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The Drosophila Cell Corpse Engulfment Receptor Draper Mediates Glial Clearance of Severed Axons
Abstract
Neuron-glia communication is central to all nervous system responses to trauma, yet neural injury signaling pathways remain poorly understood. Here we explore cellular and molecular aspects of neural injury signaling in Drosophila. We show that transected Drosophila axons undergo injury-induced degeneration that is morphologically similar to Wallerian degeneration in mammals and can be suppressed by the neuroprotective mouse Wlds protein. Axonal injury elicits potent morphological and molecular responses from Drosophila glia: glia upregulate expression of the engulfment receptor Draper, undergo dramatic changes in morphology, and rapidly recruit cellular processes toward severed axons. In draper mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the CNS. Thus Draper appears to act as a glial receptor for severed axon-derived molecular cues that drive recruitment of glial processes to injured axons for engulfment.
... The circuit-localized glial projection infiltration and coincident dose-dependent loss of OSN innervation based on critical period experience led to the hypothesis that glia are mediating synaptic glomeruli pruning. Drosophila glial phagocytosis is well characterized following neuronal injury [43][44][45] , and in early developmental neuronal remodeling 16,[46][47][48] , but it is unknown whether glia act in a critical period pruning mechanism. Drosophila Draper (mammalian MEGF10/Jedi) is an engulfment receptor for glial phagocytosis 43,47,49 . ...
... Drosophila glial phagocytosis is well characterized following neuronal injury [43][44][45] , and in early developmental neuronal remodeling 16,[46][47][48] , but it is unknown whether glia act in a critical period pruning mechanism. Drosophila Draper (mammalian MEGF10/Jedi) is an engulfment receptor for glial phagocytosis 43,47,49 . To test whether glial phagocytosis is required for critical period pruning, we use both a draper null mutant (draper Δ5 ) and glial-targeted repo-Gal4 draper RNAi 43 compared to matched genetic background and transgenic driver controls respectively. ...
... We discover critical period sensory experience drives very striking Basket translocation into remodeling glial nuclei (Fig. 5). The glial nuclei remain outside of the synaptic glomeruli 44,76 , and extend infiltrating membrane projections 43,77 into the neuropil to mediate experience-dependent pruning. We discover circuit-localized signaling around EB-responsive VM7 glomeruli (Fig. 5). ...
Critical periods are temporally-restricted, early-life windows when sensory experience remodels synaptic connectivity to optimize environmental input. In the Drosophila juvenile brain, critical period experience drives synapse elimination, which is transiently reversible. Within olfactory sensory neuron (OSN) classes synapsing onto single projection neurons extending to brain learning/memory centers, we find glia mediate experience-dependent pruning of OSN synaptic glomeruli downstream of critical period odorant exposure. We find glial projections infiltrate brain neuropil in response to critical period experience, and use Draper (MEGF10) engulfment receptors to prune synaptic glomeruli. Downstream, we find antagonistic Basket (JNK) and Puckered (DUSP) signaling is required for the experience-dependent translocation of activated Basket into glial nuclei. Dependent on this signaling, we find critical period experience drives expression of the F-actin linking signaling scaffold Cheerio (FLNA), which is absolutely essential for the synaptic glomeruli pruning. We find Cheerio mediates experience-dependent regulation of the glial F-actin cytoskeleton for critical period remodeling. These results define a sequential pathway for experience-dependent brain synaptic glomeruli pruning in a strictly-defined critical period; input experience drives neuropil infiltration of glial projections, Draper/MEGF10 receptors activate a Basket/JNK signaling cascade for transcriptional activation, and Cheerio/FLNA induction regulates the glial actin cytoskeleton to mediate targeted synapse phagocytosis.
... Drosophila ORNs have been widely used for modelling axonopathy. Ablation of ORN axons can be caused by the non-lethal severing of the third antennal segment or maxillary palps; alternatively, different ORN-specific Gal4 lines (table 3) are used to misexpress or knockdown certain axonal degeneration related proteins in the ORNs (MacDonald et al. 2006). Toxin-based conditional ablation of different classes of olfactory receptor neurons has also been performed (Berdnik et al. 2006). ...
... Expression of Wallerian degeneration slow (Wld s ) in different groups of ORNs shows axo-protective effects (Hoopfer et al. 2006;MacDonald et al. 2006). Expression of Drosophila nicotinamide mononucleotide adenyltransferase (dNmnat) (MacDonald et al. 2006) and deubiquitinating yeast enzyme ubiquitinbinding protein 2 (UBP2) (Hoopfer et al. 2006) in injured ORNs protect against axonal loss. ...
... Expression of Wallerian degeneration slow (Wld s ) in different groups of ORNs shows axo-protective effects (Hoopfer et al. 2006;MacDonald et al. 2006). Expression of Drosophila nicotinamide mononucleotide adenyltransferase (dNmnat) (MacDonald et al. 2006) and deubiquitinating yeast enzyme ubiquitinbinding protein 2 (UBP2) (Hoopfer et al. 2006) in injured ORNs protect against axonal loss. This model system was helpful in understanding overlaps and distinctions between injury-induced axonal loss and developmental pruning (Hoopfer et al. 2006). ...
The fruit fly, Drosophila melanogaster, has been one of the finest systems for decoding myriad puzzles across different domains of biology. Beyond addressing the fundamental problems, it has been used as a fantastic model organism for human disease research. Being an insect, Drosophila has a robust and advanced olfactory system that has been used many times as a model neuronal circuit to study fundamental questions in neurobiology. The circuit is well-explored at anatomical, physiological, and functional levels. It provides several advantages for the study of neurobiological disorders, such as spatiotemporally regulated misexpression or knockdown of disease proteins, genetic tractability, well-studied neuroanatomy, simple behavioural training paradigms, and quantifiable assays. Hence, Drosophila olfaction has been a favourite choice for the study of several neurodegenerative and neurodevelopmental disorders including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, fragile X syndrome, etc. This review aims to discuss earlier progress and future scope in using the Drosophila olfactory system for modelling human neurological pathophysiology for conducting fundamental and applied research. A major goal of research in biological science is to alleviate human disease burden. Diverse experimental systems are required to address different aspects of disease aetiology. Drosophila is one of the finest in vivo systems; its olfactory system is arguably the most well-characterized circuit for modelling human neurological disorders. A vast amount of research has been conducted to decipher cellular, molecular, and even cognitive aspects of human disorders using the Drosophila olfactory system. This review aims at summarizing such research progress to date and critically analysing the suitability of this system for modelling more complex neurological conditions.
... For this purpose, we utilized a well-established nerve injury model. 51 Olfactory receptor neurons in the antennal lobe of the fly brain have their cell bodies at the tips of the maxillary palps and extend long axons into the antennal lobe 52 ( Figure 4A). Severing the maxillary palps removes the cell bodies, causes axonal degeneration in the antennal lobe, and triggers host defense responses and tissue repair programs, such as the engulfment of cellular debris by surrounding glial cells. ...
... Severing the maxillary palps removes the cell bodies, causes axonal degeneration in the antennal lobe, and triggers host defense responses and tissue repair programs, such as the engulfment of cellular debris by surrounding glial cells. 51,53 Using this model, we compared mRNA levels of Toll-9 and other Toll-related genes in the fly brain before and 6 h after acute neurodegeneration. Toll-9 expression was specifically and significantly increased after severing the maxillary palps ( Figure 4B). ...
... This study also revealed that Toll-9 expression was induced in the model of acute nerve injury. 51,54 In Drosophila, degenerated axons were removed by glial cells through activation of draper, a mammalian ortholog of MEGF10/11. 51,54 More recently, Toll-6/dSarm signaling in glial cells was shown to promote drapermediated phagocytic clearance of apoptotic neurons during development. ...
Drosophila Toll-9 is most closely related to mammalian Toll-like receptors, however, physiological functions of Toll-9 remain elusive. We examined the roles of Toll-9 in fly brains in aging and neurodegeneration. Toll-9 mRNA levels were increased in aged fly heads accompanied by activation of nuclear factor-kappa B (NF-kB) and stress-activated protein kinase (SAPK) signaling, and many of these changes were modulated by Toll-9 in glial cells. The loss of Toll-9 did not affect lifespan or brain integrity, whereas it exacerbated hydrogen peroxide-induced lethality. Toll-9 expression was also induced by nerve injury but did not affect acute stress response or glial engulfment activity, suggesting Toll-9 may modulate subsequent neurodegeneration. In a fly tauopathy model, Toll-9 deficiency enhanced neurodegeneration and disease-related tau phosphorylation with reduced SAPK activity, and blocking SAPK enhanced tau phosphorylation and neurodegeneration. In sum, Toll-9 is induced upon aging and nerve injury and affects neurodegeneration by modulating stress kinase signaling.
... For the nervous system, injury is a strong trigger of innate plasticity mechanisms, many of which are highly conserved across animal life. The Drosophila nervous system has been probed for its responses to many different kinds of injuries (Fang and Bonini 2012;Rooney and Freeman 2014;Hao and Collins 2017;Brace and DiAntonio 2020), including models of headimpact brain injury in adult flies (Katzenberger et al. 2013;Saikumar et al. 2020), removal of body structures that contain neurons (antennae, legs, or part of the wings) (MacDonald et al. 2006;Fang et al. 2013;Neukomm et al. 2014;Purice et al. 2017), peripheral nerve crush (Xiong et al. 2010), and focused laser microsurgery to axons and/or dendrites in defined locations (Stone et al. 2010;Ghannad-Rezaie et al. 2012;Song et al. 2012;Soares et al. 2014;DeVault et al. 2018). Many of these studies have revealed cellular pathways triggered by injury shared in vertebrates and mammals. ...
... Coupled with this cell-autonomous and local mechanism for destruction is the conversion of damaged axons and dendrites into debris that can be effectively cleared by surrounding glial cells or epithelial cells, depending on the location (Doherty et al. 2009;Han et al. 2014). Drosophila have proven to be a powerful model for unraveling the cellular pathways that are engaged by cells that recognize and phagocytose neuronal debris (MacDonald et al. 2006;Sapar and Han 2019) in addition to Wallerian degeneration. ...
A fundamental feature of nervous systems is a highly specified synaptic connectivity between cells and the ability to adaptively change this connectivity through plasticity mechanisms. Plasticity mechanisms are highly relevant for responding to nervous system damage, and studies using nervous system injury paradigms in Drosophila (as well as other model organisms) have revealed conserved molecular pathways that are triggered by axon damage. Simple assays that introduce injuries to axons in either adult flies or larvae have proven to be particularly powerful for uncovering mechanisms of axonal degeneration and clearance. They have also been used to reveal requirements for regrowth of axons and dendrites, as well as signaling pathways that regulate cellular responses to nerve injury. Here we review commonly used and simple to carry out techniques that enable experimenters to study responses to axonal damage in either adult flies (following antennal transection) or larvae (following nerve crush to segmental nerves). Because axons and dendrites in the larval peripheral nervous system can be readily visualized through the translucent cuticle, another versatile method to probe injury responses is to focus high-energy laser light to a small and specific location in the animal. We therefore discuss a method for immobilizing intact larvae for imaging through the cuticle to carry out injury by pulse dye laser, which can be used to generate many different kinds of injuries and directed ablations within intact larvae. These techniques, combined with powerful genetic tools in Drosophila , make the fruit fly an excellent model system for studying the effects of injury and the mechanisms of axon degeneration, synapse plasticity, and immune response.
... We recommend injuring cohorts of flies that will be processed at multiple time points after the injury (e.g., 1, 3, 5, 7, and 10 d, as shown in Fig. 1D). Fragmentation and loss of axon tracts occur 1-3 d postinjury, while near-complete terminal loss in the glomeruli is apparent 5 d postinjury (MacDonald et al. 2006). ...
... Second, ORN synaptic terminals are readily visualized at defined glomeruli in the brain. Glial cells do not normally enter this region; however, their processes can be observed to enter this synaptically dense region as the injured terminals are cleared (MacDonald et al. 2006). Hence, this assay enables excellent visualization of the loss and clearance of synapses, the interactions of glial cells, and the role of glial pathways in the clearance mechanism (Sapar and Han 2019). ...
Neurons extend their axons and dendrites over long distances and rely on evolutionarily conserved mechanisms to maintain the cellular structure and function of neurites at a distance from their cell body. Neurites that lose connection with their cell body following damage or stressors to their cytoskeleton undergo a programmed self-destruction process akin to apoptosis but using different cellular machinery, termed Wallerian degeneration. While first described for vertebrate axons by Augustus Waller in 1850, key discoveries of the enzymes that regulate Wallerian degeneration were made through forward genetic screens in Drosophila melanogaster . Powerful techniques for genetic manipulation and visualization of single neurons combined with simple methods for introducing axotomy (neuron severing) to certain neuron types in Drosophila have enabled the discovery and study of the cellular machinery responsible for Wallerian degeneration, in addition to mechanisms that enable clearance of the resulting debris. This protocol describes how to study the degeneration and clearance of axons from olfactory receptor neurons (ORNs). These peripheral neurons reside in the antennae and project axons to olfactory glomeruli of the anterior brain. Simple and nonlethal removal of antennae from adult flies causes axotomy of ORNs, and the fate of the injured axons can be readily visualized in a whole-mount dissected brain. This assay takes advantage of well-characterized genetic methods to robustly and specifically label subsets of ORNs. This method of neurite labeling and axotomy was the first axon injury paradigm to be developed in flies and is still regularly used due to its simplicity to perform, dissect, image, and analyze.
... The classical and most studied phagocytic receptors have an immunoreceptor tyrosine-based activation motif (ITAM) region in their intracellular domain, which is phosphorylated by SFKs upon ligand-receptor interaction, allowing for the recruitment of SYK kinases and triggering of engulfment (Uribe-Querol and Rosales, 2020). In Drosophila, the ITAM receptor Draper (Drpr) is required for follicle cells to recognize the germline during engulfment of late-stage nurse cells or in nutrient-deprived conditions (MacDonald et al., 2006;Serizier et al., 2022;Timmons et al., 2017). To test whether Src-CA-expressing border cells require Drpr for polar cell internalization, we expressed Src-CA in a drpr null mutant background. ...
... Hyperactivated Src is sufficient to drive live cell engulfment SRC-family kinases are necessary for phagocytosis in cells as diverse as mammalian innate immune cells (Lowell, 2011;Wetzel et al., 2016), Drosophila glia (MacDonald et al., 2006), and their respective crop images are shown in A'-G'. (A''-G'') Schematic representations of the images shown in A'-G'. ...
