InteractiveFly: GeneBrief

pretaporter: Biological Overview | References

Previous work has shown that the Arf1-mediated lipolysis pathway sustains stem cells and cancer stem cells (CSCs); its ablation resulted in necrosis of stem cells and CSCs, which further triggers a systemic antitumor immune response. This study shows that knocking down Arf1 in intestinal stem cells (ISCs) causes metabolic stress, which promotes the expression and translocation of ISC-produced damage-associated molecular patterns (DAMPs; Pretaporter [Prtp] and calreticulin [Calr]). DAMPs regulate macroglobulin complement-related (Mcr) expression and secretion. The secreted Mcr influences the expression and localization of enterocyte (EC)-produced Draper (Drpr) and LRP1 receptors (pattern recognition receptors [PRRs]) to activate autophagy in ECs for ATP production. The secreted ATP possibly feeds back to kill ISCs by activating inflammasome-like pyroptosis. This study identified an evolutionarily conserved pathway that sustains stem cells and CSCs, and its ablation results in an immunogenic cascade that promotes death of stem cells and CSCs as well as antitumor immunity (Aggarwal, 2022).

Previous work showed that the Arf1-mediated lipolysis pathway is specifically activated in stem cells and sustains stem cells in adult Drosophila (Singh, 2016). Arf1 is one of the most evolutionarily conserved genes between Drosophila and mouse, with an amino acid identity of 95.6% between the two species. It was found recently that Arf1-mediated lipid metabolism sustains cancer stem cells (CSCs) and that its ablation triggers immunogenic-like death (immunogenic cell death [ICD]) of CSCs and induces antitumor immunity by exposing damage-associated molecular patterns (DAMPs; calreticulin [Calr], high-mobility group box 1 [HMGB1], and ATP) (Aggarwal, 2022).

However, the molecular mechanism that coordinates stem cells/CSCs with neighboring cells to execute the biological processes (stem cell necrosis or anti-tumor immunity) is still unclear. This study dissected the molecular mechanism using the Drosophila genetic system. Knockdown of the pathway was found to promote stem cell death through an immunogenic-like and aging cascade. Ablation of Arf1-mediated lipid metabolism in Drosophila ISCs resulted in several aging-like hallmarks, including lipid droplet (LD) accumulation, Reactive oxygen species (ROS) accumulation, mitochondrial defects, mitophagy activation, and lysosomal protein aggregates, followed by an immunogenic-like cell death (Aggarwal, 2022).

ICD is a process that releases DAMPs and activates immune responses to destroy damaged or stressed cells in the absence of microbial components. These molecules are often present in a given cell compartment and are not expressed or are only somewhat expressed under physiological conditions but strongly induced and then translocated to the cell surface or extracellular space under conditions of stress, damage, or injury. The most important DAMPs are (1) pre-apoptotic exposure of the ER-sessile molecular chaperone Calr on the cell surface, (2) release of the non-histone nuclear protein HMGB1 into the extracellular space, and (3) active secretion of ATP. With respect to tumors, the surface-exposed Calr facilitates engulfment of tumor-associated antigens by binding to LRP1/CD91 receptors (pattern recognition receptors [PRRs]) on dendritic cells (DCs). During ICD, Calr interacts with another protein, ERp57, and the two are rapidly translocated to the cell surface from the ER lumen before the cells exhibit any sign of apoptosis. ERp57 is a disulfide isomerase that has several thioredoxin-like domains and regulates cell redox homeostasis. Knocking down Arf1-mediated lipolysis in ISCs was found to promote the expression and translocation of ISC-produced DAMPs (Pretaporter [Prtp] and Calr). Like ERp57, Prtp is a disulfide isomerase with several thioredoxin-like domains. The DAMPs may then regulate the expression and secretion of the protein macroglobulin complement-related (Mcr; a complement C5 homolog). The secreted Mcr possibly further controls the expression and localization of EC-produced Draper [Drpr] and LRP1 receptors (PRRs) to activate autophagy in ECs for ATP production. The secreted ATP likely feeds back to kill ISCs by activating inflammasome-like pyroptosis. Therefore, Arf1-mediated lipid metabolism is crucial for stem cell maintenance, and its ablation promotes stem cell decay and anti-tumor immunity through an immunogenic aging cascade (Aggarwal, 2022).

Stem cell functional decay or decline may be one of the important causes of organismal aging and disease. This study demonstrated that Arf1-mediated lipid metabolism sustains stem cells and that its ablation triggers an immunogenic-like stem cell death cascade. The dying stem cells display the following features: LD accumulation, mitochondrial defects, ROS production, ER stress and release of DAMPs to activate PRRs in neighboring ECs, mitophagy activation, lysosomal protein aggregations, and ISC necrosis through inflammasome-like pyroptosis. These features are similar to hallmarks of aging. Arf1 ablation in ISCs might trigger a stem cell aging and death cascade. The gold standard method for evaluating ICD is in vivo tumor vaccination. Previously an experiment of vaccination was performed in Arf1-ablated mice. The current study has demonstrated that many of the factors that contribute to ICD are expressed and function in Arf1-ablated flies, indicating that the pathway is partially conserved between Drosophila and mammals. However, it is important to confirm conserved biological functions of the ICD in Drosophila in future experiments. Similarly, inflammasome pyroptosis is only partially conserved between Drosophila and mammals. It is important to confirm the pathway by using inflammasome markers and demonstrate conserved biological functions of the pathway in Drosophila in future experiments (Aggarwal, 2022).

A previous report demonstrated that Mcr, through Drpr, cell non-autonomously regulates autophagy during wound healing and salivary gland cell death in Drosophila and that Prtp is not involved in this Mcr-Drpr-mediated autophagy induction. Mcr is an analog of mammalian C1q/C5. C1q binds to the Calr-LRP1 coreceptor in mammals, and Mcr binds to LRP1 (Flybase) in Drosophila. This study found that Calr and Prtp function in parallel or downstream of the Arf1-lipolysis pathway and regulate the expression of Mcr and LRP1. Mcr and LRP1 further regulate each other and control the expression of Drpr. Calr and Prtp also regulate the expression of their respective receptors, LRP1 and Drpr. This information suggests that two interconnected complexes, Calr-Mcr-LRP1 and Prtp-Drpr, function downstream of the Arf1-lipolysis pathway and coordinately regulate ISC death (Aggarwal, 2022).

