurons during metamorphosis. In that context, orion is expressed in the remodeling MB neurons and functions as a 'find-me' signal for glia to penetrate the axon bundles and engulf axonal debris. In contrast, in the larval peripheral nervous system (PNS), orion is supplied by many nonneural tissues and functions as a permissive signal for phagocytosis of sick or broken dendrites. This distinction is likely due to two differences between the larval PNS and the remodeling CNS. First, in the PNS, the dendrites of da neurons are exposed to the hemolymph and are readily accessible to orion secreted from other tissues, whereas in the CNS, the axons are more tightly packed and may be harder for extrinsic orion to access. Neuron-derived orion would thus be more effective than extrinsic orion for promoting phagocytosis in the CNS. Intriguingly, orion expression was detected in a small number of neurons in the larval ventral nerve cord. It will be interesting to find out whether these neurons are particularly subject to degeneration. Second, compared to the larval PNS where degenerative events are rare, the nervous system undergoing metamorphosis has a much greater demand for phagocytosis. Turning on orion expression in neurons may thus be required for efficient clearance of all pruned neurites. Consistent with this idea, orion expression was also in a subset of da neurons during metamorphosis (Ji, 2023).

Fluorescent PS probes based on AV and Lact are widely used to visualize PS exposure in cell culture and live animals and have been crucial for many discoveries in PS biology. LactC1C2 is known to have a higher affinity to PS than AV. Previously additional differences were observed between these two proteins. Compared to LactC1C2, which coats the dendrite surface well, AV tends to traffic to endocytic vesicles inside PS-exposing dendrites, consistent with the ability of the AV complex to induce endocytosis upon PS binding. Importantly, unlike AV, which does not alter the kinetics of neurite degeneration, LactC1C2 binding to PS-exposing dendrites potentiates Drpr-dependent degeneration. This latter observation previously led us to hypothesize that LactC1C2 may contain unknown sequences that interact with Drpr. This study showed that the effect of LactC1C2 in exacerbating dendrite degeneration depends on orion, suggesting instead that LactC1C2 may indirectly promote phagocyte/dendrite interactions by enhancing orion function. One possible mechanism is that LactC1C2 binding on the plasma membrane further disrupts the membrane and causes more PS exposure that can subsequently be detected by endogenous orion (Ji, 2023).

Compared to AV and LactC1C2, orion displays unique lipid-binding properties. The in vivo evidence suggests that orion may have a higher affinity for PS than AV, as orion efficiently outcompetes AV in binding to injured dendrites. In addition, overexpressed orion binds to healthy epidermal cells and dendrites (albeit the latter only in wandering 3rd instar larvae), while AV and LactC1C2 do not. Althoughr in vitro evidence suggests that orion may have an intrinsic affinity for phospholipid bilayers, in vivo orion binding to epidermal cells and WT dendrites largely depends on PS exposure. One possibility is that other endogenous factors modify orion-lipid interactions and make orion more specific to PS in vivo (Ji, 2023).

The surprising finding that overexpressed orion binds to peripheral tissues and dendrites suggests that these cells may expose PS under physiological conditions, perhaps at a level too low to detect by AV and LactC1C2. Thus, low levels of PS exposure may be much more prevalent in vivo than previously thought. Intriguingly, binding of overexpressed orion induces degeneration of wild-type dendrites but does not cause obvious phagocytosis of other nonneural tissues, suggesting that neurons may be more vulnerable to PS-induced phagocytosis than other cell types (Ji, 2023).

Recently, many human chemokines were found to bind to PS exposed on apoptotic vesicles and serve as 'find-me' signals to attract phagocytes (Pontejo, 2021). orion shares the CX3C motif with the mammalian chemokine CXC3L1 and has also three glycosaminoglycan putative binding sequences, a hallmark of chemokine activity. Even though direct binding to PS has not been demonstrated for CXC3L1, this chemokine is required for microglia-mediated synapse elimination after whisker lesioning (Gunner, 2019), a process likely involving PS exposure. In addition, orion contains a RRY motif commonly found in human neutrophil peptides, small antimicrobial peptides important for innate immunity. Both CX3C and RRY motifs are important for orion function in mediating phagocytosis of neurons. Although orion does not show global sequence homologies to mammalian immunomodulatory proteins, its interaction partner Drpr has mammalian homologs that are involved in the phagocytosis of neurons through unknown mechanisms . Thus, the common features between orion and human proteins indicate that a functional conservation may exist between PS-sensing mechanisms in insects and humans (Ji, 2023).