Src family kinases (SFKs) are evolutionarily conserved proteins acting downstream of receptors and regulating cellular processes including proliferation, adhesion, and migration. Elevated SFK expression and activity correlate with progression of a variety of cancers. Here, using the Drosophila melanogaster border cells as a model, we report that localized activation of a Src kinase promotes an unusual behavior: engulfment of one cell by another. By modulating Src expression and activity in the border cell cluster, we found that increased Src kinase activity, either by mutation or loss of a negative regulator, is sufficient to drive one cell to engulf another living cell. We elucidate a molecular mechanism that requires integrins, the kinases SHARK and FAK, and Rho family GTPases, but not the engulfment receptor Draper. We propose that cell cannibalism is a result of aberrant phagocytosis, where cells with dysregulated Src activity fail to differentiate between living and dead or self versus non-self, thus driving this malignant behavior.
... Their cell bodies are housed in 3 rd antennal segments and project their axons into the antennal lobe of the central nervous system (CNS) (Vosshall et al., 2000). The bilateral ablation of 3 rd antennal segments results in the removal of the cell bodies and the degeneration of ipsi-and contralateral axonal projections (MacDonald et al., 2006). We observed the degeneration of wild-type axons and subsequent progressive clearance of the resulting debris within 14 days post axotomy (dpa; e.g., post antennal ablation) ( Figure 1A). ...
... In Drosophila, a previous study has demonstrated that the over-expression of dNmnat preserves axonal morphology for ~5 days after injury, by the use of an N-terminal Myc tagged dNmnat (Myc::dNmnat) (MacDonald et al., 2006). Here, we used an untagged dNmnat transgene (Zhai et al., 2006). ...
After injury, the axon separated from the soma activates an evolutionarily conserved pathway to initiate its degeneration within a day. Attenuation of this pathway results in morphologically preserved severed axons and synapses for weeks after injury. When evoked, these projections remain able to elicit a postsynaptic behavior, a phenomenon that we define as preserved circuit integrity. The mechanism enabling the preservation of circuit integrity is currently unknown. Here we demonstrate that dNmnat-mediated over-expression potently blocks programmed axon degeneration in Drosophila . Thus, severed axons and their synapses of distinct sensory neuron populations remain morphologically preserved and circuit-integrated for at least a week. We used ribosomal pulldown to isolate translatomes from these severed projections. Transcriptional profiling revealed several enriched biological classes. Identified candidates were assessed by the quantification of evoked antennal grooming behavior as proxy for preserved circuit integrity. We identified mediators of local protein synthesis, and specifically candidates involved in protein ubiquitination and calcium homeostasis required for the preservation of circuit integrity by RNAi-mediated knock-down. Our dataset uncovered several uncharacterized genes linked to human disease and may therefore offer insights into avenues for therapeutic treatments.
... 147 However, long before they were grouped within the Nimrod family, Draper and SIMU had been implicated in the engulfment of cellular debris in multiple Drosophila phagocytes. 42,60,78,148 Both of these receptors have been reported to bind phosphatidylserine, the classic "Eat-me" signal exposed on the outer membrane of apoptotic corpses. 149,150 However, these two receptors have very different intracellular domains. ...
... 78,98 However, this is not clear cut and Draper does appear to contribute to corpse uptake in other contexts and developmental stages. 148,151,152 Nevertheless, for the phagocytes of the embryo, it is SIMU that appears to be more generally required for apoptotic corpse uptake. 78 Given the absence of any intracellular domain in SIMU, there is presumably a need for as yet unidentified docking receptor(s) to promote corpse uptake. ...
The clearance of dead and dying cells, termed efferocytosis, is a rapid and efficient process and one that is critical for organismal health. The extraordinary speed and efficiency with which dead cells are detected and engulfed by immune cells within tissues presents a challenge to researchers who wish to unravel this fascinating process, since these fleeting moments of uptake are almost impossible to catch in vivo. In recent years, the fruit fly (Drosophila melanogaster) embryo has emerged as a powerful model to circumvent this problem. With its abundance of dying cells, specialist phagocytes and relative ease of live imaging, the humble fly embryo provides a unique opportunity to catch and study the moment of cell engulfment in real‐time within a living animal. In this review, we explore the recent advances that have come from studies in the fly, and how live imaging and genetics have revealed a previously unappreciated level of diversity in the efferocytic program. A variety of efferocytic strategies across the phagocytic cell population ensure efficient and rapid clearance of corpses wherever death is encountered within the varied and complex setting of a multicellular living organism.
... Glial cells are an attractive candidate because of their close association with neurons and essential roles in neural development, synaptic plasticity, and injury responses 22,23 . During development and metamorphosis when substantial axon and dendrite pruning occurs, and during injury induced neuronal degeneration, glial cells detect and engulf neuronal debris through a conserved signaling pathway mediated by an engulfment receptor, MEGF10 and Jedi (vertebrates) 24,25 /CED-1 (C-elegans) 26 /Draper (Drosophila) [27][28][29] . Draper contains an ITAM domain found in many mammalian immunoreceptors which can be phosphorylated by Src42a to allow binding of an SH2 domain kinase, Shark 30 . ...
... The Drosophila engulfment receptor, Draper, is implicated in axonal debris clearance in several Drosophila nerve injury models including axotomy of olfactory and wing sensory neurons 9,28,34 . However, whether Draper is required for the clearance of axonal debris generated by programmed cell death and the subsequent cross-neuron plasticity is not clear. ...
Neuronal cell death and subsequent brain dysfunction are hallmarks of aging and neurodegeneration, but how the nearby healthy neurons (bystanders) respond to the death of their neighbors is not fully understood. In the Drosophila larval neuromuscular system, bystander motor neurons can structurally and functionally compensate for the loss of their neighbors by increasing their terminal bouton number and activity. We term this compensation as cross-neuron plasticity, and in this study, we demonstrate that the Drosophila engulfment receptor, Draper, and the associated kinase, Shark, are required for cross-neuron plasticity. Overexpression of the Draper-I isoform boosts cross-neuron plasticity, implying that the strength of plasticity correlates with Draper signaling. In addition, we find that functional cross-neuron plasticity can be induced at different developmental stages. Our work uncovers a role for Draper signaling in cross-neuron plasticity and provides insights into how healthy bystander neurons respond to the loss of their neighboring neurons.
... The observed glial membrane expansion indicates that the glia may be hyperactive when S1P is elevated. 71 It has been previously shown that hyperactive glial phagocytosis is suffi-cient to induce neuronal loss of TH (tyrosine hydroxylase) expressing neurons and to reduce the life span of flies. 72 Since glial phagocytosis is mediated by Draper, a phagocytic receptor present on glia, 73,74 we assessed if the elevation of S1P in glia affects Draper expression in the adult CNS. ...
... In contrast, decreasing the levels of Draper result in less enlarged glial membrane morphology than the control (Repo>mCherry), and the number of axons is not affected (Figures S4A and S4B). Since Draper transcription is increased in glia when axons are injured, 71 the data indicate that expression of ELOVL1 or SK1 in glia leads to an elevation of S1P and an increased level of Draper that promotes glial phagocytosis. ...
VLCFAs (very-long-chain fatty acids) are the most abundant fatty acids in myelin. Hence, during demyelination or aging, glia are exposed to higher levels of VLCFA than normal. We report that glia convert these VLCFA into sphingosine-1-phosphate (S1P) via a glial-specific S1P pathway. Excess S1P causes neuroinflammation, NF-κB activation, and macrophage infiltration into the CNS. Suppressing the function of S1P in fly glia or neurons, or administration of Fingolimod, an S1P receptor antagonist, strongly attenuates the phenotypes caused by excess VLCFAs. In contrast, elevating the VLCFA levels in glia and immune cells exacerbates these phenotypes. Elevated VLCFA and S1P are also toxic in vertebrates based on a mouse model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Indeed, reducing VLCFA with bezafibrate ameliorates the phenotypes. Moreover, simultaneous use of bezafibrate and fingolimod synergizes to improve EAE, suggesting that lowering VLCFA and S1P is a treatment avenue for MS.
... MEGF10 is an ortholog of Drosophila Draper and the C. elegans protein CED-1, which help to mediate axon pruning by Drosophila glial cells and phagocytosis of apoptotic cells by worms, respectively.86,87 MEGF10 has also been shown to mediate phagocytosis through the involvement of other proteins, such as GULP1 and ABCA1, but little is known about the function of GULP1 in this pathway.88-90 ...
Background
Stroke is an acute cerebrovascular disease in which brain tissue is damaged due to sudden obstruction of blood flow to the brain or the rupture of blood vessels in the brain, which can prompt ischemic or hemorrhagic stroke. After stroke onset, ischemia, hypoxia, infiltration of blood components into the brain parenchyma, and lysed cell fragments, among other factors, invariably increase blood–brain barrier (BBB) permeability, the inflammatory response, and brain edema. These changes lead to neuronal cell death and synaptic dysfunction, the latter of which poses a significant challenge to stroke treatment.
Results
Synaptic dysfunction occurs in various ways after stroke and includes the following: damage to neuronal structures, accumulation of pathologic proteins in the cell body, decreased fluidity and release of synaptic vesicles, disruption of mitochondrial transport in synapses, activation of synaptic phagocytosis by microglia/macrophages and astrocytes, and a reduction in synapse formation.
Conclusions
This review summarizes the cellular and molecular mechanisms related to synapses and the protective effects of drugs or compounds and rehabilitation therapy on synapses in stroke according to recent research. Such an exploration will help to elucidate the relationship between stroke and synaptic damage and provide new insights into protecting synapses and restoring neurologic function.
... immune-responsive fat body tissue within the head, evidence of neuroinflammation was probed in the Gba1b -/fly brain by examining the highly conserved Draper-dependent glial immune pathway. Draper is a phagocytic recognition receptor on the surface of engulfing glia in flies and is required for the phagocytic removal of cellular debris following axonal injury [24,25]. Consistent with the widespread innate immune activation, we observed increased Draper gene expression in the heads of 3-week-old Gba1b -/flies (Fig 2A). ...
Mutations in the GBA1 gene cause the lysosomal storage disorder Gaucher disease (GD) and are the greatest known genetic risk factors for Parkinson’s disease (PD). Communication between the gut and brain and immune dysregulation are increasingly being implicated in neurodegenerative disorders such as PD. Here, we show that flies lacking the Gba1b gene, the main fly orthologue of GBA1 , display widespread NF -k B signalling activation, including gut inflammation, and brain glial activation. We also demonstrate intestinal autophagic defects, gut dysfunction, and microbiome dysbiosis. Remarkably, modulating the microbiome of Gba1b knockout flies, by raising them under germ-free conditions, partially ameliorates lifespan, locomotor and immune phenotypes. Moreover, we show that modulation of the immune deficiency (IMD) pathway is detrimental to the survival of Gba1 deficient flies. We also reveal that direct stimulation of autophagy by rapamycin treatment achieves similar benefits to germ-free conditions independent of gut bacterial load. Consistent with this, we show that pharmacologically blocking autophagosomal-lysosomal fusion, mimicking the autophagy defects of Gba1 depleted cells, is sufficient to stimulate intestinal immune activation. Overall, our data elucidate a mechanism whereby an altered microbiome, coupled with defects in autophagy, drive chronic activation of NF -k B signaling in a Gba1 loss-of-function model. It also highlights that elimination of the microbiota or stimulation of autophagy to remove immune mediators, rather than prolonged immunosuppression, may represent effective therapeutic avenues for GBA1- associated disorders.
... First, activation of most remodeling events occurs by glial release of the Transforming Growth Factor-β (TGFβ) family member Myoglianin, which activates expression of EcR via TGFβ receptors on target neurons, thereby establishing their competence to prune upon receipt of steroid hormonal signals (Awasaki, Huang et al. 2011, Hakim, Yaniv et al. 2014, Yu, Gutman et al. 2013). After cell death or neurite/synapse degeneration has occurred, glial cells act as phagocytes to recognize and phagocytose neuronal debris through conserved signaling pathways like Draper/MEGF10 (MacDonald, Beach et al. 2006, Doherty, Logan et al. 2009, Hakim, Yaniv et al. 2014 or Fractalkine/Orion (Boulanger, Thinat et al. 2021, Ji, Wang et al. 2023, Perron, Carme et al. 2023. ...
Neuronal remodeling is extensive and mechanistically diverse across the nervous systems of complex metazoans. To explore circuit refinement mechanisms, we screened for new neuronal subtypes in the Drosophila nervous system that undergo remodeling early in metamorphosis. We find Beat-Va M neurons elaborate a highly branched neurite network during larval stages that undergoes local neurite pruning during early metamorphosis. Surprisingly, Beat-Va M neurons remodel their branches despite blockade of steroid hormone signaling and instead depend on signaling from astrocytes to activate pruning. We show Beat-Va L neurons undergo steroid hormone-dependent cell death in posterior but not anterior abdominal segments. Correct activation of apoptotic cell death relies on segment-specific expression of the hox gene Abd-B , which is capable of activating cell death in any Beat-Va L neuron. Our work provides new model cells in which to study neuronal remodeling, highlights an important role for astrocytes in activating local pruning in Drosophila independent of steroid signaling, and defines a Hox gene-mediated mechanism for segment-specific cell elimination.
Summary
Lehmann et al. characterize two new populations of neurons that undergo remodeling during Drosophila metamorphosis. Beat-Va M neurons undergo drastic neurite pruning that is largely independent of ecdysone signaling and instead is driven by astrocytes. Beat-Va L neurons undergo Abd-B mediated, caspase driven cell death in a segmentally restricted manner.
... Following axonal injury, the Drosophila glial surface receptor Draper initiates the phagocytic pathway involved in severed axon clearance (MacDonald et al., 2006). Using appendage axotomy to robustly activate glial injury responses, the evolutionarily conserved signaling pathway downstream of Draper was recently identified (Lu et al., 2017;Purice et al., 2017). ...
The degeneration of axons and their terminals occurs following traumatic, toxic, or genetically-induced insults. Common molecular mechanisms unite these disparate triggers to execute a conserved nerve degeneration cascade. In this review, we will discuss how models of peripheral nerve injury and neuropathy in Drosophila have led the way in advancing molecular understanding of axon degeneration and nerve injury pathways. Both neuron-intrinsic as well as glial responses to injury will be highlighted. Finally, we will offer perspective on what additional questions should be answered to advance these discoveries toward clinical interventions for patients with neuropathy.
... In Drosophila, Draper (Drpr) is the best-known receptor responsible for phagocytosis of neurons (15). As a homolog of the Caenorhabditis elegans engulfment receptor CED-1 (16) and the mammalian engulfment receptors Jedi-1 and MEGF10 (17), Drpr is involved in many contexts of neuronal phagocytosis, including the clearance of apoptotic neurons during embryonic development (15,18), axon and dendrite pruning during neuronal remodeling (19,20), injury-induced neurite degeneration (21,22), and removal of destabilized boutons at neuromuscular junctions (23). Despite the well-known importance of Drpr in sculpting the nervous system, how Drpr recognizes degenerating neurons in vivo is still unclear. ...