In the mammalian immune system, DCs are activated after DAMPs bind to PRRs on their surface. The activated DCs present antigens to T cells, and the activated T cells kill damaged cells. The current study found that ablation of the COPI/Arf1-mediated lipolysis-β-oxidation pathway in stem cells induced expression of DAMPs, which then activate the phagocytic ECs through PRRs (LRP1 and Drpr) on the ECs to kill the stem cells. These findings suggest that such a coordinated cell death process is not limited to mammalian immune responses. In another naturally occurring example, Drpr pathway phagocytosis genes in follicle cells (FCs) non-autonomously promote nurse cell (NC) death in the developing Drosophila ovary. Although it is not clear how the stretch FCs time the precise developmental death of NCs, in light of the present findings, it is possible that a metabolic or stress signal during this developmental stage increases DAMPs in NCs to activate the Drpr pathway in FCs and non-autonomously promote NC death. DAMPs are also induced in organs during organ transplantation as a result of ischemic damage from the interrupted blood supply while the organ is outside of the body. The DAMPs induced in a graft stimulate immune responses mediated by host innate cells at the site of the graft and the donor's innate immune system and contribute to graft rejection. Drpr-mediated phagocytosis is also an essential process during development and in maintenance of tissue homeostasis in several systems. As mentioned above, the Mcr-Drpr pathway is involved in autophagy induction during wound healing and salivary gland cell death in Drosophila. It is proposed that such a coordinated cell death (CCD) is a novel and general cell death process in which death of abnormal or altered cells occurs by first sending danger signals (such as DAMPs) and then activating neighboring cells to execute the death process. The abnormality or alteration can be metabolic stress (such as disruption of Arf1-mediated lipid metabolism in stem cells), developmental changes (such as NC death during Drosophila ovary development or salivary gland cell death during metamorphosis), or damage during wound healing or circulation blockage during ischemic damage or pathogen infection. The danger signals then activate phagocytes and other cells (such as T cells) to cell non-autonomously promote targeted cell death. CCD may mediate cell aging/death and organ degeneration under physiological conditions or CSC death and anti-tumor immunity under pathological conditions (Aggarwal, 2022).

The finding that the DAMP-Mcr-LRP1/Drpr pathway connects metabolically stressed stem cells after Arf1 ablation to activation of phagocytic ECs to kill the stem cells will enable further dissection of the CCD mechanism in Drosophila. Arf1 is one of the most evolutionarily conserved genes, and the DAMP-Mcr/C1q-LRP1/Drpr pathway is well conserved throughout evolution. CCD involves coordination or communication of two or more different cells. Model organisms such as Drosophila, with their advanced genetic tractability and well-characterized cellular histology, will serve as valuable in vivo models for dissecting the detailed cellular and molecular mechanisms of CCD. These findings may lead to new therapeutic strategies for many human diseases, such as induction of anti-tumor immunity in individuals with cancer and the blocking of neuronal death in individuals with neurodegenerative conditions (Aggarwal, 2022).

This study has identified an evolutionarily conserved pathway that sustains stem cells, and its ablation results in an ICD cascade that promotes death of stem cells through inflammasome-like pyroptosis. It was demonstrated that many of the factors that contribute to ICD and inflammasome-like pyroptosis are expressed and function in Arf1-ablated flies. However, the gold standard method for evaluating ICD is in vivo tumor vaccination. The components of ICD and inflammasome-like pyroptosis are only partially conserved between Drosophila and mammals. It is important to further confirm the pathway by using inflammasome markers and demonstrate conserved biological functions of the pathway in Drosophila in future experiments (Aggarwal, 2022).

Fragile X mental retardation protein coordinates neuron-to-glia communication for clearance of developmentally transient brain neurons

In the developmental remodeling of brain circuits, neurons are removed by glial phagocytosis to optimize adult behavior. Fragile X mental retardation protein (FMRP) regulates neuron-to-glia signaling to drive glial phagocytosis for targeted neuron pruning. This study finds that FMRP acts in a mothers against decapentaplegic (Mad)-insulin receptor (InR)-protein kinase B (Akt) pathway to regulate pretaporter (Prtp) and amyloid precursor protein-like (APPL) signals directing this glial clearance. Neuronal RNAi of Drosophila fragile X mental retardation 1 (dfmr1) elevates mad transcript levels and increases pMad signaling. Neuronal dfmr1 and mad RNAi both elevate phospho-protein kinase B (pAkt) and delay neuron removal but cause opposite effects on InR expression. Genetically correcting pAkt levels in the mad RNAi background restores normal remodeling. Consistently, neuronal dfmr1 and mad RNAi both decrease Prtp levels, whereas neuronal InR and akt RNAi increase Prtp levels, indicating FMRP works with pMad and insulin signaling to tightly regulate Prtp signaling and thus control glial phagocytosis for correct circuit remodeling. Neuronal dfmr1 and mad and akt RNAi all decrease APPL levels, with the pathway signaling higher glial endolysosome activity for phagocytosis. These findings reveal a FMRP-dependent control pathway for neuron-to-glia communication in neuronal pruning, identifying potential molecular mechanisms for devising fragile X syndrome treatments (Song, 2023).

Neuron-to-glia communication has critical roles in controlling brain circuit remodeling. Neuronal signaling induces glial phagocytosis from synapses to whole neurons, crucial in normal brains and in neurodevelopmental disorder conditions. Disruption of neuron-to-glia communication causes aberrant neuronal pruning, resulting in defects ranging from defective synaptic transmission to impaired circuit wiring to transient neuroinflammation. A key case is fragile X syndrome (FXS), a leading intellectual disability and autism spectrum disorder, typically caused by the epigenetic loss of fragile X mental retardation protein (FMRP) owing to expanded CGG repeats in the 5' -untranslated region of fragile X mental retardation 1 (fmr1). In neurons, FMRP binds to specific transcripts to regulate protein translation during brain circuit development and later plasticity, including synaptic connectivity remodeling and intercellular signaling mechanisms In the Drosophila FXS disease model, FMRP loss blocks removal of the developmentally transient pigment-dispersing factor (PDF)-Tri peptidergic neurons from the juvenile brain. Cell-specific RNAi studies show that FMRP is required only in neurons, not glia, to transcellularly activate glial phagocytosis driving PDF-Tri neuron clearance. Thus, FMRP-dependent neuron-to-glia communication drives targeted neuron pruning, but the molecular mechanisms remain largely unknown (Song, 2023).

