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With a little help from my friends: how intercellular communication shapes neuronal remodeling
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ScienceDirect With a little help from my friends: how intercellular communication shapes neuronal remodeling Hagar Meltzer and Oren Schuldiner Developmental neuronal remodeling shapes the mature connectivity of the nervous system in both vertebrates and invertebrates. Remodeling often combines degenerative and regenerative events, and defects in its normal progression have been linked to neurological disorders. Here we review recent progress that highlights the roles of cell–cell interactions during remodeling. We propose that these are fundamental to elucidating how spatiotemporal control of remodeling and coordinated circuit remodeling are achieved. We cover examples spanning various neuronal circuits in vertebrates and invertebrates and involving interactions between neurons and different cell types. Address Dept of Molecular Cell Biology, Weizmann Institute of Science, Israel Corresponding authors: Meltzer, Hagar ([email protected]), Schuldiner, Oren ([email protected])
Current Opinion in Neurobiology 2020, 63:xx–yy This review comes from a themed issue on Cellular neuroscience Edited by Thomas Schwarz and Holly Cline
https://doi.org/10.1016/j.conb.2020.01.018 0959-4388/ã 2020 Elsevier Ltd. All rights reserved.
instance, glia closely associate with neurons throughout the nervous system, while PNS neurons intermingle with the tissue they inhabit. So far, most studies of neuronal remodeling have focused on cell-autonomous processes, such as cytoskeletal organization and degradation pathways [2,6–8]. While the surrounding cells were never ignored, advances were relatively modest. Several recent studies began to shift the focus back towards cell–cell communication. An emerging theme from these studies suggests that a deeper understanding of circuit remodeling, and specifically its precise spatiotemporal control, must integrate both neuronal intrinsic processes and dynamic interactions with the environment. In this review we explore the roles of cell–cell interactions during neuronal remodeling and circuit maturation. We relate to various aspects of cell communication in diverse neural systems and model organisms, and raise open questions in the field. We review the existing literature based on the type of interaction between the communicating cells; however, this is mainly for convenience, as the different interaction types are not mutually exclusive, and may heavily depend on one another. Importantly, we aim to focus on recent findings, and do not cover all available literature. Topics that are extensively reviewed elsewhere were excluded from the scope of this review, as mentioned below.
Neuronal activity coordinates remodeling Introduction Precise connectivity of mature nervous systems depends on regressive and progressive events, often including elimination of exuberant connections followed by regrowth to adult-specific targets. Collectively known as developmental neuronal remodeling, such processes are used to refine neural circuits throughout evolution [1,2]. Remodeling sculpts central and peripheral nervous systems (CNS and PNS, respectively), and occurs in different scales — from pruning of single synapses or short axonal segments (normally by retraction), to elimination of long axonal stretches or entire dendritic trees (generally via local fragmentation). Defects in remodeling have been associated with neurodevelopmental disorders such as autism and schizophrenia [3–5]. A remodeling neuron is often part of a complex tissue encompassing both neurons and non-neuronal cells. For www.sciencedirect.com
Interaction via chemical synapses is probably the most unique mode of inter-neuronal communication, and not surprisingly, plays important roles during remodeling in both vertebrates and invertebrates. In mammals, classic examples of activity-dependent remodeling include the development of the neuromuscular junction (NMJ), in which motor axons compete for muscle fiber innervation, as well as the refinement of the cerebellar climbing fiber (CF) circuit, in which several CFs compete for the innervation of a single Purkinje cell (PC). In both processes [thoroughly reviewed elsewhere; 9–11] refinement involves strengthening of ‘winners’ and pruning of ‘losers’ to promote transition from poly-innervation to mono-innervation. Specifically, in the refinement of the CF-PC circuit, elimination of CF synapses requires, at different stages, both GABAergic inhibition [originating in basket cells; 12] as well as glutamatergic signaling [from parallel fibers; 13,14] onto PC somata. Notably, while this process essentially involves neuron–neuron interactions, Nakayama et al. recently suggested that microglia promote GABAergic Current Opinion in Neurobiology 2020, 63:1–8
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inhibition on PCs, in a phagocytosis-independent mechanism whose precise nature remains to be uncovered [15]. In Drosophila, olfactory sensory neurons (OSNs) synapse onto discrete glomeruli to form elaborate sensory maps [16]. Early adult sensory experience was reported to induce non-stereotypic remodeling to expand glomerulus innervation by the activated OSN [17]. However, Golovin et al. recently demonstrated an opposite effect, in which activation of a specific OSN led to volume reduction in its corresponding glomerulus due to synapse elimination [18]. This contradiction resembles the apparent conflict in the CF-PC circuit, in which both inhibitory and excitatory signals are required within the same cell to promote synapse pruning. Thus, more studies are required to dissect the precise relations between activity and pruning in different cellular contexts. Particularly fascinating is the study of neuronal networks that remodel together. In the mammalian retinogeniculate circuit, extensive remodeling refines initially overlapping projections to form segregated eye-specific domains. Retinal ganglion cells (RGCs) transmit retinal input to the dorsal lateral geniculate nucleus (dLGN) in the thalamus, which is mainly comprised of relay cells that project to the visual cortex and thalamic interneurons. During early postnatal stages, RGCs locally prune their axon arbors to generate eye-specific domains within the dLGN, in an activity-dependent manner [19]. Remarkably, two recent studies demonstrated that retinal activity also drives the remodeling of relay cells and interneurons. In the absence of retinal input, relay cells demonstrate reduced dendritic surface area within the dLGN [20], and interneurons fail to branch and thus maintain a simple architecture [21]. Coordinated circuit remodeling is also evident in the Drosophila mushroom body (MB), a complex CNS neuropil, in which one neuronal type, the g-Kenyon Cells (g-KCs), undergo stereotypic remodeling during metamorphosis, including pruning of larval axons followed by regrowth of adult-specific axons [8]. g-KCs receive input from projection neurons (PNs), and modulatory signals mainly from GABAergic and dopaminergic neurons (DANs). Interestingly, embryonically born PNs and the GABAergic anterior paired lateral (APL) neuron undergo developmental remodeling in a similar timeframe as g-KCs [22,23]. In the case of the APL, Mayseless et al. used advanced genetic tools, which allow simultaneous manipulation of two distinct neuronal populations, to demonstrate that cell-autonomous inhibition of g-KC pruning inhibits APL pruning. However, once activitybased communication between unpruned g-KCs and the APL was disrupted, APL neurites pruned independently of KCs. This implies coordinated circuit remodeling by mechanisms that rely, at least in part, on g-KCs activity, which is mediated by APL nuclear calcium signaling. Current Opinion in Neurobiology 2020, 63:1–8
Taken together, these examples demonstrate that complex neuronal circuits coordinate remodeling using activitybased mechanisms that are yet to be fully explored. In this field, we anticipate that Drosophila could provide important insights, due to the existence of well-characterized circuits, combined with powerful genetic tools.