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, we show 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, our results uncover a missing link in PS-mediated phagocytosis in Drosophila and imply conserved mechanisms of phagocytosis of neurons.
... In Drosophila, the primary glial phagocytosis receptor is Draper which has considerable homology to mammalian Jedi-1 and MEGF10, as well as CED-1 in C. elegans (Zhou et al., 2001;Suzuki and Nakayama, 2007;Wu et al., 2009). Drosophila glia require Draper to phagocytose neuronal debris (Freeman et al., 2003;Awasaki et al., 2006;MacDonald et al., 2006). Cortex glia engulf dying neurons during development, astrocytes prune mushroom body γ neurites during metamorphosis, and ensheathing glia phagocytose degenerating axonal debris after injury, all in a Draper-dependent manner (Marin et al., 2005;Doherty et al., 2009;Tasdemir-Yilmaz and Freeman, 2014;McLaughlin et al., 2019;Herrmann et al., 2022). ...
Glial phagocytic activity refines connectivity, though molecular mechanisms regulating this exquisitely sensitive process are incompletely defined. We developed the Drosophila antennal lobe as a model for identifying molecular mechanisms underlying glial refinement of neural circuits in the absence of injury. Antennal lobe organization is stereotyped and characterized by individual glomeruli comprised of unique olfactory receptor neuronal (ORN) populations. The antennal lobe interacts extensively with two glial subtypes: ensheathing glia wrap individual glomeruli, while astrocytes ramify considerably within them. Phagocytic roles for glia in the uninjured antennal lobe are largely unknown. Thus, we tested whether Draper regulates ORN terminal arbor size, shape, or presynaptic content in two representative glomeruli: VC1 and VM7. We find that glial Draper limits the size of individual glomeruli and restrains their presynaptic content. Moreover, glial refinement is apparent in young adults, a period of rapid terminal arbor and synapse growth, indicating that synapse addition and elimination occur simultaneously. Draper has been shown to be expressed in ensheathing glia; unexpectedly, we find it expressed at high levels in late pupal antennal lobe astrocytes. Surprisingly, Draper plays differential roles in ensheathing glia and astrocytes in VC1 and VM7. In VC1, ensheathing glial Draper plays a more significant role in shaping glomerular size and presynaptic content; while in VM7, astrocytic Draper plays the larger role. Together, these data indicate that astrocytes and ensheathing glia employ Draper to refine circuitry in the antennal lobe before the terminal arbors reach their mature form and argue for local heterogeneity of neuron-glia interactions.
... LAP is initiated by receptor-mediated internalization of extracellular ligands such as apoptotic cells, immune complexes, and pathogens. In drpr homozygous null mutant flies, glial cells fail to even contact axon debris 8,10 . Importantly, heterozygosity for drpr is enough to strongly interfere with axon debris phagocytosis 44 , so we decided to use this sensitized genetic background to test if halving the level of Draper has any further effect on axon debris clearance in LAP mutants. ...
Glial engulfment of neuron-derived debris after trauma, during development, and in neurodegenerative diseases supports nervous system functions. However, mechanisms governing the efficiency of debris degradation in glia have remained largely unexplored. Here we show that LC3-associated phagocytosis (LAP), an engulfment pathway assisted by certain autophagy factors, promotes glial phagosome maturation in the Drosophila wing nerve. A LAP-specific subset of autophagy-related genes is required in glia for axon debris clearance, encoding members of the Atg8a (LC3) conjugation system and the Vps34 lipid kinase complex including UVRAG and Rubicon. Phagosomal Rubicon and Atg16 WD40 domain-dependent conjugation of Atg8a mediate proper breakdown of internalized axon fragments, and Rubicon overexpression in glia accelerates debris elimination. Finally, LAP promotes survival following traumatic brain injury. Our results reveal a role of glial LAP in the clearance of neuronal debris in vivo, with potential implications for the recovery of the injured nervous system.
... Arsenic affects nearly every cellular process and organ function [78]. Drosophila, a standard olfactory system model, provides an excellent opportunity to examine a lack of sensing and induction signals that impact the health of sensory neurons [79][80][81][82]. Earlier studies of acute arsenic toxicity by Muñiz Ortiz et al. [83] on transgenic Drosophila demonstrated effects of the methylation of arsenic into a +3-oxidation state on organismal longevity. ...
Millions of people in developing countries are affected by arsenic (As) toxicity and its prevalence. Arsenic’s detrimental effects on humans have been amplified by an unacceptable level of exposure to food and drinking water, the ongoing rise in industrial usage, and several other occupational conditions. Due to increased cellular absorption and the ability to cross the blood–brain barrier (BBB), inorganic arsenic (iAs) is extremely hazardous to living organisms in its trivalent form. Arsenic toxicity damages an organism’s tissues and organs, resulting in skin cancer, circulatory system abnormalities, and central nervous system disorders. However, a competent model system is required to investigate the acute effects of arsenic on the brain, cognition ability, and to assess any behavioral impairment. Hence, Drosophila, with its short generation time, genomic similarities with humans, and its availability for robust behavioral paradigms, may be considered an ideal model for studying arsenic toxicity. The present study helps to understand the toxic effects of acute arsenic treatment on the behavior, cognition, and development of Drosophila in a time-dependent manner. We found that the exposure of fruit flies to arsenic significantly affected their locomotor abilities, pupae size, cognitive functions, and neurobehavioral impairment. Hence, providing a better understanding of how arsenic toxicity affects the brain leading to acute behavioral disorders and neurological alterations, this study will lead to a better understanding of the mechanisms.
... To test whether OST antagonizes microglial activation induced by other pathogens besides LPS in other organisms besides mice, we utilized the Alzheimer's disease (AD) Drosophila model, whose neural system harbors the over-expression of human amyloid beta (Aβ), with symptoms of shortened survival, diminished climbing ability, defects of learning ability, and a higher oxidative burden [28]. By treating the AD Drosophila model with OST, we found that markers of microglial activation, such as the gene expression of Drpr and Ced [29], decreased ( Figure 3D) and that AD symptoms, including survival ( ...
Microglia are neuroglia in the brain with an innate immune function and participate in the progress of neurodegenerative diseases. Osthole (OST) is a coumarin derivative extracted from Cnidium monnieri and bears a microglia-antagonizing ability. However, the underlying mechanism of the antagonism is not clear. The lipopolysaccharides-induced microglial BV2 cell line and amyloid-overexpressing fruit fly were used as models to study OST treatment. We found that OST treatment is sufficient to evoke NRF2 cascade under an LPS-induced inflammatory environment, and silencing NRF2 is sufficient to abolish the process. Moreover, we found that OST is sufficient to antagonize microglial activation in both LPS-induced BV2 cells and Aβ-overexpressing fruit flies, and silencing NRF2 abolishes OST’s antagonism. Furthermore, OST treatment rescued survival, climbing, and the learning ability of Aβ-overexpressing fruit flies and relieved oxidative stress. In conclusion, we proved that OST antagonizes microglial activation induced by either LPS or Aβ and that NRF2 is necessary for OST’s antagonism.
... However, myelin, which is produced by oligodendrocytes, is not formed in the CNS of insects (Yildrim et al., 2018). In the adult fly brain, ENG responds to injury and clean the axonal debris in the neuropil (MacDonald et al., 2006). Similarly, in vertebrates, Schwann cells surrounding axons clear cellular debris and coordinate repair processes after peripheral nerve injury (Zuchero and Barres, 2015). ...
Food scarcity represents a major metabolic challenge for the brain and processesthat ensure its adequate functioning are considered crucial for organism survival (Mattsonet al, 2017). The brain is the central regulator of energy homeostasis, and it gives priority toits own supply over peripheral organs. However, the mechanisms through which theenergy status regulates brain plasticity remains largely unknown. We have previouslyrevealed using Drosophila melanogaster that the brain is subjected to adaptive plasticityand that under a metabolic challenge, such as starvation (i.e when the level of brain’smajor fuel, the glucose, scarce), the brain disables the costly formation of protein synthesisdependent long-term memory (LTM) to favor survival, but concomitantly upregulatedanother form of consolidated long-lasting memory called LT-ARM (Placais and Preat,2013). Recently, we have demonstrated that flies double their sugar intake and upregulatetheir mitochondrial pyruvate flux in the fly olfactory memory center, the Mushroom Body(MB), after LTM formation (Placais et al., 2017). Nevertheless, in absence of glucosederivatives, how does the brain regulate memory formation to withstands periods of foodscarcity? Moreover, what are the metabolic fuels used by the brain to sustain the formationof persistent memory upon starvation? And eventually which are the brain cell typesinvolved in this process?Ketone Bodies (KBs) are high-energy rich metabolites, derived from fatty acidmetabolism and used by the brain under metabolic challenging conditions such asstarvation (Owen et al. 1967). However, it is currently unknown whether neurons are able touse KBs to sustain neuronal physiology and in particular memory formation. In my thesis, Iaddressed the in vivo role of KBs metabolism in aversive olfactory memory formation uponstarvation (sLT-ARM), the metabolic pathways required and the cell types involved in thisprocess by combining genetics, behavioral assays, fluorescent lipid staining and in vivotwo photon imaging using the model organism Drosophila melanogaster.By using cell type specific gene knockdown restricted to adulthood associated tobehavioral and in vivo imaging experiments, I have demonstrated that KBs import andmitochondrial oxidation are required in MB neurons to sustain sLT-ARM formationspecifically. Then I have identified the cortex glia cells as the ones providing KBs toneurons upon sLT-ARM formation. Eventually using behavioral assays and fluorescent lipidstaining, I have shown that sLT-ARM formation is dependent on ketogenesis in cortex gliafrom their own lipid store and that this process is likely regulated by the master energysensor AMPK.Based on these data, we can propose a model of cortex glia-MB neuronsinteractions specific to starvation in which KB synthetized by glia from their own lipid storeare shuttled to neurons to sustain persistent memory formation. Our data suggest that, atleast in glial cells, AMPK could be one of the keys to activate this model upon starvation.This work presented here is of particular relevance in the frame of recent evidences thatfood deprivation periods such as caloric restriction or intermittent fasting might havebeneficial effects on cognition in age-related cognitive impairments.
... Given the presence of immune-responsive fat body tissue within the head, evidence of neuroinflammation was probed in the Gba1b -/fly brain by examining the highly conserved glial immune pathway involving Draper. Draper is a phagocytic recognition receptor on the surface of engulfing glia in flies and is required for the glial phagocytic removal of axonal debris following axonal injury (Doherty et al., 2009, MacDonald et al., 2006. Consistent with the widespread innate immune activation, increased Draper gene expression was observed in the heads of 3-week-old Gba1b -/flies ( Figure 2A). ...
Mutations in the GBA1 gene cause the lysosomal storage disorder Gaucher disease (GD) and are the greatest genetic risk factor for Parkinson disease (PD). Communication between gut and brain and immune dysregulation are increasingly being implicated in neurodegenerative disorders such as PD. Here, we show that flies lacking the Gba1b gene, the main fly orthologue of GBA1, display widespread innate immune up-regulation, including gut inflammation and brain glial activation. We also demonstrate gut dysfunction in flies lacking Gba1b, with increased intestinal transit time, gut barrier permeability and microbiome dysbiosis. Remarkably, modulating the microbiome of Gba1b knockout flies, by raising them under germ-free conditions, can partially ameliorate lifespan, locomotor and some neuropathological phenotypes. Lastly, direct stimulation of autophagy by rapamycin treatment achieves similar beneficial effects. Overall, our data reveal that the gut microbiome drives systemic immune activation in Gba1b knockout flies and that reducing innate immune response activation either by eliminating the microbiota or clearance of immunogens by autophagy may represent potential therapeutic avenues for GBA1-associated neurodegenerative disease.
... To more specifically target glia→axon support mechanisms (as opposed to direct support of the cell body), we developed a system where axons would survive for weeks without their cell body. Briefly, severing wild-type axons causes the portion of the axon distal to the injury site to undergo Wallerian degeneration and dies/is eliminated (Fig. 2 A ;MacDonald et al., 2006). However, this can be blocked by overexpressing Wld S in neurons, which suppresses Wallerian degeneration and allows distal severed axons to remain intact for weeks after axotomy (Fig. 2 A; Glass and Griffin, 1991). ...
Maintaining long, energetically demanding axons throughout the life of an animal is a major challenge for the nervous system. Specialized glia ensheathe axons and support their function and integrity throughout life, but glial support mechanisms remain poorly defined. Here, we identified a collection of secreted and transmembrane molecules required in glia for long-term axon survival in vivo. We showed that the majority of components of the TGFβ superfamily are required in glia for sensory neuron maintenance but not glial ensheathment of axons. In the absence of glial TGFβ signaling, neurons undergo age-dependent degeneration that can be rescued either by genetic blockade of Wallerian degeneration or caspase-dependent death. Blockade of glial TGFβ signaling results in increased ATP in glia that can be mimicked by enhancing glial mitochondrial biogenesis or suppressing glial monocarboxylate transporter function. We propose that glial TGFβ signaling supports axon survival and suppresses neurodegeneration through promoting glial metabolic support of neurons.
... Among the most well-studied scavenger receptors in Drosophila is Draper, which stimulates glial phagocytosis and clears apoptotic cells [67][68][69][70] . Evidence suggests that the upregulation of draper promotes the loss of neurons as well as locomotor dysfunction 68 . ...
The hallmark of Parkinson’s disease (PD) is the loss of dopaminergic (DA) neurons in the brain. However, little is known about why DA neurons are selectively vulnerable to PD. We previously completed a screen identifying genes associated with the progressive degeneration of DA neurons. Here we describe the role of a previously uncharacterized gene, CG42339, in the loss of DA neurons using Drosophila Melanogaster. CG42339 mutants display a progressive loss of DA neurons and locomotor dysfunction, along with an accumulation of advanced glycation end products (AGEs) in the brain. Based on this phenotype, we refer to CG42339 as vexed. We demonstrate that vexed is specifically required within cortex glia to maintain neuronal viability. Loss of vexed function results in excessive activation of the innate immune response in the brain, leading to loss of DA neurons. We show that activation of the innate immune response leads to increased nitric oxide signaling and accumulation of AGEs, which ultimately result in neurodegeneration. These results provide further insight into the relationship between the role of the immune response in the central nervous system and how this impacts neuronal viability.
... Alternatively, some terminals might arise via ascending projections from T2 (midleg) or T3 (hindleg) and thus are not directly affected by foreleg amputation. Drosophila has been previously used in numerous studies as a model of nerve injury 52 , where it has been shown that axonal degradation following injury can be similar to Wallerian degeneration in mammals 53 . Thus, our long-term imaging toolkit might in the future be used to test for pharmacological interventions that can slow down or prevent injury-induced axonal degeneration in motor circuits. ...