FMRP regulates bone morphogenic protein (BMP) and insulin-like peptide (ILP) signaling. Activated Drosophila BMP receptors phosphorylate Mothers against decapentaplegic (pMad) to control gene transcription, including the Insulin receptor (InR). InRs phosphorylate protein kinase B (pAkt) to suppress dendrite pruning. Importantly, mouse FMRP binds smad messenger ribonucleic acid (mRNA) (Drosophila mad homologue), and Drosophila FMRP regulates pMad signaling levels in neurons. Moreover, FMRP loss elevates InR-dependent signaling. In neuron-to-glia communication, pretaporter (Prtp) and amyloid precursor protein like (APPL) from neurons both activate glial phagocytosis. Drosophila Prtp traffics to the neuron surface to bind the glial phagocytotic receptor Draper (Drpr). Loss of neuronal FMRP decreases glial Drpr expression, consistent with FMRP-dependent Prtp signaling. Drosophila APPL has a cleavable N terminus, and APPL release from neurons activates glial phagocytosis. Glia take up secreted APPL to maintain Drpr expression and up-regulate Rab GTPases, activating the glial endolysosomal network for the neuron clearance mechanism. Taken together, these studies suggest that neuronal FMRP interacts with neuronal InR, pMad, and pAkt signaling cascades to tightly regulate Prtp and APPL neuron-to-glia communication controlling the glial phagocytosis of target neurons during circuit remodeling in the juvenile brain (Song, 2023).

This study used Drosophila brain PDF-Tri neuron removal via glial phagocytosis to study the neuron-to-glia communication remodeling mechanism, assaying both the early pruning steps at 1 day post-eclosion (dpe) and end-stage clearance (5 dpe). It was discovered that neuronal FMRP binds Mad mRNA to restrict pMad signaling in neurons. Surprisingly, however, it was found that both neuronal Drosophila Fragile X mental retardation 1 (Dfmr1) and Mad RNAi similarly block PDF-Tri neuron removal, indicating a more complex regulatory mechanism. Consistently, it was found that pMad is a positive transcription factor for InRs driving downstream pAkt signaling but that pMad also indirectly inhibits pAkt, inducing the phenocopy between neuronal dfmr1 and Mad RNAi conditions. Both neuronal dfmr1 and Mad RNAi similarly decrease Prtp neuron-to-glia signaling, resulting in reduced glial phagocytic activity and a block of PDF-Tri neuron clearance, whereas loss of neuronal InR and pAkt has that opposite phenotype of elevating Prtp to accelerate neuronal removal. It was also discovered that the FMRP-pMad-InR-pAkt pathway positively regulates neuronal APPL signaling to induce glial Rab7-driven endolysomal activation for PDF-Tri neuron clearance. Taken together, it is concluded that neuronal FMRP-pMad and InR-pAkt cascades coordinate an integrated regulatory decision network governing neuron-to-glia communication via neuronal Prtp and APPL signaling ligands that drive glial phagocytosis for targeted neuron pruning from brain circuits (Song, 2023).

This study has discovered an integrated mechanism of neuronal FMRP-dependent network signaling that regulates neuron-to-glia communication to drive the glial phagocytic removal of targeted neurons from an otherwise maintained brain circuit. Specifically, within the neurons, RNA-binding FMRP restricts the translation of bound Mad transcripts to limit phosphorylated Mad (pMad) signaling, which, in turn, inhibits phosphorylated Akt (pAkt) to promote glial phagocytosis for neuron removal. In parallel, the neuronal insulin receptor (InR) regulates pAkt signaling in a second intersecting cascade controlling the neuronal clearance mechanism. This bone morphogenic protein (BMP) and insulin-like peptide (ILP) neural decision-making network controls neuron-to-glia communication regulating glial phagocytosis function for targeted neuron removal. Neuronal pretaporter (Prtp) is a ligand for the Draper (Drpr) engulfment receptor on glia. The FMRP-pMad pathway promotes neuron-to-glia Prtp signaling to induce glial phagocytosis for neuron clearance, whereas the InR-pAkt pathway suppresses Prtp signaling to repress glia-mediated neuron removal. Neuronal amyloid precursor protein like (APPL) is released via a cleavable N terminus to activate Rab7 GTPase endolysomes in glia. The FMRP-pMad regulatory pathway promotes this neuron-to-glia APPL signaling, consistent with inducing glial phagocytosis for neuron clearance, and the intersecting InR-pAkt pathway also up-regulates this signaling, suggesting additional roles in neuron removal. Overall, these findings indicate neuronal FMRP coordinates signal transduction cascades to provide cross talk downstream of two signaling inputs (BMP and ILP), which provides output in the form of two neuron-to-glia signaling ligands (Prtp and APPL) that regulate glial phagocytosis for the clearance of targeted neurons from the juvenile brain (Song, 2023).

It is suggested that RNA-binding FMRP limits pMad signaling levels by reducing the number of Mad transcripts available for translation. FMRP is established to maintain protein translational homeostasis by modulating RNA target stability. Ribosome profiling and transcriptome sequencing demonstrate imbalanced FMRP-targeted mRNA levels are common in the mouse FXS model brain due to reduced stabilization. In mouse RNA immunoprecipitation (RIP) sequencing, FMRP binds Smad (Drosophila Mad homologue) transcripts widely in the 5' -UTR, within the coding region, and in the 3'-UTR. With both RIP and qPCR measurements, this study found Drosophila FMRP likewise binds Mad mRNA and that FMRP loss increases both mad transcripts and pMad protein levels within brain neurons. It is therefore suggest a causal effect correlation, although other indirect regulatory mechanisms are also possible. Loss of neuronal pMad decreases InR levels but elevates InR-dependent pAkt signaling, suggesting cross talk inhibition from pMad to pAkt. The pMad transcription factor positively regulates InR expression. Downstream of the InR, pAkt regulates numerous targets, including target of rapamycin complexes and the transcription factor forkhead box O, to regulate cell fate decisions. In the brain neuronal fate decision, it is suggested that pMad inhibits pAkt signaling as a major cross talk regulation within the neuron-to-glia signaling network. With western blot assays, this study found reducing neuronal pMad levels causes no changes in Akt protein levels but a approximately twofold increase in pAkt signaling. Thus, FMRP-dependent pMad signaling modulates InR-pAkt signaling at two levels: as a positive transcription factor regulating InR expression and as an inhibitor of downstream pAkt signaling. It is concluded that this cross talk provides critical regulation for targeted neuron pruning from the juvenile brain (Song, 2023).