Chemokine-like orion is involved in the transformation of glial cells into phagocytes in different developmental neuronal remodeling paradigms

During animal development, neurons often form exuberant or inappropriate axons and dendrites at early stages, followed by the refinement of neuronal circuits at late stages. Neural circuit refinement leads to the production of neuronal debris in the form of neuronal cell corpses, fragmented axons and dendrites, and pruned synapses requiring disposal. Glial cells act as predominant phagocytes during neuronal remodeling and degeneration, and crucial signaling pathways between neurons and glia are necessary for the execution of phagocytosis. Chemokine-like mushroom body neuron-secreted orion is essential for astrocyte infiltration into the γ axon bundle leading to γ axon pruning. This study shows a role of orion in debris engulfment and phagocytosis in Drosophila. Interestingly, orion is involved in the overall transformation of astrocytes into phagocytes. In addition, analysis of several neuronal paradigms demonstrates the role of orion in eliminating both peptidergic vCrz+ and PDF-Tri neurons via additional phagocytic glial cells like cortex and/or ensheathing glia. These results suggest that orion is essential for phagocytic activation of astrocytes, cortex and ensheathing glia, and point to orion as a trigger of glial infiltration, engulfment and phagocytosis (Perron, 2023).

Developmental remodeling of neural circuitry is a key strategy employed to prune redundant, inappropriate or interfering neurons to optimize connectivity. Signals sent from dying neurons or neurites to be removed are received by appropriate glial cells. After receiving these signals, glia infiltrate degenerating sites, engulf, and clear neuronal debris through phagocytic mechanisms. The chemokine-like orion has been identified as ligand, secreted by Drosophila MB γ neurons of the central brain, inducing astrocytic infiltration into the γ axon bundle (Boulanger, 2021). Thus, in orion-lacking flies, astrocytes are unable to infiltrate the γ axon bundle and, consequently, axon fragmentation-resulting debris is not eliminated during metamorphosis. Previous observations have shown a high elimination of neuronal processes and synaptic terminals along the VNC early in development by astrocytes, which already infiltrate the neuropil before neuronal remodeling, differing from what occurs in MB bundles. This study shows that orion has a crucial role in engulfment and phagocytosis of VNC neuronal debris. These data suggest that orion not only orchestrates glia infiltration into the axonal bundle fated to degenerate but is also required to engulf remnant debris and for phagocytosis (Perron, 2023).

Little is known about the pathways involved in glial activation. Astrocyte activation into phagocytes has been previously documented. Glia activation is characterized by enlarged processes and abundant phagocytic vacuoles displaying phagolysosomal activity in the astrocyte cytoplasm. This activation depends on the expression of steroid hormone 20-hydroxyecdysone (ecdysone) receptor (EcR), which regulates the expression of drpr. Thus, loss of EcR signaling is sufficient to cell-autonomously suppress the transformation of astrocytes into phagocytes at pupariation. Interestingly, a similar phenotype of astrocyte vacuolated appearance and thick extensions was observed in orion-lacking flies, suggesting that orion mediates the overall transformation of astrocytes into phagocytes leading to engulfment and phagocytosis (Perron, 2023).

MB γ neurons prune their medial and dorsal axon branches and dendrites at an early pupal stage, while their cell bodies remain. Later, at mid-pupal stages, they re-extend medial axon branches to establish adult-specific connectivity. In contrast, peptidergic vCrz+ and PDF-Tri neurons exhibit complete neurite degeneration, and their cell bodies undergo apoptotic death and are eventually eliminated. This elimination occurs at different developmental stages: the vCrz+ neuron apoptosis initiates in early pupae, whereas the PDF-Tri neuron apoptosis starts early after adult eclosion. This study shows that orion is involved in eliminating both types of peptidergic neurons, extending the role of orion to the elimination of apoptotic neurons. Interestingly, orion is needed not only for the elimination of vCrz+ and PDF-Tri neurites, but also cell bodies. This suggests that orion can also be presented to phagocytic cells by soma to be removed. Furthermore, as PDF-Tri neurons are eliminated in newly eclosed flies, the results formally extend the role of orion to young adult stages (Perron, 2023).