The dynamics and connectivity of neural circuits continuously change on timescales ranging from milliseconds to an animal’s lifetime. Therefore, to understand biological networks, minimally invasive methods are required to repeatedly record them in behaving animals. Here we describe a suite of devices that enable long-term optical recordings of the adult Drosophila melanogaster ventral nerve cord (VNC). These consist of transparent, numbered windows to replace thoracic exoskeleton, compliant implants to displace internal organs, a precision arm to assist implantation, and a hinged stage to repeatedly tether flies. To validate and illustrate our toolkit we (i) show minimal impact on animal behavior and survival, (ii) follow the degradation of chordotonal organ mechanosensory nerve terminals over weeks after leg amputation, and (iii) uncover waves of neural activity caffeine ingestion. Thus, our long-term imaging toolkit opens up the investigation of premotor and motor circuit adaptations in response to injury, drug ingestion, aging, learning, and disease.
... S7, C and D), as would be expected of glia. The cluster is also enriched in the expression of genes associated with signaling, adhesion, sugar transport, and axon pruning in the context of neuron-glia interactions in Drosophila [rau, uzip, Tret1-2, and draper; (64)(65)(66)(67)(68)]. Putative sensory organ accessory cells Cell clusters 3 and 21 are associated with the large epidermal supercluster described above. ...
Animals can regenerate complex organs, yet this process frequently results in imprecise replicas of the original structure. In the crustacean Parhyale, embryonic and regenerating legs differ in gene expression dynamics but produce apparently similar mature structures. We examine the fidelity of Parhyale leg regeneration using complementary approaches to investigate microanatomy, sensory function, cellular composition, and cell molecular profiles. We find that regeneration precisely replicates the complex microanatomy and spatial distribution of external sensory organs and restores their sensory function. Single-nuclei sequencing shows that regenerated and uninjured legs are indistinguishable in terms of cell-type composition and transcriptional profiles. This remarkable fidelity highlights the ability of organisms to achieve identical outcomes via distinct processes.
... In Drosophila, aggregates formed by mHTT ex1 proteins transfer from presynaptic ORNs to postsynaptic PNs in the fly olfactory system only after passage through the cytoplasm of phagocytic glial cell intermediates ( Figure 3J) (Donnelly et al., 2020). This circuitous route for mHTT spreading requires expression of Draper (Pearce et al., 2015;Donnelly et al., 2020), a scavenger receptor that regulates key phagocytic pathways in fly glia and other cell types ( Figure 3G) (MacDonald et al., 2006;Etchegaray et al., 2016;Ray et al., 2017). The mammalian homolog of Draper, MEGF10, is highly expressed in astrocytes and mediates phagocytic clearance of synapses in healthy and diseased adult mouse brains (Chung et al., 2013;Iram et al., 2016;Shi et al., 2021). ...
The hypothesis that pathogenic protein aggregates associated with neurodegenerative diseases spread from cell-to-cell in the brain in a manner akin to infectious prions has gained substantial momentum due to an explosion of research in the past 10–15 years. Here, we review current evidence supporting the existence of prion-like mechanisms in Huntington’s disease (HD), an autosomal dominant neurodegenerative disease caused by expansion of a CAG repeat tract in exon 1 of the huntingtin (HTT) gene. We summarize information gained from human studies and in vivo and in vitro models of HD that strongly support prion-like features of the mutant HTT (mHTT) protein, including potential involvement of molecular features of mHTT seeds, synaptic structures and connectivity, endocytic and exocytic mechanisms, tunneling nanotubes, and nonneuronal cells in mHTT propagation in the brain. We discuss mechanisms by which mHTT aggregate spreading and neurotoxicity could be causally linked and the potential benefits of targeting prion-like mechanisms in the search for new disease-modifying therapies for HD and other fatal neurodegenerative diseases.
... Neuronal expression of the mouse Wallerian degeneration slow (Wld S ) protein was found to delay Wallerian degeneration, which is an evolutionarily conserved process to clear distal axons after axon injury, in both mice and flies (23)(24)(25). Indeed, expression of Wld S can also protect axon degeneration or neuronal survival in several models of neurodegeneration (26,27). Recently, the roles of Wld S in dendrites have started to be revealed. ...
Neurodegeneration arising from aging, injury, or diseases has devastating health consequences. Whereas neuronal survival and axon degeneration have been studied extensively, much less is known about how neurodegeneration affects dendrites, in part due to the limited assay systems available. To develop an assay for dendrite degeneration and repair, we used photo-switchable caspase-3 (caspase-Light-Oxygen-Voltage-sensing [caspase-LOV]) in peripheral class 4 dendrite arborization (c4da) neurons to induce graded neurodegeneration by adjusting illumination duration during development and adulthood in Drosophila melanogaster. We found that both developing and mature c4da neurons were able to survive while sustaining mild neurodegeneration induced by moderate caspase-LOV activation. Further, we observed active dendrite addition and dendrite regeneration in developing and mature c4da neurons, respectively. Using this assay, we found that the mouse Wallerian degeneration slow (WldS) protein can protect c4da neurons from caspase-LOV-induced dendrite degeneration and cell death. Furthermore, our data show that WldS can reduce dendrite elimination without affecting dendrite addition. In summary, we successfully established a photo-switchable assay system in both developing and mature neurons and used WldS as a test case to study the mechanisms underlying dendrite regeneration and repair.
... | Megf10 in the central nervous systemIn the eye, MEGF10 contributes to the formation of retinal mosaics.67 Elsewhere in the central nervous system, MEGF10 binds to dead neurons, contributing to the engulfment activities of glial cells 74 and phagocytic activities of neurons,75 indicating a key role in neuronal apoptosis.Parallel functions have been identified for the Drosophila orthologDrpr.[76][77][78] The ATP binding cassette transporter ABCA1 contributes to the engulfment activity of MEGF10. ...
The Notch signaling pathway is a key regulator of skeletal muscle development and regeneration. Over the past decade, the discoveries of three new muscle disease genes have added a new dimension to the relationship between the Notch signaling pathway and skeletal muscle: MEGF10, POGLUT1, and JAG2. We review the clinical syndromes associated with pathogenic variants in each of these genes, known molecular and cellular functions of their protein products with a particular focus on the Notch signaling pathway, and potential novel therapeutic targets that may emerge from further investigations of these diseases. The phenotypes associated with two of these genes, POGLUT1 and JAG2, clearly fall within the realm of muscular dystrophy, whereas the third, MEGF10, is associated with a congenital myopathy/muscular dystrophy overlap syndrome classically known as early‐onset myopathy, areflexia, respiratory distress, and dysphagia. JAG2 is a canonical Notch ligand, POGLUT1 glycosylates the extracellular domain of Notch receptors, and MEGF10 interacts with the intracellular domain of NOTCH1. Additional genes and their encoded proteins relevant to muscle function and disease with links to the Notch signaling pathway include TRIM32, ATP2A1 (SERCA1), JAG1, PAX7, and NOTCH2NLC. There is enormous potential to identify convergent mechanisms of skeletal muscle disease and new therapeutic targets through further investigations of the Notch signaling pathway in the context of skeletal muscle development, maintenance, and disease.
... Recent studies indicate that a sufficient amount of CED-1/Draper is critical for its engulfment function (Hilu-Dadia et al., 2018;Kurant et al., 2008;MacDonald et al., 2006;Manaka et al., 2004). Whether appropriate levels of CED-1 are maintained for executing engulfment function remains unknown. ...
The phagocytic receptor CED-1 mediates apoptotic cell recognition by phagocytic cells, enabling cell corpse clearance in Caenorhabditis elegans . Whether appropriate levels of CED-1 are maintained for executing the engulfment function remains unknown. Here, we identified the C. elegans E3 ubiquitin ligase tripartite motif containing-21 (TRIM-21) as a component of the CED-1 pathway for apoptotic cell clearance. When the NPXY motif of CED-1 was bound to the adaptor protein CED-6 or the YXXL motif of CED-1 was phosphorylated by tyrosine kinase SRC-1 and subsequently bound to the adaptor protein NCK-1 containing the SH2 domain, TRIM-21 functioned in conjunction with UBC-21 to catalyze K48-linked poly-ubiquitination on CED-1, targeting it for proteasomal degradation. In the absence of TRIM-21, CED-1 accumulated post-translationally and drove cell corpse degradation defects, as evidenced by direct binding to VHA-10. These findings reveal a unique mechanism for the maintenance of appropriate levels of CED-1 to regulate apoptotic cell clearance.
CED-1 is a transmembrane receptor involved in the recognition of “eat-me” signals displayed on the surface of apoptotic cells and thus central for the subsequent engulfment of the cell corpse in C. elegans. The roles of CED-1 in engulfment are well established, as are its downstream effectors. The latter includes the adapter protein CED-6/GULP and the ABC family homolog CED-7. However, how CED-1 is maintained on the plasma membrane in the absence of engulfment is currently unknown. Here, we show that CED-6 and CED-7 have a novel role in maintaining CED-1 correctly on the plasma membrane. We propose that the underlying mechanism is via endocytosis as CED-6 and CED-7 act redundantly with clathrin and its adaptor, the AP2 complex, in ensuring correct CED-1 localization. In conclusion, CED-6 and CED-7 impact other cellular processes than engulfment of apoptotic cells.
The long length of axons makes them vulnerable to damage; hence, it is logical that nervous systems have evolved adaptive mechanisms for responding to axon damage. Studies in Drosophila melanogaster have identified evolutionarily conserved molecular pathways that enable axonal degeneration and regeneration of damaged axons and/or dendrites. This protocol describes a simple method for inducing nerve crush injury to motoneuron and sensory neuron axons in the peripheral (segmental) nerves in second- or early third-instar larvae. Small forceps are used to pinch the cuticle at a location that overlays the segmental nerves. Although the connective tissue of the nerves remains intact and the larva survives the injury, single motoneuron and sensory neuron axons incur a break in continuity at the damage site and then undergo Wallerian degeneration distal to the break. This degeneration includes the dismantling of neuromuscular junction (NMJ) synapses formed by the axons that incurred damage. With stereotyped anatomy and accessibility to structural and electrophysiological studies, the larval NMJ is a good model to characterize the cellular changes that occur in synapses undergoing degeneration and to identify conditions that can protect axons and synapses from degeneration.
Protein misfolding, aggregation, and spread through the brain are primary drivers of neurodegenerative disease pathogenesis. Phagocytic glia are responsible for regulating the load of pathological proteins in the brain, but emerging evidence suggests that glia may also act as vectors for aggregate spread. Accumulation of protein aggregates could compromise the ability of glia to eliminate toxic materials from the brain by disrupting efficient degradation in the phagolysosomal system. A better understanding of phagocytic glial cell deficiencies in the disease state could help to identify novel therapeutic targets for multiple neurological disorders. Here, we report that mutant huntingtin (mHTT) aggregates impair glial responsiveness to injury and capacity to degrade neuronal debris in male and female adult Drosophila expressing the gene that causes Huntington's disease (HD). mHTT aggregate formation in neurons impairs engulfment and clearance of injured axons and causes accumulation of phagolysosomes in glia. Neuronal mHTT expression induces upregulation of key innate immunity and phagocytic genes, some of which were found to regulate mHTT aggregate burden in the brain. A forward genetic screen revealed Rab10 as a novel component of Draper-dependent phagocytosis that regulates mHTT aggregate transmission from neurons to glia. These data suggest that glial phagocytic defects enable engulfed mHTT aggregates to evade lysosomal degradation and acquire prion-like characteristics. Together, our findings uncover new mechanisms that enhance our understanding of the beneficial and harmful effects of phagocytic glia in HD and other neurodegenerative diseases.
Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. Here, using a Drosophila model, we show that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure down-regulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial Draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and Draper expression. Significantly, we show that genetically stimulating phosphoinositide 3-kinase (Pi3k), a downstream effector of insulin receptor (IR) signaling, rescues high-sugar diet (HSD)-induced glial defects. Hence, we establish that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris.
In nervous system development, disease, and injury, neurons undergo programmed cell death, leaving behind cell corpses that are removed by phagocytic glia. Altered glial phagocytosis has been implicated in several neurological diseases including Alzheimer’s disease. To untangle the links between glial phagocytosis and neurodegeneration, we investigated Drosophila mutants lacking the phagocytic receptor Draper. Loss of Draper leads to persistent neuronal cell corpses and age-dependent neurodegeneration. Here we investigate whether the phagocytic defects observed in draper mutants lead to chronic increased immune activation that promotes neurodegeneration. We found that the antimicrobial peptide Attacin-A is highly upregulated in the fat body of aged draper mutants and that the inhibition of the Immune deficiency (Imd) pathway in the glia and fat body of draper mutants led to reduced neurodegeneration. Taken together, these findings indicate that phagocytic defects lead to neurodegeneration via increased immune signaling, both systemically and locally in the brain.
Wallerian axonal degeneration (WD) does not occur in the nematode C. elegans, in contrast to other model animals. However, WD depends on the NADase activity of SARM1, a protein that is also expressed in C. elegans (ceSARM/ceTIR-1). We hypothesized that differences in SARM between species might exist and account for the divergence in WD. We first show that expression of the human (h)SARM1, but not ceTIR-1, in C. elegans neurons is sufficient to confer axon degeneration after nerve injury. Next, we determined the cryoelectron microscopy structure of ceTIR-1 and found that, unlike hSARM1, which exists as an auto-inhibited ring octamer, ceTIR-1 forms a readily active 9-mer. Enzymatically, the NADase activity of ceTIR-1 is substantially weaker (10-fold higher Km) than that of hSARM1, and even when fully active, it falls short of consuming all cellular NAD+. Our experiments provide insight into the molecular mechanisms and evolution of SARM orthologs and WD across species.
Childhood neglect and/or abuse can induce mental health conditions with unknown mechanisms. Here, we identified stress hormones as strong inducers of astrocyte-mediated synapse phagocytosis. Using in vitro, in vivo, and human brain organoid experiments, we showed that stress hormones increased the expression of the Mertk phagocytic receptor in astrocytes through glucocorticoid receptor (GR). In post-natal mice, exposure to early social deprivation (ESD) specifically activated the GR-MERTK pathway in astrocytes, but not in microglia. The excitatory post-synaptic density in cortical regions was reduced in ESD mice, and there was an increase in the astrocytic engulfment of these synapses. The loss of excitatory synapses, abnormal neuronal network activities, and behavioral abnormalities in ESD mice were largely prevented by ablating GR or MERTK in astrocytes. Our work reveals the critical roles of astrocytic GR-MERTK activation in evoking stress-induced abnormal behaviors in mice, suggesting GR-MERTK signaling as a therapeutic target for stress-induced mental health conditions.