Neuron removal from brain circuits is a normal mechanism in neurodevelopment, often mediated by glial phagocytosis. During the clearance process, glia engulf neurons and degrade internalized debris in endolysosomes. In glia, the engulfed neuronal debris is first sorted in Rab5 GTPase early endosomes, then trafficked from early-to-late endosomes with accompanying increased intravacuolar acidification, and finally delivered to Rab7 GTPase lysosomes where acid hydrolases complete the degradation process. In both rodent and Drosophila FXS disease models, this glial phagocytosis mechanism is severely impaired, resulting in a failure to prune neurons in brain circuits. In the Drosophila FXS model, loss of neuronal FMRP decreases the glial Drpr engulfment receptor, and loss of glial Drpr blocks glial phagocytosis to prevent neuron removal. This previous study generated the hypothesis that the impaired brain circuit neuron removal in the FXS disease model is caused by the loss of neuron-to-glia communication driving glial phagocytosis. With neuron-specific RNAi trials, this study found the neuronal FMRP-pMad pathway triggers Drpr ligand Prtp signaling from neurons to mediate glial engulfment and phagocytosis, whereas the neuronal InR-pAkt pathway represses Prtp to oppose the glial removal mechanism. Furthermore, targeted knockdown of neuronal FMRP, pMad, and Prtp impedes glial endolysosomal activation with reduced Rab7 expression, showing the neuronal FMRP-dependent network is required for glial phagocytosis function. These findings are consistent with the impaired glia-mediated neuron pruning in FXS disease models and provide insight into the molecular mechanisms of this defect. Clearly, the decision to eliminate neurons by glial phagocytosis rests on multiple, distinct signaling inputs and is executed via parallel avenues of neuron-to-glia communication (Song, 2023).

Loss of either neuronal Prtp or APPL was found to hampers glial phagocytosis neuron removal from the juvenile brain circuit but that loss of either signal alone causes a less severe impairment than blocking the neuronal FMRP-pMad pathway. These results suggest that Prtp and APPL act combinatorially downstream of FMRP-pMad, consistent with both neuronal Prtp and APPL driving the glial phagocytic clearance of targeted neurons. Prtp is a transmembrane protein trafficked from the endoplasmic reticulum (ER) to the cell surface of a neuron thus marked for removal, where it binds the Drpr engulfment receptor on glia. Through this mechanism, Prtp surface presentation downstream of the neuronal FMRP-pMad pathway is proposed to directly signal glial engulfment. In contrast, the cleaved extracellular domain of transmembrane APPL from neurons activates the glial endolysosomal network. This provides a separable function for APPL, suggesting why two signaling ligands mediate neuron removal by the glial phagocytosis mechanism. The Prtp signaling changes closely match the neuron clearance phenotypes of all neuronal dfmr1, mad, InR, and akt RNAi experiments. Likewise, the APPL signaling changes are consistent with FMRP-pMad regulation but not the InR-pAkt pathway. It id therefore proposed that Prtp and APPL coregulate glial phagocytic activity with different strengths or independently participate in glial phagocytosis via other regulatory mechanisms. To test the possibility that Prtp interacts with APPL in this clearance mechanism, this study could test the putative cross talk interaction at molecular and/or genetic levels. Conversely, neuronal Prtp and APPL may be independent signals in neuron-to-glial communication linked predominantly or solely through upstream FMRP regulation. Future studies will dissect these combinatorial signals downstream of inputs targeting the clearance of neurons from the juvenile brain (Song, 2023).

The pigment-dispersing factor (PDF) brain circuit mediates circadian clock functions, with the developmentally transient central PDF-Tri neurons presumably driving eclosion timing. Multiple signals appear to coordinate the targeted clearance of PDF-Tri neurons from the juvenile brain, including insulin-like peptide (ILP) signaling . The insulin receptor (InR) is present in both neurons and glia, acting to coordinate intercellular communication and neuronal remodeling. For glial signaling, InRs are proposed to be activated by secreted ILPs from the remodeling neurons to stimulate transduction cascades promoting glial phagocytosis. Glial InR phosphorylation triggers pAkt production, which, in turn, elevates Drpr engulfment receptor levels to drive glial phagocytosis. Consistently, genetically increasing glial InR levels elevates glia-dependent neuron removal of the PDF-Tri neurons. In the Drosophila FXS disease model, loss of FMRP represses InR phosphorylation in glia, whereas glial InR activation restores PDF-Tri neuron pruning. For neuron signaling, it. is proposed that InR-mediated pAkt signal transduction regulates neuron-to-glia communication in this same remodeling process. Similarly, InR signaling in Drosophila dendrite arborization neurons inhibits their developmental pruning. This work shows neuronal InR loss elevates Prtp signaling to drive glial phagocytosis, thus accelerating PDF-Tri neuron clearance from the juvenile brain. Neuronal InRs may be responding to autocrine ILP signaling from the remodeling neurons or ILPs from another source which is coordinating neuron and glia functions to properly regulate the glial phagocytosis neuron removal process. Future work will investigate how the different intercellular signals act on both neurons and glia to ensure the exact targeting and timing of the coordinated PDF-Tri neuron clearance from the juvenile brain (Song, 2023).

Cortex glia clear dead young neurons via Drpr/dCed-6/Shark and Crk/Mbc/dCed-12 signaling pathways in the developing Drosophila optic lobe

The molecular and cellular mechanism for clearance of dead neurons was explored in the developing Drosophila optic lobe. During development of the optic lobe, many neural cells die through apoptosis, and corpses are immediately removed in the early pupal stage. Most of the cells that die in the optic lobe are young neurons that have not extended neurites. This study shows that clearance was carried out by cortex glia via a phagocytosis receptor, Draper (Drpr). drpr expression in cortex glia from the second instar larval to early pupal stages was required and sufficient for clearance. Drpr that was expressed in other subtypes of glia did not mediate clearance. Shark and Ced-6 mediated clearance of Drpr. The Crk/Mbc/dCed-12 pathway was partially involved in clearance, but the role was minor. Suppression of the function of Pretaporter, CaBP1 and phosphatidylserine delayed clearance, suggesting a possibility for these molecules to function as Drpr ligands in the developing optic lobe (Nakano, 2019).