Cortex glia appears to be essential for eliminating both types of neuronal cell bodies, as revealed by the high level of cortex glia engulfment around the vCrz+ and PDF-Tri cell bodies in wild types compared with orion mutants. Interestingly, a high number of ensheathing glia extensions was observed along the PDF-Tri neurites on both the MDBL and SEZ regions. The proximity of this type of glia to PDF-Tri axons in wild-type and orion mutant flies does not allow determination of the engulfment degree of these axons. Nevertheless, as the lack of orion induces a high amount of uncleared axonal PDF-Tri debris and based on the observations showing PDF-Tri debris surrounded by ensheathing glia in wild-type flies during PDF-Tri neuronal remodeling, it can be anticipated that this type of glia is also a target of orion. This idea is reinforced by a study suggesting that ensheathing glia drive the clearance of PDF-Tri neurons. Together, these data provide evidence that orion is able to signal via astrocyte, cortex and ensheathing glia, depending on the neuronal remodeling paradigm (Perron, 2023).

Recent studies have shown that orion is required for the elimination of larval dendritic arborization (da) dendrites by phagocytic epidermal cells after laser ablation at larval stages (Ji, 2023). To extend the role of orion to axonal injury paradigms, this study explored two distinct antennal and wing axotomy models in Drosophila. orion is not involved in the clearance of the ORN axonal debris, as a similar level of axon debris clearance is observed in controls and orion1 mutants after ORN axotomy. In the Drosophila L1 wing vein, wrapping glia eliminate the debris from injured axons. After axotomy, orion is not involved in the communication between severed axons and wrapping glia, suggesting that orion is not required for proper glial clearance after axotomy in adult flies, which contrasts with the dendrite injury model in larvae. Concerning the lack of orion effect in NMJ dismantling, it could be explained by the fact that this is a retraction process not implying phagocytosis. Thus, orion plays a crucial role during development, but not in adult flies (Perron, 2023).

orion expression in MB is necessary and sufficient for γ neuron remodeling (Boulanger, 2021). In contrast, orion expression is not required in da neurons for their remodeling (Ji, 2023). Moreover, expression of orion in the fat body is sufficient to rescue da remodeling phenotypes, suggesting, in this case, sources of orion external from the neurons to be remodeled. Similarly, the data led to a hypothesis that secretion of orion from sources other than vCrz and PDF-Tri neurons is sufficient to rescue vCrz and PDF-Tri neuron remodeling phenotypes seen in an orion mutant (elav-GAL4; UAS-orion). The specific orion expression in both types of remodeling neurons, which does not rescue orion mutant phenotypes (Crz- and PDF-GAL4; UAS-orion), and the specific UAS-orion-RNAi expression, which does not block their pruning, are in accordance with this hypothesis (Perron, 2023).

orion is required in neurons other than Crz for vCrz neuronal remodeling (elav-GAL4; UAS-orion-RNAi). This implies that other neuron-secreted orion targets vCrz neurons to be remodeled. For the PDF-Tri neuronal remodeling,it was not possible conclude about the source of orion for technical reasons (leakiness of the UAS-orion-RNAi at adult stage) (Perron, 2023).

One can note that, even if neuronal sources of orion are required for vCrz neuronal remodeling, it does not preclude that other sources of orion are not also required. Multiple sources of orion could also be the case for the PDF-Tri neuronal remodeling. In accordance with this hypothesis, it is reported that a strong expression of orion was observed during development in many tissues, such as fat body, epidermal cells, trachea, hemocytes and glia. Therefore, MBs could be a particular case in which orion expression is required in the neurons to be remodeled. This could be due to the requirement of glia infiltration into the MB bundles, an initial step of pruning not required for remodeling of individual neurons. Consequently, this suggests that orion has two functions: one specific for glia infiltration in MB bundles and one general for phagocytosis of neuronal debris (Perron, 2023).