Traumatic brain injury (TBI) caused by external mechanical forces is a major health burden worldwide, but the underlying mechanism in glia remains largely unclear. We report herein that Drosophila adults exhibit a defective blood-brain-barrier (BBB), elevated innate immune responses, and hypertrophy of astrocytes upon consecutive strikes with a high-impact trauma device. RNA sequencing (RNA-seq) analysis of these astrocytes revealed upregulated expression of genes encoding PDGF and VEGF receptor-related (Pvr, a receptor tyrosine kinase (RTK)), adaptor protein complex 1 (AP-1, a transcription factor complex of the c-Jun N-terminal Kinase (JNK) pathway) composed of Jun-related antigen (Jra) and kayak (kay), and matrix metalloproteinase 1 (Mmp1) following TBI. Interestingly, Pvr is both required and sufficient for AP-1 and Mmp1 upregulation, while knockdown of AP-1 expression in the background of Pvr overexpression in astrocytes rescued Mmp1 upregulation upon TBI, indicating that Pvr acts as the upstream receptor for the downstream AP-1–Mmp1 transduction. Moreover, dynamin-associated endocytosis was found to be an important regulatory step in downregulating Pvr signaling. Our results identify a new Pvr–AP-1–Mmp1 signaling pathway in astrocytes in response to TBI, providing potential targets for developing new therapeutic strategies of TBI.
Phosphatidylserine (PS) is a lipid component of the plasma membrane. It is asymmetrically distributed to the inner leaflet in live cells. In cells undergoing apoptosis, phosphatidylserine is exposed to the outer surfaces. The exposed phosphatidylserine acts as an evolutionarily conserved “eat-me” signal that attracts neighboring engulfing cells in metazoan organisms, including the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and mammals. During apoptosis, the exposure of phosphatidylserine to the outer surface of a cell is driven by the membrane scramblases and flippases, the activities of which are regulated by caspases. Cells undergoing necrosis, a kind of cell death frequently associated with cellular injuries and morphologically distinct from apoptosis, were initially believed to allow passive exposure of phosphatidylserine through membrane rupture. Later studies revealed that necrotic cells actively expose phosphatidylserine before any rupture occurs. A recent study in C. elegans further reported that the calcium ion (Ca²⁺) plays an essential role in promoting the exposure of phosphatidylserine on the surfaces of necrotic cells. These findings indicate that necrotic and apoptotic cells, which die through different molecular mechanisms, use common and unique mechanisms for promoting the exposure of the same “eat me” signal. This article will review the mechanisms regulating the exposure of phosphatidylserine on the surfaces of necrotic and apoptotic cells and highlight their similarities and differences.
Neuronal cell death and subsequent brain dysfunction are hallmarks of aging and neurodegeneration, but how the nearby healthy neurons (bystanders) respond to the cell death of their neighbors is not fully understood. In the Drosophila larval neuromuscular system, bystander motor neurons can structurally and functionally compensate for the loss of their neighbors by increasing their axon terminal size and activity. We termed this compensation as cross-neuron plasticity, and in this study, we demonstrated that the Drosophila engulfment receptor, Draper, and the associated kinase, Shark, are required in glial cells. Surprisingly, overexpression of the Draper-I isoform boosts cross-neuron plasticity, implying that the strength of plasticity correlates with Draper signaling. Synaptic plasticity normally declines as animals age, but in our system, functional cross-neuron plasticity can be induced at different time points, whereas structural cross-neuron plasticity can only be induced at early stages. Our work uncovers a novel role for glial Draper signaling in cross-neuron plasticity that may enhance nervous system function during neurodegeneration and provides insights into how healthy bystander neurons respond to the loss of their neighboring neurons.
Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. Here, using a Drosophila model, we show that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure downregulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila Insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and draper expression. Significantly, we show that genetically stimulating Phosphoinositide 3-kinase (PI3K), a downstream effector of Insulin receptor signaling, rescues HSD-induced glial defects. Hence, we establish that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris.
Tauopathy is characterised by neuronal dysfunction and degeneration occurring as a result of changes to the microtubule associated protein tau. The neuronal changes evident in Tauopathy bear striking morphological resemblance to those reported in models of Wallerian degeneration. The mechanisms underpinning Wallerian degeneration are not fully understood although it can be delayed by the expression of the slow Wallerian degeneration (WldS) protein, which has also been demonstrated to delay axonal degeneration in some models of neurodegenerative disease. Given the morphological similarities between tauopathy and Wallerian degeneration, this study investigated whether tau-mediated phenotypes can be modulated by co-expression of WldS.
In a Drosophila model of tauopathy in which expression of human 0N3R tau protein leads to progressive age-dependent phenotypes, WldS was expressed with and without activation of the downstream pathway. The olfactory receptor neuron circuit OR47b was used for these studies in adults and the larval motor neuron system was employed in larvae. Tau phenotypes studied included neurodegeneration, axonal transport, synaptic deficits and locomotor behaviour. Impact on total tau was ascertained by assessing total, phosphorylated and misfolded tau levels by immunohistochemistry.
Activation of the pathway downstream of WldS completely suppressed tau-mediated degeneration. This protective effect was evident even if the pathway downstream of WldS was activated several weeks after tau-mediated degeneration had become established. Though total tau levels were not altered, the protected neurons displayed significantly reduced MC1 immunoreactivity suggestive of clearance of misfolded tau, as well as a trend for a decline in phosphorylated tau species phosphorylated at the AT8 and PHF1 epitopes. In contrast, WldS expression without activation of the downstream protective pathway did not rescue tau-mediated degeneration in adults or improve tau-mediated neuronal dysfunction including deficits in axonal transport, synaptic alterations and locomotor behaviour in tau–expressing larvae.
This collectively implies that the pathway mediating the protective effect of WldS intersects with the mechanism(s) of degeneration initiated by tau and can effectively halt tau-mediated degeneration at both early and late stages. Understanding the mechanisms underpinning this protection could identify much-needed disease-modifying targets for tauopathies.
The importance of glial cells has become increasingly apparent over the past 20 years, yet compared to neurons we still know relatively little about these essential cells. Most critical glial cell functions are conserved in Drosophila glia, often using the same key molecular players as their vertebrate counterparts. The relative simplicity of the Drosophila nervous system, combined with a vast array of powerful genetic tools, allows us to further dissect the molecular composition and functional roles of glia in ways that would be much more cumbersome or not possible in higher vertebrate systems. Importantly, Drosophila genetics allow for in vivo manipulation, and their transparent body wall enables in vivo imaging of glia in intact animals throughout early development. Here we discuss recent advances in Drosophila glial development detailing how these cells take on their mature morphologies and interact with neurons to perform their important functional roles in the nervous system.
The evolutionary conservation of glial cells has been appreciated since Ramon y Cajal and Del Rio Hortega first described the morphological features of cells in the nervous system. We now appreciate that glial cells have essential roles throughout life in most nervous systems. The field of glial cell biology has grown exponentially in the last ten years. This new wealth of knowledge has been aided by seminal findings in non-mammalian model systems. Ultimately, such concepts help us to understand glia in mammalian nervous systems. Rather than summarizing the field of glial biology, I will first briefly introduce glia in non-mammalian models systems. Then, highlight seminal findings across the glial field that utilized non-mammalian model systems to advance our understanding of the mammalian nervous system. Finally, I will call attention to some recent findings that introduce new questions about glial cell biology that will be investigated for years to come.
Axon degeneration contributes to the disruption of neuronal circuit function in diseased and injured nervous systems. Severed axons degenerate following the activation of an evolutionarily conserved signaling pathway, which culminates in the activation of SARM1 in mammals to execute the pathological depletion of the metabolite NAD ⁺ . SARM1 NADase activity is activated by the NAD ⁺ precursor nicotinamide mononucleotide (NMN). In mammals, keeping NMN levels low potently preserves axons after injury. However, it remains unclear whether NMN is also a key mediator of axon degeneration and dSarm activation in flies. Here, we demonstrate that lowering NMN levels in Drosophila through the expression of a newly generated prokaryotic NMN-Deamidase (NMN-D) preserves severed axons for months and keeps them circuit-integrated for weeks. NMN-D alters the NAD ⁺ metabolic flux by lowering NMN, while NAD ⁺ remains unchanged in vivo . Increased NMN synthesis, by the expression of mouse nicotinamide phosphoribosyltransferase (mNAMPT), leads to faster axon degeneration after injury. We also show that NMN-induced activation of dSarm mediates axon degeneration in vivo . Finally, NMN-D delays neurodegeneration caused by loss of the sole NMN-consuming and NAD ⁺ -synthesizing enzyme dNmnat. Our results reveal a critical role for NMN in neurodegeneration in the fly, which extends beyond axonal injury. The potent neuroprotection by reducing NMN levels is similar to the interference with other essential mediators of axon degeneration in Drosophila .
Development of the brain and the emergence of the mind constitute some of the most important concerns of contemporary biology. Disturbances during fetal life may have profound implications for a child's future neurological and psychological development, which can in turn impact society. The new edition of this highly respected work presents a comprehensive review of the basic mechanisms of brain development and the pathophysiology of disorders of the infant brain, written by a team of distinguished neuroscientists, neonatologists, and neuropediatricians. The book follows the main milestones of brain development, from formation of the neural tube and wiring of the neurons in the brain. Neurotrophic factors, neurotransmitters, glial cell biology, cerebral circulation development of sensory functions are all described in detail. Furthermore, there are more philosophical chapters on the evolution of the brain and the emergence of consciousness. Clinical considerations are highlighted where relevant.
Development of the brain and the emergence of the mind constitute some of the most important concerns of contemporary biology. Disturbances during fetal life may have profound implications for a child's future neurological and psychological development, which can in turn impact society. The new edition of this highly respected work presents a comprehensive review of the basic mechanisms of brain development and the pathophysiology of disorders of the infant brain, written by a team of distinguished neuroscientists, neonatologists, and neuropediatricians. The book follows the main milestones of brain development, from formation of the neural tube and wiring of the neurons in the brain. Neurotrophic factors, neurotransmitters, glial cell biology, cerebral circulation development of sensory functions are all described in detail. Furthermore, there are more philosophical chapters on the evolution of the brain and the emergence of consciousness. Clinical considerations are highlighted where relevant.
Development of the brain and the emergence of the mind constitute some of the most important concerns of contemporary biology. Disturbances during fetal life may have profound implications for a child's future neurological and psychological development, which can in turn impact society. The new edition of this highly respected work presents a comprehensive review of the basic mechanisms of brain development and the pathophysiology of disorders of the infant brain, written by a team of distinguished neuroscientists, neonatologists, and neuropediatricians. The book follows the main milestones of brain development, from formation of the neural tube and wiring of the neurons in the brain. Neurotrophic factors, neurotransmitters, glial cell biology, cerebral circulation development of sensory functions are all described in detail. Furthermore, there are more philosophical chapters on the evolution of the brain and the emergence of consciousness. Clinical considerations are highlighted where relevant.
The last step of cell death is cell clearance, a process critical for tissue homeostasis. For efficient cell clearance to occur, phagocytes and dead cells need to reciprocally signal to each other. One important phenomenon that is under-investigated, however, is that phagocytes not only engulf corpses but contribute to cell death progression. The aims of this study were to determine how the phagocytic receptor Draper non-autonomously induces cell death, using the Drosophila ovary as a model system. We found that Draper, expressed in epithelial follicle cells, requires its intracellular signaling domain to kill the adjacent nurse cell population. Kinases Src, Shark and JNK were required for Draper-induced nurse cell death. Signs of nurse cell death occurred prior to apparent engulfment and required the caspase Dcp-1, indicating that it uses a similar apoptotic pathway as starvation-induced cell death. These findings indicate that active signaling by Draper is required to kill nurse cells via the caspase Dcp-1, providing novel insights into mechanisms of phagoptosis driven by non-professional phagocytes.
Glial engulfment of dead neurons and neurites after trauma, during development and in neurodegenerative diseases plays a crucial role in nervous system maintenance. Axon debris generated after traumatic injury is cleared by phagocytic glia via Draper receptor signalling in Drosophila. However, mechanisms governing the efficiency of axon debris phagocytosis and degradation have remained largely unexplored. Here we show that glial LC3-associated phagocytosis (LAP), an engulfment pathway assisted by certain components of the macroautophagy machinery, promotes clearance of degenerating axons in the Drosophila wing nerve. A LAP-specific subset of autophagy-related (Atg) genes is required in glia for efficient debris elimination, which includes members of the Atg8a (LC3) conjugation system and the Vps34 lipid kinase complex subunits UVRAG and Rubicon but not Atg14 or the Atg1 kinase complex. Atg8a and Rubicon are recruited to Rab7-positive phagosomes and Atg8a lipid conjugation is essential for debris-containing phagosome maturation. Finally, Rubicon overexpression in glia accelerates axon debris elimination. Our results reveal the critical role of LAP in glia in the clearance of neuronal debris in vivo, with important implications for the recovery of the injured nervous system.
Using mAbs and immunocytochemistry we have examined the response of macrophages (M phi) after crush injury to the sciatic or optic nerve in the mouse and rat. We have established that large numbers of M phi enter peripheral nerves containing degenerating axons; the M phi are localized to the portion containing damaged axons, and they phagocytose myelin. The period of recruitment of the M phi in the peripheral nerve is before and during the period of maximal proliferation of the Schwann cells. In contrast, the degenerating optic nerve attracts few M phi, and the removal of myelin is much slower. These results show the clearly different responses of M phi to damage in the central and peripheral nervous systems, and suggest that M phi may be an important component of subsequent repair as well as myelin degradation.
The object of the present observations is to describe certain alterations which take place in the elementary fibres of the nerve after they have been removed from their connection with the brain or spinal marrow. The following is a brief summary of the opinions and researches of modern physiologists on alterations of the nerve-tubes.
Exons of three genes were identified within the 85-kilobase tandem
triplication unit of the slow Wallerian degeneration mutant mouse,
C57BL/WldS. Ubiquitin fusion
degradation protein 2 (Ufd2) and a previously
undescribed gene, D4Cole1e, span the proximal and distal
boundaries of the repeat unit, respectively. They have the same
chromosomal orientation and form a chimeric gene when brought together
at the boundaries between adjacent repeat units in
WldS. The chimeric mRNA is
abundantly expressed in the nervous system and encodes an in-frame
fusion protein consisting of the N-terminal 70 amino acids of
Ufd2, the C-terminal 302 amino acids of
D4Cole1e, and an aspartic acid formed at the junction.
Antisera raised against synthetic peptides detect the expected 43-kDa
protein specifically in WldS brain.
This expression pattern, together with the previously established role
of ubiquitination in axon degeneration, makes the chimeric gene a
promising candidate for Wld. The third gene altered by
the triplication, Rbp7, is a novel member of the
cellular retinoid-binding protein family and is highly expressed in
white adipose tissue and mammary gland. The whole gene lies within the
repeat unit leading to overexpression of the normal transcript in
WldS mice. However, it is
undetectable on Northern blots of
WldS brain and seems unlikely to be
the Wld gene. These data reveal both a candidate gene
for Wld and the potential of the
WldS mutant for studies of ubiquitin
and retinoid metabolism.
Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/Wld
S mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in Wld
S tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.
Upon transection of a peripheral nerve, axons distal to the transection degenerate. As a consequence of this axonal degeneration, myelin-forming Schwann cells cease biosynthesis of new myelin membrane, contribute to phagocytosis of previously formed myelin, and markedly down-regulate expression of myelin-specific markers. Among the most prominent of these down-regulated markers are the major structural proteins of peripheral myelin, Po and myelin basic protein (MBP). We have used slot blot and in situ hybridization techniques to demonstrate that for actively myelinating Schwann cells, down-regulation of the Po and MBP genes occurs primarily at the level of mRNA expression. Together with other recent data, these findings strongly argue for axonal modulation of Po and MBP gene transcription during active myelination.
We report the identification of RK2, a glial-specific homeodomain protein. RK2 is localized to the nucleus of virtually all embryonic and imaginal glial cells, with the exception of midline glia. Embryos mutant for the gene encoding RK2 are embryonic lethal but normal for early gliogenesis (birth, initial divisions and migration of glia) and axonogenesis (neuronal pathfinding and fasciculation). However, later in development, there are significantly fewer longitudinal glia that are spatially disorganized; in addition, there is a slight disorganization of axon fascicles and a defective nerve cord condensation. This suggests that RK2 is not required for early glial determination, but rather for aspects of glial differentiation or function that are required for embryonic viability.
We have examined the glial cell response, the possible expression of compounds associated with the complement cascade, including the putative complement inhibitor clusterin, and their cellular association during Wallerian degeneration in the central nervous system. Examination of the proliferation pattern revealed an overall greater mitotic activity after rhizotomy, an exclusive involvement of microglia in this proliferation after peripheral nerve injury, but, in addition, a small fraction of proliferating astrocytes after rhizotomy. Immunostaining with the phagocytic cell marker ED1 gradually became very prominent after rhizotomy, possibly reflecting a response to the extensive nerve fiber disintegration. Lumbar dorsal rhizotomy did not induce endogenous immunoglobulin G (IgG) deposition or complement expression in the spinal cord dorsal horn, dorsal funiculus, or gracile nucleus. This is in marked contrast to the situation after peripheral nerve injury, which appears to activate the entire complement cascade in the vicinity of the central sensory processes. Clusterin, a multifunctional protein with complement inhibitory effects, was markedly upregulated in the dorsal funiculus in astrocytes. In addition, there was an intense induction of clusterin expression in the degenerating white matter in oligodendrocytes, possibly reflecting a degeneration process in these cells. The findings suggest that 1) complement expression by microglial cells is intimately associated with IgG deposition; 2) axotomized neuronal perikarya, but not degenerating central fibers, undergo changes which induce such deposition; and 3) clusterin is not related to complement expression following neuronal injury but participates in regulating the state of oligodendrocytes during Wallerian degeneration.
The selective degeneration of an axon, without the death of the parent neuron, can occur in response to injury, in a variety of metabolic, toxic, and inflammatory disorders, and during normal development. Recent evidence suggests that some forms of axon degeneration involve an active and regulated program of self-destruction rather than a passive "wasting away" and in this respect and others resemble apoptosis. Here we investigate whether selective axon degeneration depends on some of the molecular machinery that mediates apoptosis, namely, the caspase family of cysteine proteases. We focus on two models of selective axon degeneration: Wallerian degeneration of transected axons and localized axon degeneration induced by local deprivation of neurotrophin. We show that caspase-3 is not activated in the axon during either form of degeneration, although it is activated in the dying cell body of the same neurons. Moreover, caspase inhibitors do not inhibit or retard either form of axon degeneration, although they inhibit apoptosis of the same neurons. Finally, we cannot detect cleaved substrates of caspase-3 and its close relatives immunocytochemically or caspase activity biochemically in axons undergoing Wallerian degeneration. Our results suggest that a neuron contains at least two molecularly distinct self-destruction programs, one for caspase-dependent apoptosis and another for selective axon degeneration.
All the higher mental and cognitive functions unique to humans depend on the neocortex ('new' cortex, referring to its relatively recent appearance in evolution), which is divided into discrete areas that subserve distinct functions, such as language, movement and sensation. With a few notable exceptions, all neocortical areas have six layers of neurons and a remarkably similar thickness and overall cell density, despite subtle differences in their cellular architecture. Furthermore, all neocortical areas are formed over roughly the same time period during development and provide little hint at early developmental stages of the rich functional diversity that becomes apparent as development comes to an end. How these areas are formed has long fascinated developmental neuroscientists, because the formation of new cortical areas, with the attendant appearance of new cortical functions, is what must have driven the evolution of mammalian behavior.
The prompt clearance of cells undergoing apoptosis is critical during embryonic development, normal tissue turnover, as well as inflammation and autoimmunity. The molecular details of the engulfment of apoptotic cells are not fully understood. ced-6 and its human homologue gulp, encode an adapter protein, whose function in engulfment is highly evolutionarily conserved; however, the upstream and downstream components of CED-6 mediated signaling are not known. Recently, ced-1 has been shown to encode a transmembrane protein on phagocytic cells, with two functional sequence motifs in its cytoplasmic tail that are important for engulfment. In this study, using a combination of biochemical approaches and yeast two-hybrid analysis, we present evidence for a physical interaction between GULP/CED-6 and one of the two motifs (NPXY motif) in the cytoplasmic tail of CED-1. The phosphotyrosine binding domain of GULP was necessary and sufficient for this interaction. Since the precise mammalian homologue of CED-1 is not known, we undertook a database search for human proteins that contain the motifs shown to be important for CED-1 function and identified CD91/LRP (low density lipoprotein receptor-related protein) as one candidate. Interestingly, recent studies have also identified CD91/LRP as a receptor involved in the phagocytosis of apoptotic cells in mammals. The GULP phosphotyrosine binding domain was able to specifically interact with one specific NPXY motif in the CD91 cytoplasmic tail. During these studies we have also identified the mouse GULP sequence. These studies suggest a physical link between CED-1 or CD91/LRP and the adapter protein CED-6/GULP during engulfment of apoptotic cells and further elucidate the pathway suggested by the genetic studies.
Neurons seem to have at least two self-destruct programs. Like other cell types, they have an intracellular death program
for undergoing apoptosis when they are injured, infected, or not needed. In addition, they apparently have a second, molecularly
distinct self-destruct program in their axon. This program is activated when the axon is severed and leads to the rapid degeneration
of the isolated part of the cut axon. Do neurons also use this second program to prune their axonal tree during development
and to conserve resources in response to chronic insults?
Differentiation of the Drosophila oocyte takes place in a cyst of 16 interconnected germ cells and is dependent on a network of microtubules that becomes polarized as differentiation progresses (polarization). We have investigated how the microtubule network polarizes using a GFP-tubulin construct that allows germ-cell microtubules to be visualized with greater sensitivity than in previous studies. Unexpectedly, microtubules are seen to associate with the fusome, an asymmetric germline-specific organelle, which elaborates as cysts form and undergoes complex changes during cyst polarization. This fusome-microtubule association occurs periodically during late interphases of cyst divisions and then continuously in 16-cell cysts that have entered meiotic prophase. As meiotic cysts move through the germarium, microtubule minus ends progressively focus towards the center of the fusome, as visualized using a NOD-lacZ marker. During this same period, discrete foci rich in gamma tubulin that very probably correspond to migrating cystocyte centrosomes also associate with the fusome, first on the fusome arms and then in its center, subsequently moving into the differentiating oocyte. The fusome is required for this complex process, because microtubule network organization and polarization are disrupted in hts(1) mutant cysts, which lack fusomes. Our results suggest that the fusome, a specialized membrane-skeletal structure, which arises in early germ cells, plays a crucial role in polarizing 16-cell cysts, at least in part by interacting with microtubules and centrosomes.
In the C57BL/WldS mouse, a dominant mutation dramatically delays Wallerian degeneration in injury and disease, possibly by influencing multi-ubiquitination. Studies on this mouse show that axons and synapses degenerate by active and regulated mechanisms that are akin to apoptosis. Axon loss contributes to neurological symptoms in disorders as diverse as multiple sclerosis, stroke, traumatic brain and spinal cord injury, peripheral neuropathies and chronic neurodegenerative diseases, but it has been largely neglected in neuroprotective strategies. Defects in axonal transport, myelination or oxygenation could trigger such mechanisms of active axon degeneration. Understanding how these diverse insults might initiate an axon-degeneration process could lead to new therapeutic interventions.
To accumulate information on the coding sequences of unidentified genes, we have carried out a sequencing project of human
cDNA clones which encode large proteins. We herein present the entire sequences of 100 cDNA clones of unidentified human genes,
named KIAA1776 and KIAA1780-KIAA1878, from size-fractionated cDNA libraries derived from human fetal brain, adult whole brain,
hippocampus and amygdala. Most of the cDNA clones to be entirely sequenced were selected as cDNAs which were shown to have
coding potentiality by in vitro transcription/translation experiments, and some clones were chosen by using computer-assisted analysis of terminal sequences
of cDNAs. Three of these clones (fibrillin3/KIAA1776, MEGF10/KIAA1780 and MEGF11/KIAA1781) were isolated as genes encoding proteins with multiple EGF-like domains by
motif-trap screening. The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here
reached 4.7 kb and 2.4 kb (785 amino acid residues), respectively. From the results of homology and motif searches against
the public databases, the functional categories of the predicted gene products of 54 genes were determined; 93% of these predicted
gene products (50 gene products) were classified as proteins related to cell signaling/communication, nucleic acid management,
or cell structure/motility. To collect additional information on these genes, their expression profiles were also studied
in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase
chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.
The role of Schwann cells (SC) during Wallerian degeneration has been controversial. The consensus opinion is that monocytes/macrophages are mainly responsible for myelin removal although SC were shown to phagocytose myelin in vitro and in vivo. In the present study, we correlate proto-oncogene expression with phenotypic changes in SC during Wallerian degeneration using immunohistochemistry and electron microscopy. The proliferative cells are labeled with proliferative cell nuclear antigen (PCNA), and macrophages are labeled with ED1 macrophage marker. We demonstrate c-fos expression in SC at the onset of axon disintegration 12 hours post-axotomy followed by expression of ED1, basic fibroblast growth factor (bFGF) and PCNA after 1 day. The myelin sheath fragments at the nodal region and forms ellipsoids. Schwann cells move to internodes and undergo nuclear and cytoplasmic hypertrophy; they phagocytose myelin ellipsoids with thick vimentin-rich processes and undergo mitosis. Resident macrophages express c-fos and phagocytose myelin debris sequestered into endoneurium by SC after 3 days, but they do not enter the tube until the fifth day. We believe that SC are induced by signals from injured axons to express c-fos which activates downstream genes that lead to the acquisition of phagocytic and proliferative activities. Myelin debris processed by SC may act as an inducer and a chemoattractant for resident macrophages. Cytokines produced by macrophages may stimulate SC proliferation and production of neurotrophic factors.
The cytokine leukemia inhibitory factor (LIF) favors the survival and growth of axons in vitro and in vivo. Fibronectin has been shown to enhance nerve regeneration when added in combination with various growth factors including LIF. The goal of this study was to evaluate the effect of LIF plus fibronectin on the regeneration of transected nerve and functional recovery of reinnervated skeletal muscle, in one experimental model of peripheral nerve repair, at two recovery times. The rat sciatic nerve was cut at mid-thigh level and a silicone cuff containing either saline (control), LIF, or LIF plus fibronectin (L + F) was used to bridge the proximal and distal nerve stumps leaving a 1 cm gap between them. Rats were then explored at 6 or 12 weeks following the initial surgery. Regenerating nerves were assessed by measuring the diameter of myelinated axons, conduction velocity, and number of myelinated fibers. Muscle reinnervation was assessed by measuring muscle mass, force of contraction, and histologically for changes in muscle fiber type (type I and type II). In this report we demonstrate that at 6 weeks there were significant increases in 1) nerve conduction velocity, 2) myelinated axon diameter, and 3) number of myelinated axons over that of control (saline-treated) animals. Both LIF groups demonstrated a shift in type II muscle fiber area compared to saline-treated controls, with the L + F group having a significant increase in muscle mass. At 12 weeks there was an improved recovery over and above that demonstrated at 6 weeks. Muscle mass was 65% and 42% greater than control for LIF and L + F, respectively. Force of contraction, conduction velocity, myelinated fiber number, and diameter were also significantly greater for both LIF- and L + F- treated rats than saline-treated rats. These results demonstrate that LIF significantly improves the regeneration of damaged peripheral nerves and the preservation of muscle viability, resulting in greatly enhanced recovery of skeletal muscle function. J. Neurosci. Res. 47:208–215, 1997. © 1997 Wiley-Liss, Inc.
Entorhinal lesion leads to anterograde degeneration of perforant path fibers in their main termination zone in the outer molecular layers of the dentate gyrus. Concomitantly, astrocytes become hypertrophic, and microglial cells alter their phenotype, suggesting participation in anterograde degeneration. This study analyzes the involvement of these lesion-induced activated glial cells in the process of phagocytosis of degenerated axonal debris. We established a phagocytosis-dependent labeling technique that allows for direct and simultaneous visualization of both labeled incorporated axonal debris and incorporating glial cells. Stereotaxic application of small crystals of the biotin- and rhodamine-conjugated dextran amine Mini Ruby (MR) into the entorhinal cortex led to strong and stable axonal staining of perforant path axons. Following entorhinal lesion, labeled terminals and fibers condensed and formed small granules. Incorporation of these rhodamine-fluorescent granules resulted in a phagocytosis-dependent cell labeling. During the first 3 days, we were able to identify these cells as microglia by using double-fluorescence and confocal microscopy. The first unequivocally double-labeled astrocytes were found 6 days post lesion (dpl). Whereas in all stages a subpopulation of microglial cells remained devoid of MR-labeled granules, all astrocytes in the middle molecular layer were double-labeled after long survival times (20 dpl). On the ultrastructural level, labeled granules appeared to be perforant path axons containing the tracer. Both terminals and myelinated fibers could be seen inside the cytoplasm of microglial cells and astrocytes. Thus, anterograde degeneration is a sufficient stimulus to induce axon incorporation by both astrocytes and a subpopulation of microglial cells. GLIA 20:145–154, 1997. © 1997 Wiley-Liss Inc.