Many studies have explored the cellular and molecular mechanisms for clearance of dead neurons in the developing Drosophila CNS. During embryonic development, dead neurons are phagocytosed by subperineurial glia. Draper (Drpr) acts as a phagocytosis receptor on the glial membrane to clear dead neurons in the embryo. Another receptor, Six-microns-under (SIMU), works in cortex glia to allow recognition and engulfment of apoptotic cells, whereas Drpr works to degrade apoptotic cells in the embryonic CNS. During metamorphosis, dead neurons are engulfed by glia in the CNS. Elimination of neurites of vCrz neurons during metamorphosis is performed by astrocyte-like glia via the Crk/Mbc/dCed-12 signaling pathway but not the Drpr pathway. In contrast, elimination of cell bodies of vCrz neurons, a group of neurons that express neuropeptide Corazonin, requires Drpr, but its expression is not required in astrocyte-like glia. However, recent studies have reported inconsistent results on the requirement of Drpr for dead cell clearance and the glia subtypes that work for clearance in the brain during metamorphosis. It has been reported that dead neurons that died in the central brain before the beginning of the third larval instar and in the optic lobe before the late third larval instar are cleared by cortex glia via the Drpr pathway, but neurons that die thereafter are efficiently cleared without Drpr. Drpr has been shown to be required for apoptotic cell clearance during metamorphosis and its expression is required in ensheathing glia and astrocyte-like glia, but not in cortex glia (Nakano, 2019).

One of the causes of inconsistency among previous studies may be differences in the cellular materials to be phagocytosed, and different mechanisms could work for phagocytosis of different materials in the CNS during metamorphosis. Three types of neurons need to be phagocytosed during metamorphosis. Obsolete larval neurons die, and their cell bodies and neurites are removed by phagocytosis. Larval neurons of another type are respecified from larval to adult neurons via pruning of larval neurites and extension of new adult neurites. Pruned neurites are removed by phagocytosis. Adult-specific neurons are produced by precursor cells during post-embryonic development and differentiate during metamorphosis. A number of these young neurons die during development before extending neurites. Therefore, studies on a single type of neuron or specifically defined neurons are needed to define the molecular and cellular mechanisms for clearance of dead neurons. Moreover, clearance of neurites and cell bodies of dead neurons should be studied independently (Nakano, 2019).

In this study, clearance of dead neurons in the developing optic lobe was examined. The Drosophila optic lobe is a unique center in which a large number of dying cells are observed during its development. Most dying neurons in the optic lobe are young neurons that had just started to differentiate into adult neurons. One of paired neurons derived from intermediate precursors (GMCs) is eliminated by apoptosis under the control of Notch signaling. Neurons that die in the developing optic lobe have not yet extended neurites at the time they die. Therefore, cellular materials to be cleared after the cell death include nuclei and general cytoplasm, but not neurites in the developing optic lobe (Nakano, 2019).

The adult optic lobe develops from the primordium during metamorphosis. Optic lobe neurons are produced by two proliferation centers, the outer proliferation center (OPC) and inner proliferation center (IPC). Neurons differentiate, extend neurites, and produce four types of neuropil, the lamina, medulla, lobula plate, and lobula. Then, the optic lobe consists of four types of neuropil and surrounding cortices of neuronal cell bodies. According to previous studies, many neurons and a small number of precursor cells undergo cell death during optic lobe development. This cell death does not occur randomly in the optic lobe but occurs in clusters in a specific temporal and spatial pattern. The number of dead cells in the optic lobe starts to increase at the puparium formation, reaches a peak at 24 h after puparium formation (24 h APF), and decreases to almost zero by 48 h APF. Two types of cell death are involved in this process: ecdysone dependent and independent. Both types of cell death are apoptosis and involve the Drosophila effector caspases, DrIce and Dcp-1. DrIce plays an important role in dead cell clearance as well. The role of cell death is to prevent the emergence of abnormal neural structures in the optic lobe (Nakano, 2019).

This study explored the cellular and molecular mechanisms for clearance of dead young neurons in the developing optic lobe. The results showed that clearance was carried out by cortex glia via a phagocytosis receptor, Drpr. Drpr expression in cortex glia from the second instar larval to early pupal stages was required and sufficient for clearance. Signaling molecules, Shark and Ced-6 mediated clearance downstream of Drpr. The Crk/Mbc/dCed-12 pathway was partially involved in clearance, but the role was minor. Suppression of the function of Pretaporter, CaBP1 and phosphatidylserine delayed clearance, suggesting a possibility for these molecules to function as Drpr ligands in the developing optic lobe (Nakano, 2019).

This study revealed that Drpr expressed in cortex glia were required for dead cell clearance in the MLpL region of the developing optic lobe, and that Drpr in other subtypes of glia did not mediate clearance. This is the first study that showed clearance of dead young neurons in the developing optic lobe required Drpr expression in the cortex glia. In the lamina region, lamina distal cortex glia work for dead cell clearance (Nakano, 2019).

The expression pattern of Drpr agreed with the alteration in the activity of dead cell clearance in the optic lobe during metamorphosis. At early pupal stages, Drpr is expressed weakly in a mesh-like pattern and strongly in a centripetal pattern in cortex glia in the MLpL region. This expression weakened thereafter, and only weak expression was seen in a mesh-like pattern during the last half of the pupal period. Moreover, no protrusion was seen of Drpr expressing glial cytoplasm into the neuropil from neuropil glia (NG) at early pupal stages. This agrees with the fact that cell death in the developing optic lobe occurs mainly in young neurons before they extend neurites or in abnormal neurons with no neurites (Nakano, 2019).

After 48 h APF, cell death was rarely observed and thus activity of dead cell clearance was low. However, this does not mean that glia lost potential ability to clear corpses at late pupal stages. Forced expression of wild-type drpr on the drpr mutant background at 48 or 72 h APF resulted in clearance of accumulated TUNEL-positive cells. This indicates that the components of the mechanism for dead cell clearance except Drpr are retained until late pupal stages. Therefore, if some cells died at late pupal stages and Drpr expression was induced in cortex glia, the dead cells would be cleared via Drpr pathway. Moreover, the fact that accumulated TUNEL-positive cells were removed when wild-type drpr was forcibly expressed in late pupal stages on the drpr mutant background suggests that 'eat me' signals were secreted or displayed by accumulated TUNEL-positive cells in drpr mutants not only at early pupal stages, when the cells died, but also at late pupal stages long after cell death (Nakano, 2019).