Axonal chemokine-like orion induces astrocyte infiltration and engulfment during mushroom body neuronal remodeling

The remodeling of neurons is a conserved fundamental mechanism underlying nervous system maturation and function. Astrocytes can clear neuronal debris and they have an active role in neuronal remodeling. Developmental axon pruning of Drosophila memory center neurons occurs via a degenerative process mediated by infiltrating astrocytes. However, how astrocytes are recruited to the axons during brain development is unclear. Using an unbiased screen, the gene requirement of orion/CG2206, encoding for a chemokine-like protein, was identified in the developing mushroom bodies. Functional analysis shows that orion is necessary for both axonal pruning and removal of axonal debris. orion performs its functions extracellularly and bears some features common to chemokines, a family of chemoattractant cytokines. It is proposed that orion is a neuronal signal that elicits astrocyte infiltration and astrocyte-driven axonal engulfment required during neuronal remodeling in the Drosophila developing brain (Boulanger, 2021).

Neuronal remodeling is a widely used developmental mechanism, across the animal kingdom, to refine dendrite and axon targeting necessary for the maturation of neural circuits. Importantly, similar molecular and cellular events can occur during neurodevelopmental disorders or after nervous system injury. A key role for glial cells in synaptic pruning and critical signaling pathways between glia and neurons has been identified. In Drosophila, the mushroom body (MB), a brain memory center, is remodeled at metamorphosis and MB γ neuron pruning occurs by a degenerative mechanism. Astrocytes surrounding the MB have an active role in the process: blocking their infiltration into the MBs prevents remodeling. MB γ neuron remodeling relies on two processes: axon fragmentation and the subsequent clearance of axonal debris. Importantly, it has been shown that astrocytes are involved in these two processes and that these two processes can be decoupled. Altering the ecdysone signaling in astrocytes, during metamorphosis, results both in a partial axon pruning defect, visualized as either some individual larval axons or as thin bundles of intact larval axons remaining in the adults, and also in a strong defect in clearance of debris, visualized by the presence of clusters of axonal debris. Astrocytes have only a minor role in axon severing as evidenced by the observation that most of the MB γ axons are correctly pruned when ecdysone signaling is altered in these cells. When astrocyte function is blocked, the γ axon-intrinsic fragmentation process remains functional and the majority of axons degenerate (Boulanger, 2021).

It has been widely proposed that a 'find-me/eat-me' signal emanating from the degenerating γ neurons is necessary for astrocyte infiltration and engulfment of the degenerated larval axons. However, the nature of this glial recruitment signal is unclear (Boulanger, 2021).

This study has identified a gene (orion), not previously described, by screening for viable ethyl methanesulfonate (EMS)-induced mutations and not for lethal mutations in MB clones as was done previously. This allowed the identification of genes involved in glial cell function by directly screening for defects in MB axon pruning. It was found that orion¹, a viable X-chromosome mutation, is necessary for both the pruning of some γ axons and removal of the resulting debris. orion is secreted from the neurons, remains near the axon membranes where it associates with infiltrating astrocytes, and is necessary for astrocyte infiltration into the γ bundle. This implies a role for an as-yet-undefined orion receptor on the surface of the astrocytes. orion bears some chemokine features, for example, a CX3C motif, three glycosaminoglycan (GAG) binding consensus sequences that are required for its function. Altogether, these results identify a neuron-secreted extracellular messenger, which is likely to be the long-searched-for signal responsible for astrocyte infiltration and engulfment of the degenerated larval axons and demonstrate its involvement for neuronal remodeling (Boulanger, 2021).

Adult orion¹ individuals showed a clear and highly penetrant MB axon pruning phenotype as revealed by the presence of some adult unpruned vertical γ axons as well as the strong presence of debris (100% of mutant MBs). Astrocytes, visualized with alrm-GAL4, are the major glial subtype responsible for the clearance of the MB axon debris. The presence of γ axon debris is a landmark of defective astrocyte function, as has been described, and is also further shown in this study. The unpruned axon phenotype was particularly apparent during metamorphosis. At 24 h after puparium formation (APF), although γ axon branches were nearly completely absent in the wild-type control, they persisted in the orion¹ mutant brains, where a significant accumulation of debris was also observed. The number of unpruned axons at this stage is lower in orion¹ than in Hr39C13 where the γ axon-intrinsic process of pruning is blocked. In addition, the MB dendrite pruning was clearly affected in orion¹ individuals (Boulanger, 2021).