Glial fibrillary acidic protein (GFAP) immunocytochemistry was used to monitor the response of astrocytes in the rat spinal cord to either dorsal root or sciatic nerve lesions. Image analysis methods were used to provide a quantitative correlate of the reactive gliosis. Multiple dorsal root section elicited a rapid increase in GFAP immunoreactivity of astrocytes unilaterally within the spinal cord along the pathway of the degenerating dorsal root axons in the dorsal and ventral horns and this gliosis persisted in the dorsal horn beyond the time at which active phagocytosis of degenerative debris occurred. Labeling of proliferating cells using [3H]thymidine revealed that none of the dividing cells contained detectable GFAP, suggesting that the increased GFAP labeling represents primarily a hypertrophy rather than a proliferation of astrocytes. Comparison of animals that had been deafferented in the early neonatal period with those deafferented as adults indicated that the GFAP immunoreactive response persisted following neonatal lesions but that it was markedly less intense than after adult lesions. Sciatic nerve section in adults does not result in extensive frank degeneration but it does evoke a rapid and marked increase in staining of astrocytes both in the dorsal horn and in the ventral horn. Transganglionic changes in GFAP staining in the dorsal horn occur by 3 days post-operatively, which is much earlier than the time of dorsal root ganglion neuron death caused by the sciatic nerve lesion. These experiments indicate that astrocytes can respond to signals from a variety of changes in neurons, including not only Wallerian degeneration, but also retrograde and transganglionic changes.
The crush-injured sciatic nerve provides a model to study Schwann cell regulation of myelin gene expression during the process of demyelination and remyelination. In order to investigate the possible transcriptional regulation of myelin gene expression, the quantity, quality and translational efficiency of PO (the major myelin glycoprotein) and MBP (the myelin basic proteins) coding messages were investigated as a function of time following crush-injury of the adult rat sciatic nerve. Northern blot analysis indicated that the size of the PO and MBP transcripts remain unchanged in the distal segments of crushed sciatic nerves at 1, 2, 4, 7, 10, 14 and 21 days after crush-injury. Dot-blot analysis showed a sharp drop in levels of PO and MBP coding transcripts 1 day after crush-injury with the lowest steady-state levels at 4-7 days. Message levels were found to increase after 7 days, the highest increase in levels of message was found to be between 10 and 14 days. The highest steady-state level of both transcripts was observed at 21 days. In vitro translation and immunoprecipitation of PO-translated products from various stages of crush-injury also indicated this trend. The pattern of gene expression of PO- and MBP-coding transcripts parallel each other and follow the pattern of demyelination and remyelination. The results are also consistent with our previous interpretation which suggests that PO and MBP gene expression is regulated at the level of transcription and that these two genes might be coordinately expressed. Western blot analysis of PO protein from these stages revealed a similar decrease and then increase in the levels of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Three types of hairs were identified on the maxillary palp of Drosophila melanogaster Meigen (Diptera : Drosophilidae): (i) single-walled, multiporous sensilla basiconica, which constitute 75% of the innervated hairs; (ii) thick walled non-porous sensilla trichodea, which make up the remaining 25% of the innervated hairs; and (iii) numerous spinules, which are un-innervated. These sensilla basiconica uniformly contain 2 bipolar sense cells, whereas sensilla trichodea have a single dendrite with a tubular body at the base of each hair. A majority of the sensilla basiconica is located on the distal half of the dorsal surface, whereas sensilla trichodea are positioned on the tip and entire ventrolateral ridge of the palp. Approximately 125 axons of the sense cells join to form a single nerve. The structure of sensilla basiconica and sensilla trichodea suggests that they are olfactory and mechanosensory respectively. The contact chemoreceptors (gustatory sensilla) are conspicuously absent on the maxillary palp.Golgi silver impregnations and cobalt fills show that the primary sensory fibres from sensilla trichodea and sensilla basiconica on the maxillary palp project in the posterior suboesophageal ganglion (SOG) and the antennal lobe respectively. A single fibre projects separately either in the SOG or in the antennal lobe. In the antennal lobe, the input received from sensilla basiconica is usually bilateral and at least 5 glomeruli are innervated symmetrically on either side from both the palps.This study suggests that the sensory neurons are capable of making selective projections in the specific regions of the brain. Accordingly, the fibres from a sensillum project to the brain with respect to their functions and the individual glomeruli represent functional units of the brain, receiving inputs in a characteristic combination.
Light microscopical degeneration and ultrastructural alterations in the rat spinal dorsal horn were studied following either cutting of the sciatic nerve or rhizotomy at L4 and L5; survival time for both procedures was 3 weeks. Fink-Heimer silver methods showed minimal degeneration of afferent central processes after sciatic section, and limited ultrastructural changes were present. Both rhizotomy and nerve section resulted in degenerating terminals. Most were swollen and electron lucent, with loss of vesicles; some electron-dense terminals were present, particularly after rhizotomy. Both procedures also produced significant degeneration of postsynaptic dendrites and soma, evidenced by either increases in electron density, or loss of organelles and cavitation, or accumulation of osmiophilic floccular material. Glial processes frequently were expanded and extended to engulf single degenerating terminals and dendrites, or terminal-dendrite units; in other cases glial tongues separated terminals from their postsynaptic dendrite. Glial processes often wrapped around degenerating profiles or groups of profiles in several layers, sometimes forming complex labyrinths. These results confirm past descriptions of pre- and postsynaptic changes resulting from peripheral nerve section, but newly reveal that dendritic destruction and increased glial activity are also significant following rhizotomy. Documentation of these changes is relevant for studies of reorganization following nerve and spinal cord damage, as well as providing an ultrastructural basis for evaluation of effects of neurotoxins that affect primary afferents, as described in a companion paper.
Studies of deafferentation and regeneration, as well as studies requiring several tracing techniques, would benefit from availability of a substance that would selectively lesion the central components of a single peripheral nerve. Pronase, a combination of proteolytic enzymes, was tested for this purpose.
Three weeks following microinjection of Pronase (5–25 mg) into the rat sciatic nerve, many ganglia cells in the L3-L5 ganglia were degenerated. Degeneration of primary afferents also was evident in the dorsal horn, as detected by silver Fink-Heimer methods. Patterns of terminal fields coincided with those mapped in normal rats for the sciatic nerve by using HRP transport.
Ultrastructural changes were similar to those seen at 3 weeks following sciatic nerve section or rhizotomy, as described in our companion paper. However, degenerative changes following Pronase injection of the sciatic nerve were quantitatively greater than those following sciatic nerve section alone. Degenerating terminals were either electron lucent and swollen, electron dense, or filamentous with loss of vesicles. Postsynaptic dendrites, and occasionally somata, also showed signs of degeneration. Some became electron dense, others accumulated osmiophilic floccular material, but most became electron lucent and developed large membrane-bound cavities. Glial processes expanded around degenerating elements, wrapping around both terminals and dendrites. Glial sheets covered denervated dendritic and somatic spines, separating them from their terminals. Labyrinth formations of glial sheaths around debris were also found. Pronase appears to mimic the effects of mechanical destruction of primary afferents, but when compared to rhizotomy, is selective for the afferents of a single nerve, and, when compared to nerve section, produces a greater effect. Further, the substance is relatively safe for investigators compared to other toxins such as ricin.
Using mAbs and immunocytochemistry we have examined the response of macrophages (M phi) after crush injury to the sciatic or optic nerve in the mouse and rat. We have established that large numbers of M phi enter peripheral nerves containing degenerating axons; the M phi are localized to the portion containing damaged axons, and they phagocytose myelin. The period of recruitment of the M phi in the peripheral nerve is before and during the period of maximal proliferation of the Schwann cells. In contrast, the degenerating optic nerve attracts few M phi, and the removal of myelin is much slower. These results show the clearly different responses of M phi to damage in the central and peripheral nervous systems, and suggest that M phi may be an important component of subsequent repair as well as myelin degradation.
Leukemia inhibitory factor (LIF) is a cytokine that affects the survival and differentiation of certain neuronal populations in vitro. To identify LIF-responsive neurons in the adult rat, we have demonstrated retrograde axonal transport of 125I-LIF to sensory and motor neurons. The accumulation of 125I-LIF by both cell types was significantly increased by prior sciatic nerve crush. Retrograde transport of 125I-LIF was inhibited by excess unlabeled LIF but not by related cytokines, indicating a specific receptor-mediated mechanism. Northern blot analysis revealed LIF expression in peripheral nerve that was increased in distal segments after axotomy. The correlation between LIF expression and increased retrograde transport following injury suggests that LIF plays a role in peripheral nerve regeneration.
The role of Schwann cells (SC) during Wallerian degeneration has been controversial. The consensus opinion is that monocytes/macrophages are mainly responsible for myelin removal although SC were shown to phagocytose myelin in vitro and in vivo. In the present study, we correlate proto-oncogene expression with phenotypic changes in SC during Wallerian degeneration using immunohistochemistry and electron microscopy. The proliferative cells are labeled with proliferative cell nuclear antigen (PCNA), and macrophages are labeled with ED1 macrophage marker. We demonstrate c-fos expression in SC at the onset of axon disintegration 12 hours post-axotomy followed by expression of ED1, basic fibroblast growth factor (bFGF) and PCNA after 1 day. The myelin sheath fragments at the nodal region and forms ellipsoids. Schwann cells move to internodes and undergo nuclear and cytoplasmic hypertrophy; they phagocytose myelin ellipsoids with thick vimentin-rich processes and undergo mitosis. Resident macrophages express c-fos and phagocytose myelin debris sequestered into endoneurium by SC after 3 days, but they do not enter the tube until the fifth day. We believe that SC are induced by signals from injured axons to express c-fos which activates downstream genes that lead to the acquisition of phagocytic and proliferative activities. Myelin debris processed by SC may act as an inducer and a chemoattractant for resident macrophages. Cytokines produced by macrophages may stimulate SC proliferation and production of neurotrophic factors.
Acute inflammation plays an important role in host tissue defense against injury and infection, and also subsequent tissue repair. In the central nervous system parenchyma, following many types of insults, the acute inflammatory response to rapid neuronal degeneration or challenge with inflammatory substances differs dramatically from that of other tissues. The rapid recruitment of neutrophils is virtually absent and monocytes are only recruited after a delay of several days. It appears that the microenvironment of the central nervous system has evolved mechanisms to protect it from the potentially damaging consequences of some aspects of the acute inflammatory response.
We report the identification of a Drosophila locus, reversed polarity (repo). Weak repo alleles were viable but affected glia in the optic lobe, resulting in a reversal in polarity of the electrophysiological to light in the adult. Strong repo alleles caused defects in embryonic glia and resulted in embryonic lethality. Expression of repo appeared to be specific to glia throughout development. In the adult visual system, repo was expressed in laminal glia, medullar glia, and subretinal cells; in the embryo, repo was expressed in nearly all of the identified glia in the central and peripheral nervous systems except midline glia. The repo gene encoded a homeo domain protein suggesting that it might be a transcriptional regulator of genes required for glial development.
During the development of the nervous system extensive programmed neuronal death occurs that is regulated by neurotrophic factors. Invariably, degeneration and death of the neuronal soma as a result of trophic factor deprivation is accompanied by concurrent degeneration of the neurites. By examining the degeneration of sympathetic neurons after deprivation of their physiological trophic factor nerve growth factor, we show that the "slow Wallerian degeneration" allele (Wld6) expressed by homozygous mutant C57BL/Ola mice alters the normal time course of programmed neuronal death by selectively and dramatically delaying the onset of neurite disintegration. In contrast, degenerative events affecting the neuronal soma are not altered: Atrophy of the soma, apoptotic disintegration of the nucleus, commitment to die, and loss of viability occur normally. The enucleate neurites remaining after death of the soma have an intact plasma membrane, are metabolically active, and require an active metabolism for physical integrity. We suggest that the degeneration of neurites during developmentally occurring neuronal death is controlled by events confined to the neurites and occurs autonomously from the neuronal soma. Furthermore, programmed neuronal death of the soma proceeds independent from any influence exerted by degenerating neurites.
Transected axons in C57BL/Ola mice survive for extraordinary lengths of time as compared to those of normal rodents. The biological difference in the substrain that confers the phenotype of prolonged axonal survival is unknown. Previous studies suggest that 'defect' to be a property of the nervous system itself, rather than one of haematogenous cells. Neuronal or non-neuronal elements could be responsible for this phenotype. This study was undertaken to determine whether Schwann cells, the most numerous of the non-neuronal cells intrinsic to the peripheral nerve, are responsible for delayed degeneration of transected axons. We created sciatic nerve chimeras by transplanting nerve segments between standard C57BL/6 and C57BL/Ola mice, allowing regeneration of host axons through the grafts containing donor Schwann cells. These nerves were then transected and the time course of axonal degeneration was observed. The results show that fast or slow degeneration is a property conferred by the host, and therefore cannot be ascribed to the Schwann cells. Similarly, transected C57BL/Ola axons in explanted dorsal root ganglia cultures survived longer than transected axons from standard mice. Taken together these results indicate that the responsible abnormality is intrinsic to the C57BL/Ola axon.
Leukaemia inhibitory factor (LIF) and Interleukin-6 (IL-6) are multifunctional cytokines that are related on the basis of their predicted structural similarities and shared signal transducing receptor components. Both these factors stimulate myoblast proliferation, and whereas LIF is neurotrophic for sensory neurons, and for the motor neurons which innervate muscle, IL-6 has only been reported to act on a population of septal neurons in the brain. We have looked at the effect of peripheral nerve trauma on the expression of these factors. We show here that whereas LIF and IL-6 mRNAs are expressed in low levels in normal sciatic nerve and skeletal muscle, there is significant up-regulation in the nerve segments after injury, with proximally and distally. There is also an increase in LIF and IL-6 expression in the denervated muscle located largely in the muscle cells. In addition, while there is retrograde axonal transport of LIF by the sciatic nerve, IL-6 is not retrogradely transported, and as a result, IL-6 does not stimulate the survival of sensory neurons in vitro. Both growth factors are produced by Schwann cells. These results show a rapid response in the expression of these genes after injury and suggest that LIF and IL-6 act as trauma factors but with different roles in injured peripheral nerve.
Reactive changes in macrophages/microglia and astrocytes were evaluated following spinal cord injury in normal mice of the C57BL/6J strain and in mice carrying a mutation (WldS) which delays the onset of Wallerian degeneration in damaged axons. Crush injuries were produced at the T8 level by using an extradural approach; animals were allowed to survive for 2 days to 12 weeks, and spinal cords were prepared for immunocytochemistry using antibodies against Mac1 and glial fibrillary acidid protein (GFAP). In normal mice, Mac1-positive macrophages accumulated at the injury site by 4 days and immunostaining of these cells peaked at 6-8 days. Cells in the gray matter near the crush site and in the ascending dorsal column also exhibited increased Mac1 staining that was prominent at 1 week and remained high at 2-4 weeks. In mice carrying the WldS mutation, the accumulation of macrophages at the injury site and the increase in immunostaining of these cells were delayed, as were the increases in immunostaining in the gray matter and dorsal columns. Both normal and mutant mice exhibited pronounced increases in glial fibrillary acidic protein immunostaining at the edge of the crush site and for some distance both rostral and caudal to the injury; increased immunostaining was also prominent along the ascending dorsal columns. The center of the crush site, which contained connective tissue, remained completely unstained for GFAP. In normal mice, immunostaining for GFAP reached a peak at 1 week postinjury and then declined. In mice carrying the WldS mutation, increases in GFAP immunostaining did not reach a peak until 2-3 weeks postinjury. These results indicate that activation of macrophages, microglia, and astrocytes is delayed and prolonged in mice carrying the WldS mutation.