At late pupal stages, strong Drpr expression appeared in the cytoplasmic protrusions from the neuropil glia (NG), and astrocyte-like glia simultaneously started expressing molecular markers (specific GAL4s). This Drpr was not utilized for clearance of dead neurons, as almost no cell death arises at this stage in control conditions. In the neuropil of the optic lobe at late pupal stages, neurites extend and form synapses to make and complete neural networks. When new synapses are formed during development of the Drosophila larval neuromuscular junction, significant amounts of presynaptic membranes and a subset of immature synapses are removed from the junction by surrounding glia and postsynaptic muscle via the Drpr/dCed-6 pathway (Fuentes-Medel, 2009). Thus, the same process may arise at developing synapses in the developing optic lobe, and astrocyte-like glia expressing Drpr in the cytoplasmic protrusions may function to remove unnecessary presynaptic membranes and immature synapses (Nakano, 2019).

Previous studies have reported that astrocyte-like glia are responsible for clearance of degenerating axons of dying obsolete larval neurons in the ventral nerve cord and of pruned axons of γ neurons in the mushroom body. In contrast, degenerating axons are removed by ensheathing glia in the olfactory lobe following Wallerian degeneration of the olfactory nerve. Therefore, different subtypes of glia work to clear degenerating axons in different contexts. Astrocyte-like glia may specifically function for clearance of 'programmed' degenerating axons and ensheathing glia for clearance of 'accidently' degenerating axons. In addition, another subtype of glia, cortex glia, functions to remove dead young neurons. These young neurons had just started to differentiate into adult neurons in the developing optic lobe and have not yet extended neurites at the time they die. It has been reported that elimination of cell bodies of obsolete vCrz neurons requires Drpr, but its expression is not required in astrocyte-like glia. Young neurons in the optic lobe and cell bodies of obsolete vCrz neurons in the ventral nerve cord both locate in the cortex and almost the same cellular materials are cleared after the cell death, including nucleus and general cytoplasm, but not neurites. Therefore, as with dead young neurons in the optic lobe, the expression of Drpr in cortex glia would be required for clearance of cell bodies of dead vCrz neurons. Comparative studies are expected in the future on the mechanisms for clearance of degenerating axons of dead neurons, degenerating axons of cut nerves, dead young neurons, and cell bodies of dead obsolete neurons. Moreover, considering that 'accidently' degenerating axons are cleared by a different subtype of glia from 'programmed' degenerating axons, a possibility should be tested that cell bodies of neurons that died 'accidently' are cleared by a distinct subtype of glia (Nakano, 2019).

This is the first study to reveal that Shark mediates Drpr-dependent clearance of dead neurons in the CNS. Moreover, this study suggests that Ced-6, Crk/Mbc/dCed-12, and Rac1 are partially involved in clearance of dead young neurons. Therefore, both Drpr/Shark/dCed-6 and Crk/Mbc/dCed-12 pathways work for dead cell clearance in the developing optic lobe. As cortex glia function for clearance of dead neurons in the developing optic lobe, these pathways must work in cortex glia (Nakano, 2019).

A previous study reported that these pathways function to mediate removal of degenerating axons in ensheathing glia in the adult olfactory lobe when the olfactory nerve is cut. The same pathways work in astrocyte-like glia around the mushroom body when axons of γ neurons are pruned, and in the ventral nerve cord when neurites of dead vCrz neurons are cleared during metamorphosis. Therefore, Drpr/Shark/dCed-6 and Crk/Mbc/dCed-12 pathways generally function to clear corpses in different glia in different contexts. However, the relative role played by each pathway depends on the situation. Although drpr mutation strikingly inhibited dead cell clearance in the optic lobe, knockdown of the Crk/Mbc/dCed-12 pathway had only a moderate effect. In contrast, removal of olfactory nerve axons that have undergone Wallerian degeneration is strongly affected by both drpr mutation and Crk/Mbc/dCed-12 knockdown. In the pruning of mushroom body γ axons, mutation of drpr and knockdown of Crk/Mbc/dCed-12 additively affect clearance, although mutation of drpr has a stronger effect. In the removal of axons from dead vCrz neurons during metamorphosis, knockdown of dCed-12 causes a moderate defect, whereas drpr mutation causes no defect by itself but only enhances the defect caused by dCed-12 knockdown. How the relative role of these pathways is regulated and why remain to be defined (Nakano, 2019).

Previous studies reported that Pretaporter, CaBP1 and phosphatidylserine act as Drpr ligands when dead embryonic cells are phagocytosed in the Drosophila embryo and cultured cells. The present study suggested a possibility that these molecules mediate signaling for dead cell clearance as a Drpr ligand in the developing optic lobe. However, Pretaporter and CaBP1 are not essential for clearance and their role would be minor. Therefore, relative role of molecules that work for dead cell clearance as ligands for Drpr may be different depending on the context. As described above, different subtypes of glia work to clear corpse in the CNS: ensheathing glia for Wallerian's degenerating axons, astrocyto-like glia for pruned axons and degenerating axons of dead vCrz neurons, and cortex glia for dead young neurons in the optic lobe. Therefore, it is to be defined whether difference in ligand molecules is involved in activating different subtypes of glia (Nakano, 2019).

The present study agrees with previous results which showed that Drpr is required for elimination of cell bodies of vCrz neurons that die at 3-7 h APF. However, the current study disagrees with other studies (Nakano, 2019 and references therein).

Several possible causes may have led to these inconsistencies. One possibility is the difference in cellular materials to be cleared, that is, cell bodies or neurites, as mentioned earlier. The present study examined dead young neurons in the developing optic lobes. Cellular materials to be cleared include only nucleus and general cytoplasm, but not neurites. A previous study examined vCrz neurons and found that molecular mechanisms for clearance of cell bodies and neurites are different. However, other studies examined the central brain or the whole brain, which include cell bodies of dead neurons, neurites of dead neurons and pruned neurites. Another possibility is the difference in methods to detect dead neurons. This study used the ABC TUNEL method, which detects degraded DNA in dead cell nuclei with the streptavidin-biotin-peroxidase complex. This method is far more sensitive and reliable than the fluorescent TUNEL method (compare the number of TUNEL-positive cells between the present study and other studies). Another method to detect dead cells is anti-Dcp-1 antibody staining. This method has some problems with detection of accumulated dead cell corpses in phagocytosis-defective mutants. It detects activated Dcp-1, one of the effector caspases. However, another effector caspase, DrIce, is also expressed and is a more effective inducer of apoptosis than Dcp-1. Therefore, this method may not detect all dead cells. Another problem with this method is the unknown stability of activated Dcp-1 in dead cells. Therefore, detection of activated Dcp-1 does not show exactly how many dead cells have accumulated in phagocytosis-defective mutants. Moreover, when dendrites are pruned during remodeling of dendritic arborization sensory neurons during metamorphosis, caspase activity is detected in the dendrite. This suggests that the anti-Dcp-1 antibody may detect pruned dendrites as well as dead cells. Finally, the subtypes of glia that clear corpses are also different in the present study and previous ones. This study found that expression of GAL4 in glia subtype-specific GAL4 lines drastically changed during metamorphosis, and the expression pattern at pupal stages was different from adult stages in many GAL4 lines. However, previous studies did not examine the expression pattern of the GAL4 lines they used. Altogether, studies on a single type of dead neuron or identified neurons are required in the future. The mechanisms for clearance of dead cell bodies and degenerating neurites should be studied independently. The expression pattern of GAL4 lines in subtypes of glia should be carefully assessed before using the line as a GAL4 driver (Nakano, 2019).

Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells

Phagocytic removal of cells undergoing apoptosis is necessary for animal development and tissue homeostasis. Draper, a homologue of the C. elegans phagocytosis receptor CED-1, is responsible for the phagocytosis of apoptotic cells in Drosophila, but its ligand presumably present on apoptotic cells remains unknown. An endoplasmic reticulum protein that binds to the extracellular region of Draper was isolated. Loss of this protein, which has been named Pretaporter, led to a reduced level of apoptotic cell clearance in embryos, and the overexpression of Pretaporter in the mutant flies rescued this defect. Results from genetic analyses suggested that Pretaporter functionally interacts with Draper and the corresponding signal mediators. Pretaporter was exposed at the cell surface after the induction of apoptosis, and cells artificially expressing Pretaporter at their surface became susceptible to Draper-mediated phagocytosis. Finally, the incubation with Pretaporter augmented the tyrosine-phosphorylation of Draper in phagocytic cells. These results collectively suggest that Pretaporter relocates from the endoplasmic reticulum to the cell surface during apoptosis to serve as a ligand for Draper in the phagocytosis of apoptotic cells (Kuraishi, 2009).

Throughout the life of multi-cellular organisms, cells of particular types die and are eventually eliminated in certain places in the body and at certain developmental stages under physiological and sub-physiological conditions. Such 'unwanted cells' are mostly induced to undergo apoptosis and subjected to phagocytic elimination. Prompt and selective phagocytosis of apoptotic cells is prerequisite for morphogenesis, establishment of tissue functions, tissue renewal, avoidance of diseases, and effective progress of tissue functions (Kuraishi, 2009 and references therein).

There are two genetically identified signalling pathways that lead to the induction of phagocytosis of dying cells in C. elegans: one is made up of the proteins CED-2, CED-5, and CED-12, and the other consists of CED-1, CED-6, and CED-7. The pathways converge at CED-10, a small G-protein responsible for the rearrangement of cytoskeleton in phagocytes. The fact that all these C. elegans proteins possess counterparts in Drosophila and mammals suggests that these two partially redundant signalling pathways are evolutionally conserved. However, the mode of action of those proteins still remains to be clarified, and there are missing components in the pathways. The selectivity in the recognition of apoptotic cells by phagocytes is due to the specific interaction between receptors of phagocytes and their ligands present at the surface of target cells. Presumably, there are two phagocytosis receptors in C. elegans, and CED-1 is likely the one located upstream of CED-6 and CED-7 (Zhou, 2001; Yu, 2006; Venegas, 2007) whereas the other has not been found in genetic studies. Draper (Drpr), a Drosophila homologue of CED-1, has been shown to act as a receptor in the phagocytosis of apoptotic cells and more recently molecules that act with Drpr to accomplish the engulfment and subsequent processing of apoptotic cells in Drosophila phagocytes were reported in a previous study. However, a molecule(s) present at the surface of apoptotic cells and recognized by Drpr is yet to be identified. Another study has implied that the membrane phospholipid phosphatidylserine, the best-characterized phagocytosis marker in mammals, could be a ligand for CED-1. In contrast, the current study showed that phosphatidylserine is not required in the Drpr-mediated phagocytosis of apoptotic cells. In this study, a phagocytosis marker(s) recognized by Drpr was sought, and an endoplasmic reticulum (ER) protein was identified as a strong candidate (Kuraishi, 2009).

This study reports the identification of a Drosophila protein, Prtp, serving as a ligand for Drpr in the phagocytosis of apoptotic cells. The level of phagocytosis in embryos of the null mutants for prtp and drpr was almost the same and about half of that in controls. This suggests that Prtp is solely responsible for the Drpr-mediated phagocytosis by embryonic haemocytes, and that there are additional ligands and receptors for phagocytosis. The following pathway is proposed for the induction of the Prtp-dependent, Drpr-mediated phagocytosis of apoptotic cells: Prtp relocates from the ER to the cell surface during apoptosis; Prtp binds to Drpr of haemocytes and induces tyrosine phosphorylation; Ced-6 binds to phosphorylated Drpr and further transmits the signal leading to the activation of Rac1 and/or Rac2; and cytoskeletons are reorganized for engulfment. Additional molecules could participate in this pathway: a membrane protein named Six-microns-under reportedly acts upstream of Drpr in the phagocytosis of apoptotic neurons by glia; another signal mediator, named Shark, located proximal downstream of Drpr has been reported; and Undertaker, an intracellular membrane protein involved in the regulation of calcium homeostasis, was shown to function downstream of Drpr. In particular, Six-microns-under has been suggested to bridge glia and apoptotic neurons. The results suggested that Prtp still serves as a ligand for Drpr in the phagocytosis by glia that express Six-microns-under. Further investigation is necessary before a complete picture is obtained of the Prtp/Drpr-initiated signalling pathway for the phagocytosis of apoptotic cells. More molecules are involved in the phagocytosis of apoptotic cells in Drosophila: the receptor Croquemort, the phagocytosis marker Calreticulin, and Pallbearer, a component of ubiquitin ligase, which might be integrated into the other engulfment pathway (Kuraishi, 2009).