The orion¹ EMS mutation was localized by standard duplication and deficiency mapping as well as by whole-genome sequencing. The orion gene (CG2206) encodes two putatively secreted proteins: orion-A [664 amino acid (a.a.)] and orion-B (646 a.a.), whose messenger RNAs (mRNAs) arise from two different promoters. These two proteins differ in their N-terminal domains and are identical in the remainder of their sequences. The EMS mutation is a G to C nucleotide change inducing the substitution of the glycine (at position 629 for orion-A and 611 for orion-B) into an aspartic acid. The mutation lies in the common shared part and therefore affects both orion-A and -B functions. Both isoforms display a signal peptide at their N termini, suggesting that they are secreted. Interestingly, a CX3C chemokine signature is present in the orion common region. Chemokines are a family of chemoattractant cytokines, characterized by a CC, CXC, or CX3C motif, promoting the directional migration of cells within different tissues. Mammalian CX3CL1 (also known as fractalkine) is involved in, among other contexts, neuron-glia communication. Mammalian Fractalkines display conserved intramolecular disulfide bonds that appear to be conserved with respect to their distance from the CX3C motif present in both orion isoforms. Fractalkine and its receptor, CX3CR1, have been recently shown to be required for post-trauma cortical brain neuron microglia-mediated remodeling in a mouse whisker lesioning paradigm. This study observed that the change of the CX3C motif into CX4C or AX3C blocked the orion function necessary for the MB pruning. Similarly, the removal of the signal peptide also prevented pruning. These two results indicate that the orion isoforms likely act as secreted chemokine-like molecules. Three CRISPR/Cas9-mediated mutations in the orion gene were generated that either delete the common part (orionΔC), the A-specific part (orionΔA), or the B-specific part (orionΔB). Noticeably, orionΔC displayed the same MB pruning phenotype as orion¹, which is also the same in orion¹/Deficiency females, indicating that orion¹ and orionΔC are likely null alleles for this phenotype. In contrast, orionΔA and orionΔB have no MB phenotype by themselves, indicating the likelihood of functional redundancy between the two proteins in the pruning process (Boulanger, 2021).

Using the GAL4/UAS system, this study found that expression of wild-type orion in the orion¹ MB γ neurons (201Y-GAL4) fully rescued the MB mutant phenotype (100% of wild-type MBs n = 387), although wild-type orion expression in the astrocytes (alrm-GAL4) did not rescue. repo-GAL4 could not be used because of lethality when combined with UAS-orion. This supports the hypothesis that orion is produced by axons and, although necessary for astrocyte infiltration, not by astrocytes. Both UAS-orion-A and UAS-orion-B rescued the orion¹ pruning phenotype indicating again a likely functional redundancy between the two proteins at least in the pruning process. Complementary to the rescue results, this study found that the expression of an orion-targeting RNA interference (RNAi) in the MBs produced unpruned axons similar to that in orion¹, although the debris is not apparent likely due to an incomplete inactivation of the gene expression by the RNAi. The expression of the same RNAi in the glia had no effect. Using the mosaic analysis with a repressible cell marker (MARCM), it was found that orion¹ homozygous mutant neuroblast clones of γ neurons, in orion¹/+ phenotypically wild-type individuals, were normally pruned. Therefore, orion¹ is a non-cell-autonomous mutation that is expected if the orion proteins are secreted. orion proteins secreted by the surrounding wild-type axons rescue the pruning defects in the orion mutant clones (Boulanger, 2021).

From genetic data, orion expression is expected in the γ neurons. The fine temporal transcriptional landscape of MB γ neurons was recently described and a corresponding resource is freely accessible. Noteworthy, orion is transcribed at 0 h APF and dramatically decreases at 9 h APF with a peak at 3 h APF. The nuclear receptor EcR-B1 and its target Sox14 are key transcriptional factors required for MB neuronal remodeling. orion was found to be a likely transcriptional target of EcR-B1 and Sox14. This is also consistent with earlier microarray analysis observations. Noticeably, forced expression of UAS-EcR-B1 in the MBs did not rescue the orion mutant phenotype and EcR-B1 expression, in the MB nuclei, and is not altered in orion¹ individuals. Furthermore, the unpruned axon phenotype produced by orion RNAi is rescued by forced expression of EcR-B1 in the MBs. Therefore, the genetic interaction analyses support orion being downstream of EcR-B1 (Boulanger, 2021).