The cytokine leukemia inhibitory factor (LIF) favors the survival and growth of axons in vitro and in vivo. Fibronectin has been shown to enhance nerve regeneration when added in combination with various growth factors including LIF. The goal of this study was to evaluate the effect of LIF plus fibronectin on the regeneration of transected nerve and functional recovery of reinnervated skeletal muscle, in one experimental model of peripheral nerve repair, at two recovery times. The rat sciatic nerve was cut at mid-thigh level and a silicone cuff containing either saline (control), LIF, or LIF plus fibronectin (L+F) was used to bridge the proximal and distal nerve stumps leaving a 1 cm gap between them. Rats were then explored at 6 or 12 weeks following the initial surgery. Regenerating nerves were assessed by measuring the diameter of myelinated axons, conduction velocity, and number of myelinated fibers. Muscle reinnervation was assessed by measuring muscle mass, force of contraction, and histologically for changes in muscle fiber type (type I and type II). In this report we demonstrate that at 6 weeks there were significant increases in 1) nerve conduction velocity, 2) myelinated axon diameter, and 3) number of myelinated axons over that of control (saline-treated) animals. Both LIF groups demonstrated a shift in type II muscle fiber area compared to saline-treated controls, with the L+F group having a significant increase in muscle mass. At 12 weeks there was an improved recovery over and above that demonstrated at 6 weeks. Muscle mass was 65% and 42% greater than control for LIF and L+F, respectively. Force of contraction, conduction velocity, myelinated fiber number, and diameter were also significantly greater for both LIF- and L+F-treated rats than saline-treated rats. These results demonstrate that LIF significantly improves the regeneration of damaged peripheral nerves and the preservation of muscle viability, resulting in greatly enhanced recovery of skeletal muscle function.
Axon injury rapidly activates microglial and astroglial cells close to the axotomized neurons. Following motor axon injury, astrocytes upregulate within hour(s) the gap junction protein connexin-43, and within one day glial fibrillary acidic protein (GFAP). Concomitantly, microglial cells proliferate and migrate towards the axotomized neuron perikarya. Analogous responses occur in central termination territories of peripherally injured sensory ganglion cells. The activated microglia express a number of inflammatory and immune mediators. When neuron degeneration occurs, microglia act as phagocytes. This is uncommon after peripheral nerve injury in the adult mammal, however, and the functional implications of the glial cell responses in this situation are unclear. When central axons are injured, the glial cell responses around the affected neuron perikarya appears to be minimal or absent, unless neuron degeneration occurs.
Recent observations have provided new insight into neuronal responses to axotomy, signalling of the Schwann cell switch from 'operating' to 'proliferation' mode and temporal molecular changes in the responsiveness of Schwann cells to neuronal signals, as well as into the role of macrophages in Wallerian degeneration, nerve repair and neuropathic pain. Furthermore, promising therapeutic interventions have been developed to promote axon regeneration and to attenuate axotomy-induced neuronal cell death by means of pharmacological treatment or application of neurotrophic proteins using various strategies and routes of delivery.
Axotomy or crush of a peripheral nerve leads to degeneration of the distal nerve stump referred to as Wallerian degeneration (WD). During WD a microenvironment is created that allows successful regrowth of nerve fibres from the proximal nerve segment. Schwann cells respond to loss of axons by extrusion of their myelin sheaths, downregulation of myelin genes, dedifferentiation and proliferation. They finally aline in tubes (Büngner bands) and express surface molecules that guide regenerating fibres. Hematogenous macrophages are rapidly recruited to the distal stump and remove the vast majority of myelin debris. Molecular changes in the distal stump include upregulation of neurotrophins, neural cell adhesion molecules, cytokines and other soluble factors and their corresponding receptors. Axonal injury not only induces muscle weakness and loss of sensation but also leads to adaptive responses and neuropathic pain. Regrowth of nerve fibres occurs with high specificity with formerly motor fibres preferentially reinnervating muscle. This involves recognition molecules of the L2/HNK-1 family. Nerve regeneration occurs at a rate of 3-4 mm/day after crush and 2-3 mm/day after sectioning a nerve. Nerve regeneration can be fostered pharmacologically. Upon reestablishment of axonal contact Schwann cells remyelinate nerve sprouts and downregulate surface molecules characteristic for precursor/premyelinating or nonmyelinating Schwann cells. At present it is unclear whether axonal regeneration after nerve injury is impeded in neuropathies.
Damage to the central nervous system (CNS) elicits the activation of both astrocytes and microglia. This review is focused on the principal features that characterize the activation of microglia after CNS injury. It provides a critical discussion of concepts regarding microglial biology that include the relationship between microglia and macrophages, as well as the role of microglia as immunocompetent cells of the CNS. Mechanistic and functional aspects of microgliosis are discussed primarily in the context of microglial neuronal interactions. The controversial issue of whether reactive microgliosis is a beneficial or a harmful process is addressed, and a resolution of this dilemma is offered by suggesting different interpretations of the term 'activated microglia' depending on its usage during in vivo or in vitro experimentation.
We have isolated the "complete" repertoire of genes encoding the odorant receptors in Drosophila and employ these genes to provide a molecular description of the organization of the peripheral olfactory system. The repertoire of Drosophila odorant receptors is encoded by 57 genes. Individual sensory neurons are likely to express only a single receptor gene. Neurons expressing a given gene project axons to one or two spatially invariant glomeruli in the antennal lobe. The insect brain therefore retains a two-dimensional map of receptor activation such that the quality of an odor may be encoded by different spatial patterns of activity in the antennal lobe.
Differentiation of the Drosophila oocyte takes place in a cyst of 16 interconnected germ cells and is dependent on a network of microtubules that becomes polarized as differentiation progresses (polarization). We have investigated how the microtubule network polarizes using a GFP-tubulin construct that allows germ-cell microtubules to be visualized with greater sensitivity than in previous studies. Unexpectedly, microtubules are seen to associate with the fusome, an asymmetric germline-specific organelle, which elaborates as cysts form and undergoes complex changes during cyst polarization. This fusome-microtubule association occurs periodically during late interphases of cyst divisions and then continuously in 16-cell cysts that have entered meiotic prophase. As meiotic cysts move through the germarium, microtubule minus ends progressively focus towards the center of the fusome, as visualized using a NOD-lacZ marker. During this same period, discrete foci rich in gamma tubulin that very probably correspond to migrating cystocyte centrosomes also associate with the fusome, first on the fusome arms and then in its center, subsequently moving into the differentiating oocyte. The fusome is required for this complex process, because microtubule network organization and polarization are disrupted in hts(1) mutant cysts, which lack fusomes. Our results suggest that the fusome, a specialized membrane-skeletal structure, which arises in early germ cells, plays a crucial role in polarizing 16-cell cysts, at least in part by interacting with microtubules and centrosomes.
Patterning of the antennal lobe of adult Drosophila occurs through a complex interaction between sensory neurons, glia, and central neurons of larval and adult origin. Neurons from the olfactory sense organs are organized into distinct fascicles lined by glial cells. The glia originate from one of the three types of sensory lineages-specified by the proneural gene atonal. Gain-of-function as well as loss-of-function analysis validates a role for cells of the Atonal lineage in the ordered fasciculation of sensory neurons. Upon entry of the antennal nerve to central regions, sensory neurons at first remain closely associated with central glia which lie around the periphery of the lobe anlage. Coincident with the arrival of sensory neurons into the brain, glial precursors undergo mitosis and neural precursors expressing Dachshund appear around the lobe. Sensory neurons and glial cells project into the lobe at around the same time and are likely to coordinate the correct localization of different glomeruli. The influence of sensory neurons on the development of the olfactory lobe could serve to match and lock peripheral and central properties important for the generation of olfactory behavior.
In our series of human cDNA projects for accumulating sequence information on the coding sequences of unidentified genes, we herein present the entire sequences of 100 cDNA clones of unidentified genes, named KIAA1544 to KIAA1643, from two sets of size-fractionated human adult and fetal brain cDNA libraries. The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here reached 4.6 kb and 2.8 kb (930 amino acid residues), respectively. By computer-assisted database search of the deduced amino acid sequences, 48 predicted gene products were classified into the five functional categories of proteins relating to cell signaling/communication, nucleic acid management, cell structure/motility, protein management and metabolism. Homology search against the databases for proteins deduced from yeast, nematode and fly full genome sequences revealed only one gene (KIAA1630) was entirely conserved among human and these three organisms in the 100 genes reported here. Additionally, their chromosomal loci were determined by using human-rodent hybrid panels unless they were already assigned in the public databases. Furthermore, the expression profiles of the genes were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.
We cloned the C. elegans gene ced-1, which is required for the engulfment of cells undergoing programmed cell death. ced-1 encodes a transmembrane protein similar to human SREC (Scavenger Receptor from Endothelial Cells). We showed that ced-1 is expressed in and functions in engulfing cells. The CED-1 protein localizes to cell membranes and clusters around neighboring cell corpses. CED-1 failed to cluster around cell corpses in mutants defective in the engulfment gene ced-7. Motifs in the intracellular domain of CED-1 known to interact with PTB and SH2 domains were necessary for engulfment but not for clustering. Our results indicate that CED-1 is a cell surface phagocytic receptor that recognizes cell corpses. We suggest that the ABC transporter CED-7 promotes cell corpse recognition by CED-1, possibly by exposing a phospholipid ligand on the surfaces of cell corpses.
To accumulate information on the coding sequences of unidentified genes, we have carried out a sequencing project of human cDNA clones which encode large proteins. We herein present the entire sequences of 100 cDNA clones of unidentified human genes, named KIAA1776 and KIAA1780-KIAA1878, from size-fractionated cDNA libraries derived from human fetal brain, adult whole brain, hippocampus and amygdala. Most of the cDNA clones to be entirely sequenced were selected as cDNAs which were shown to have coding potentiality by in vitro transcription/translation experiments, and some clones were chosen by using computer-assisted analysis of terminal sequences of cDNAs. Three of these clones (fibrillin2/KIAA1776, MEGF10/KIAA1780 and MEGF11/KIAA1781) were isolated as genes encoding proteins with multiple EGF-like domains by motif-trap screening. The average sizes of the inserts and corresponding open reading frames of eDNA clones analyzed here reached 4.7 kb and 2.4 kb (785 amino acid residues), respectively. From the results of homology and motif searches against the public databases, the functional categories of the predicted gene products of 54 genes were determined; 93% of these predicted gene products (50 gene products) were classified as proteins related to cell signaling/communication, nucleic acid management, or cell structure/motility. To collect additional information on these genes, their expression profiles were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.
Wallerian degeneration of the distal stump of a severed peripheral nerve involves invasion by myelomonocytic cells, whose presence is necessary for destruction of myelin and for initiating mitosis in Schwann cells (Beuche and Friede, 1984). Degeneration of the distal ends of the axons themselves is assumed to occur by autolytic mechanisms. We describe a strain of mice (C57BL/6/Ola) in which leucocyte invasion is slow and sparse. In these mice, confirming Beuche and Friede, myelin removal is extremely slow. A new finding is that axon degeneration is also very slow. This is a consequence of lack of recruitment of myelomonocytic cells for if such recruitment is prevented in other mouse strains by a monoclonal antibody against the complement type 3 receptor (Rosen and Gordon, 1987) axon degeneration is again slowed. We have also, surprisingly, found that nerve regeneration in the C57BL/6/Ola mice is not impeded by the presence of largely intact axons in the distal stump and absence of recruited cells, myelin debris and the absence of Schwann cell mitosis.
Inflammation is a defense reaction against diverse insults, designed to remove noxious agents and to inhibit their detrimental effects. It consists of a dazzling array of molecular and cellular mechanisms and an intricate network of controls to keep them in check. In neurodegenerative diseases, inflammation may be triggered by the accumulation of proteins with abnormal conformations or by signals emanating from injured neurons. Given the multiple functions of many inflammatory factors, it has been difficult to pinpoint their roles in specific (patho)physiological situations. Studies of genetically modified mice and of molecular pathways in activated glia are beginning to shed light on this issue. Altered expression of different inflammatory factors can either promote or counteract neurodegenerative processes. Since many inflammatory responses are beneficial, directing and instructing the inflammatory machinery may be a better therapeutic objective than suppressing it.
In the C57BL/Wld(S) mouse, a dominant mutation dramatically delays Wallerian degeneration in injury and disease, possibly by influencing multi-ubiquitination. Studies on this mouse show that axons and synapses degenerate by active and regulated mechanisms that are akin to apoptosis. Axon loss contributes to neurological symptoms in disorders as diverse as multiple sclerosis, stroke, traumatic brain and spinal cord injury, peripheral neuropathies and chronic neurodegenerative diseases, but it has been largely neglected in neuroprotective strategies. Defects in axonal transport, myelination or oxygenation could trigger such mechanisms of active axon degeneration. Understanding how these diverse insults might initiate an axon-degeneration process could lead to new therapeutic interventions.
We investigate how the molecular and cellular maps of the Drosophila olfactory system are integrated. A correspondence is established between individual odor receptors, neurons, and odors. We describe the expression of the Or22a and Or22b receptor genes, show localization to dendritic membranes, and find sexual dimorphism. Or22a maps to the ab3A neuron, which responds to ethyl butyrate. Analysis of a deletion mutant lacking Or22a, along with transgenic rescue experiments, confirms the mapping and demonstrates that an Or gene is required for olfactory function in vivo. Ectopic expression of Or47a in a mutant cell identifies the neuron from which it derives and its odor ligands. Ectopic expression in a wild-type cell shows that two receptors can function in a single cell. The ab3A neuron does not depend on normal odor receptor gene expression to navigate to its target in the CNS.
CNS myelin inhibits axonal outgrowth in vitro and is one of several obstacles to functional recovery following spinal cord injury. Central to our current understanding of myelin-mediated inhibition are the membrane protein Nogo and the Nogo-66 receptor (NgR). New findings implicate NgR as a point of convergence in signal transduction for several myelin-associated inhibitors. Additional studies have identified a potential coreceptor for NgR as p75(NTR), and a second-messenger pathway involving RhoA that inhibits neurite elongation. Although these findings expand our understanding of the molecular determinants of adult CNS axonal regrowth, the physiological roles of myelin-associated inhibitors in the intact adult CNS remain ill-defined.