Prtp normally resides in the ER, most likely in its lumen, and a portion of it seems to relocate to the cell surface during apoptosis. The ER and plasma membranes exchange their components, and this might be enhanced upon the induction of apoptosis. In fact, a variety of proteins and lipids of the ER are exposed at the surface of apoptotic human cells. More recently, it was reported that the exposure of ER proteins at the surface of apoptotic mammalian cells occurs by SNARE-dependent exocytosis. It is thus probable that together with other ER components Prtp moves to the cell surface during apoptosis and serves as a ligand for the phagocytosis receptor Drpr. Prtp seemed dispensable for the removal of degenerated neural axons during metamorphosis, which requires the action of Drpr. It is speculated that Prtp is not exposed on the surface of degenerated axons because the removal of these axons occurs independently of caspases, and that another ligand for Drpr exists at the surface of degenerated axons. In contrast, Drpr appears to serve as a receptor in the phagocytosis of bacteria. Taken together, it is likely that Drpr is a multi-ligand receptor for phagocytosis responsible for the maintenance of tissue homeostasis through the removal of degenerated own cells and invading microbial pathogens (Kuraishi, 2009).

A counterpart for Prtp in C. elegans could be a ligand for CED-1, but there seems no complete homologue of prtp in its genome. The current analysis has suggested that the presence of two thioredoxin-like domains is sufficient for the binding of Prtp to Drpr. Therefore, a C. elegans protein containing such a structure, PDI for example, is a candidate for the CED-1 ligand. In contrast, the mammalian homologue of Prtp seems to exist; a mouse protein called ERp46 and human proteins belonging to the TXNDC5 family possess a domain composition similar to that of Prtp. It is important to examine if C. elegans PDI and mammalian ERp46 and TXNDC5 act as ligands for CED-1 and its mammalian homologue MEGF10, respectively, in the phagocytosis of apoptotic cells (Kuraishi, 2009).

Role of NPxY motif in Draper-mediated apoptotic cell clearance in Drosophila

Draper, a receptor responsible for the phagocytosis of apoptotic cells in Drosophila, possesses atypical epidermal growth factor (EGF)-like sequences in the extracellular region and the two phosphorylatable motifs NPxY and YxxL in the intracellular portion. It has bee suggested that Pretaporter, a ligand for Draper, binds to the EGF-like repeat and augments the tyrosine phosphorylation of Draper. This study first tested the binding of Pretaporter to various parts of the extracellular region of Draper and found that a single EGF-like sequence is sufficient for the binding. It was theb determined roles of the two intracellular motifs by forcedly expressing Draper proteins, in which tyrosine residues within the motifs had been substituted with phenylalanine, in hemocytes of Draper-lacking flies. Draper proteins with Y-to-F substitution in either motif still underwent tyrosine phosphorylation, suggesting the occurrence of phosphorylation at both motifs. The Draper protein with substitution in the YxxL motif rescued a defect of phagocytosis, as did intact Draper, but the Draper protein with substitution in the NPxY motif did not, indicating a role of the motif NPxY, but not YxxL, in Draper-mediated phagocytosis. This coincides with previous finding that Ced-6, an NPxY-binding signaling adaptor, is required for Draper's actions in apoptotic cell clearance. In summary, this study demonstrated that Draper binds to its ligand Pretaporter using EGF-like sequences, and that the NPxY motif in the intracellular region of Draper plays an essential role in its actions as an engulfment receptor (Fujita, 2012). >

Apoptosis-dependent externalization and involvement in apoptotic cell clearance of DmCaBP1, an endoplasmic reticulum protein of Drosophila

To elucidate the actions of Draper, a receptor responsible for the phagocytic clearance of apoptotic cells in Drosophila, proteins that bind to the extracellular region of Draper were isolated using affinity chromatography. One of those proteins has been identified as an uncharacterized protein called Drosophila melanogaster Calcium-binding protein 1 (DmCaBP1). This protein containing the thioredoxin-like domain resided in the endoplasmic reticulum and seemed to be expressed ubiquitously throughout the development of Drosophila. DmCaBP1 was externalized without truncation after the induction of apoptosis somewhat prior to chromatin condensation and DNA cleavage in a manner dependent on the activity of caspases. A recombinant DmCaBP1 protein bound to both apoptotic cells and a hemocyte-derived cell line expressing Draper. Forced expression of DmCaBP1 at the cell surface made non-apoptotic cells susceptible to phagocytosis. Flies deficient in DmCaBP1 expression developed normally and showed Draper-mediated pruning of larval axons, but a defect in the phagocytosis of apoptotic cells in embryos was observed. Loss of Pretaporter, a previously identified ligand for Draper, did not cause a further decrease in the level of phagocytosis in DmCaBP1-lacking embryos. These results collectively suggest that the endoplasmic reticulum protein DmCaBP1 is externalized upon the induction of apoptosis and serves as a tethering molecule to connect apoptotic cells and phagocytes for effective phagocytosis to occur (Nakayama, 2012).

Search PubMed for articles about Drosophila pretaporter

Aggarwal, P., Liu, Z., Cheng, G. Q., Singh, S. R., Shi, C., Chen, Y., Sun, L. V. and Hou, S. X. (2022). Disruption of the lipolysis pathway results in stem cell death through a sterile immunity-like pathway in adult Drosophila. Cell Rep 39(12): 110958. PubMed ID: 35732115

Fujita, Y., Nagaosa, K., Shiratsuchi, A., Nakanishi, Y. (2012). Role of NPxY motif in Draper-mediated apoptotic cell clearance in Drosophila. Drug Discov Ther, 6(6):291-297 PubMed ID: 23337816

Kuraishi, T., et al. (2009). Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells. EMBO J. 28(24): 3868-78. PubMed Citation: 19927123

Nakano, R., Iwamura, M., Obikawa, A., Togane, Y., Hara, Y., Fukuhara, T., Tomaru, M., Takano-Shimizu, T. and Tsujimura, H. (2019). Cortex glia clear dead young neurons via Drpr/dCed-6/Shark and Crk/Mbc/dCed-12 signaling pathways in the developing Drosophila optic lobe. Dev Biol 453(1):68-85. PubMed ID: 31063730

Nakayama, H., Yamamoto, N., Nakagawa, Y., Dohmae, N., Shiratsuchi, A., Nakanishi, Y. (2012). Apoptosis-dependent externalization and involvement in apoptotic cell clearance of DmCaBP1, an endoplasmic reticulum protein of Drosophila. J Biol Chem, 287(5):3138-3146 PubMed ID: 22158613

Song, C. and Broadie, K. (2023). Fragile X mental retardation protein coordinates neuron-to-glia communication for clearance of developmentally transient brain neurons. Proc Natl Acad Sci U S A 120(12): e2216887120. PubMed ID: 36920921
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date revised: 20 March 2024

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