Further molecular and cellular work focused on orion-B alone since a functional redundancy between the two isoforms was apparent. The orion-B protein was expressed in the γ neurons using an UAS-orion-B-Myc insert and the 201Y-GAL4 driver. orion-B was present along the MB lobes and extracellularly present as visualized by anti-Myc staining. Indeed, anti-Myc staining was particularly strong at the tip of the lobes indicating the presence of extracellular orion-B. Synaptic terminals are condensed in the γ axon varicosities that disappear progressively during remodeling and hole-like structures corresponding to the vestiges of disappeared varicosities can be observed at 6 h APF. The presence of Myc-labeled orion-B was noticed inside these hole-like structures. The secretion of the orion proteins should be under the control of their signal peptide and, therefore, orion proteins lacking their signal peptide (ΔSP) should not show this 'extracellular' phenotype. When UAS-orion-B-Myc-ΔSP was expressed, orion-B was not observed outside the axons or in the hole-like structures. The possibility that this 'extracellular' phenotype was due to some peculiarities of the Myc labeling was excluded by using a UAS-drl-Myc construct. Drl is a membrane-bound receptor tyrosine kinase and Drl-Myc staining, unlike orion-B, was not observed outside the axons or in the hole-like structures. In addition, the presence of Myc-labeled orion-B protein not associated with green fluorescent protein (GFP)-labeled axon membranes can be observed outside the γ axon bundle in 3D reconstructing images. Nevertheless, these signals are possibly located inside the glial compartments and not as freely diffusing orion protein. Finally, supporting the hypothesis that orion acts as a secreted protein, it has been reported to be present in biochemically purified exosomes, indicating that it may act on the glia via its presence on or in exosomes (Boulanger, 2021).

Since glial cells are likely directly involved in the orion¹ pruning phenotype, their behavior early during the pruning process was examined. At 6 h APF the axon pruning process starts and is complete by 24 h APF, but the presence of glial cells in the vicinity of the wild-type γ lobes is already clearly apparent at 6 h APF9. Glial cells visualized by a membrane-targeted GFP (UAS-mGFP) under the control of repo-GAL4 were examined, and the γ axons were co-stained with anti-Fas2. At 6 h APF, a striking difference was noted between wild-type and orion¹ brains. Unlike in the wild-type control, there is essentially no glial cell invasion of the γ bundle in the mutant. Interestingly, glial infiltration as well as engulfment of the degenerated larval axons was not observed in orion¹ neither at 12 h APF nor at 24 h APF, suggesting that glial cells never infiltrate MBs in mutant individuals. The possibility that this lack of glial cell activity was due to a lower number of astrocytes in mutant versus wild-type brains was ruled out (Boulanger, 2021).

The proximity was examined between MB orion-Myc and astrocytes, as inferred from the shape of the glial cells, labeled with the anti-Drpr antibody at 6 h APF. The distribution was examined along the vertical γ lobes (60 μm of distance) of orion-B-Myc (wild-type protein) and of orion-B-ΔSP-Myc (not secreted), in an otherwise wild-type background. Quantification was performed only from images where an astrocyte sat on the top of the vertical lobe. A peak of orion-Myc localization was always found in the axonal region close to the astrocyte (<7 μm) when wild-type orion-B-Myc was quantified. However, this was not the case (n = 9) when orion-B-ΔSP-Myc was quantified. This strongly suggests that astrocytic processes may be 'attracted' by secreted orion (Boulanger, 2021).

Moreover, it was observed that extracellularly present orion stays close to axon membranes. Protein (in particular chemokine) localization to membranes is often mediated by GAGs, a family of highly anionic polysaccharides that occurs both at the cell surface and within the extracellular matrix. GAGs, to which all chemokines bind, ensure that these signaling proteins are presented at the correct site and time in order to mediate their functions. Three consensus sequences for GAG linkage were identified in the common part of orion. These sequences were mutated individually, and the mutant proteins were examined for their ability to rescue the orion¹ pruning deficit in vivo. The three GAG sites are required for full orion function, although mutating only GAG3 produced a strong mutant phenotype (Boulanger, 2021).

These findings imply a role for an as-yet-undefined orion receptor on the surface of the glial cells. The glial receptor draper (drpr) seemed an obvious candidate, although Drpr ligands unrelated to orion have been identified. The MB remodeling phenotypes in orion¹ and drpr_Δ5 are, however, different with orion mutant phenotype being stronger than the drpr one. The use of an UAS-mGFP driven by 201Y-GAL4, instead of anti-Fas2, where the labeling of αβ axons often masks individual unpruned γ axons, allowed occasionally observation of unpruned axons in drprΔ5 1-week-old post-eclosion brains in addition to uncleared debris. This indicates a certain degree of previously undescribed unpruned axon persistence in the mutant background. Nevertheless, only orion mutant displayed a 100% penetrant phenotype of both unpruned axons and debris (strong category) in adult flies, which are still present in old flies. On the contrary, the weaker drpr mutant phenotype strongly decreases throughout adulthood. This suggests that Drpr is not an, or at least not the sole, orion receptor (Boulanger, 2021).

Independently of the possible role of Drpr as an orion receptor, it was of interest to test if orion could activate the drpr signaling pathway as it is the case for neuron-derived injury released factors and Spätzle ligands, which bind to glial insulin-like receptors and Toll-6, respectively, upregulating in turn the expression of drpr in phagocytic glia. These ligands are necessary for axonal debris elimination and act as a find-me/eat-me signal in injury and apoptosis, as orion is doing for MB pruning. The data indicate that orion does not modify either Drpr expression nor the level of the drpr transcriptional activator STAT92E in astrocytes. Consequently, orion does not seem to induce the Drpr signaling pathway in astrocytes (Boulanger, 2021).

This study has uncovered a neuronally secreted chemokine-like protein acting as a 'find-me/eat-me' signal involved in the neuron-glia crosstalk required for axon pruning during developmental neuron remodeling. Chemokine-like signaling in insects was not described previously and, furthermore, the results point to an unexpected conservation of chemokine CX3C signaling in the modulation of neural circuits. Thus, it is possible that chemokine involvement in neuron/glial cell interaction is an evolutionarily ancient mechanism (Boulanger, 2021).

Search PubMed for articles about Drosophila Orion

Boulanger, A., Thinat, C., Zuchner, S., Fradkin, L. G., Lortat-Jacob, H., Dura, J. M. (2021). Axonal chemokine-like Orion induces astrocyte infiltration and engulfment during mushroom body neuronal remodeling. Nat Commun. 12(1):1849. PubMed ID: 33758182

Gunner G., Cheadle, L., Johnson, K. M., Ayata, P., Badimon, A., Mondo, E., Nagy, M. A., Liu, L., Bemiller, S. M., Kim, K. W., Lira, S. A., Lamb, B. T., Tapper, A. R., Ransohoff, R. M., Greenberg, M. E., Schaefer, A., Schafer, D. P. (2019). Sensory lesioning induces microglial synapse elimination via ADAM10 and fractalkine signaling. Nat Neurosci. 22(7):1075-1088. PubMed ID: 31209379

Ji, H., Wang, B., Shen, Y., Labib, D., Lei, J., Chen, X., Sapar, M., Boulanger, A., Dura, J. M. and Han, C. (2023). The Drosophila chemokine-like orion bridges phosphatidylserine and Draper in phagocytosis of neurons. Proc Natl Acad Sci U S A 120(24): e2303392120. PubMed ID: 37276397

Perron, C., Carme, P., Rosell, A. L., Minnaert, E., Ruiz-Demoulin, S., Szczkowski, H., Neukomm, L. J., Dura, J. M., Boulanger, A. (2023). Chemokine-like Orion is involved in the transformation of glial cells into phagocytes in different developmental neuronal remodeling paradigms. Development150(19). PubMed ID: 37767633

Pontejo S. M., Murphy, P. M. (2021). Chemokines act as phosphatidylserine-bound "find-me" signals in apoptotic cell clearance. PLoS Biol. 19(5):e3001259. PubMed ID: 34038417

date revised: 6 December 2023

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.