Regulation of adhesion is important for remodeling Interestingly, in the aforementioned study, Mayseless et al. also demonstrated that artificially increasing adhesion between g-KCs and the APL neuron is sufficient to inhibit pruning of both neuronal populations [23]. This finding suggests that in parallel to, or in combination with, neuronal activity, coordination of g-KC-APL remodeling may also depend on adhesion regulation. Cell adhesion molecules (CAMs) are cell surface proteins that form homophilic or heterophilic interactions and were shown to mediate different aspects of neurodevelopment [e.g. 24,25]. CAMs, and regulation of their subcellular distribution, are prime candidates to provide spatial control over remodeling. Indeed, in the Drosophila MB, in addition to their putative role in coordinating g-KC-APL remodeling, CAMs mediate pruning of g-KCs in what seems to be a compartment-specific manner. Bornstein et al. showed that defasciculation of g-KCs, mediated by reduction in membrane levels of the immunoglobulin superfamily (IgSF) CAM Fasciclin II (FasII, the NCAM ortholog) facilitates axon pruning. Overexpression of FasII, or even of other CAMs, within g-KCs is sufficient to inhibit axon pruning [26; Figure 1a]. Remarkably, pruning of g-dendrites remains unaffected by FasII overexpression, suggesting that distinct mechanisms are employed within the same cell for axon and dendrite pruning. CAMs are also important during remodeling of the Drosophila dendritic arborization (da) neurons. These sensory neurons extend highly branched dendrites that innervate the body wall. Different CAM families mediate dendrite-dendrite and dendrite-ECM interactions to ensure tiling and self-avoidance for efficient coverage of the receptive field [27–29]. During metamorphosis, da neurons of specific classes prune their entire larval dendritic tree by local fragmentation, later followed by regrowth [30]. Endocytosis-mediated downregulation of the L1-type CAM Neuroglian (Nrg) was shown to be required for proper dendrite pruning [31]. However, the binding partner of Nrg in this context, and the potential cellular interactions it mediates, remain to be further investigated (Figure 1b). Interestingly, Tenenbaum et al. recently highlighted the membrane-associated protein Coracle, the single fly 4.1 protein member, as required for dendritic enclosure within the epidermis, likely by mediating adhesion via neurexins. The authors show that this enclosure restricts dendrite branching and www.sciencedirect.com
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Figure 1
a
IgSF protein (e.g., FasII)
b
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Adhesion regulation potentially impacts pruning. (a) Membranal downregulation of IgSF CAMs, such as FasII, results in axonal defasciculation thereby facilitating pruning. (b) Endocytosismediated CAM downregulation, promotes neurite pruning potentially by leading to de-adhesion of a neurite from its surrounding cells or from the ECM. Localized regulation of adhesion might provide a mechanism for spatiotemporal control.
growth, in order to coordinate receptive field innervation [32]. Since selective epidermal enclosure can potentially act as a spatiotemporal template for pruning, it would be interesting to further explore dendro-epidermal interactions during remodeling, whether mediated by Coracle or other molecules.
the tail tip region. LIN-44/Wnt, which originates in tail hypodermal cells, instructs PDB pruning via the LIN-17/ Frizzled receptor. Interestingly, expressing membranetethered LIN-44/Wnt in lin-44 mutants is sufficient for PDB neurite pruning, suggesting that Wnt in this context is gradient-independent and may mediate contactdependent interactions [37].
Cells secrete pruning-regulating factors An additional type of cell–cell communication is factor secretion, such as growth factors or axon guidance molecules, which function in short or long range. In Drosophila, glia surrounding MB g-KCs secrete the Transforming Growth Factor (TGF)-b ligand Myoglianin (Myo), whose signaling is mediated by the TGF-b receptor Baboon and facilitated by the IgSF protein Plum [33,34]. Recently, glial-derived Myo and neuronal Plum were shown to additionally mediate axon midline stopping of another KC type, the ɑ/b-KCs [35]. Interestingly, astrocyte-derived TGF-b plays a role in the retinogeniculate refinement in mammals [36], implying a conserved function. Lu and Mizumoto recently demonstrated that in Caenorhabditis elegans, Wnt signaling promotes the stereotypic pruning of the cholinergic motor neuron called PDB. During larval development, PDB undergoes asymmetrical pruning to achieve its typical V-shaped turn at www.sciencedirect.com
In mammals, innervation of the mammary gland undergoes sexually dimorphic remodeling, controlled by secretion of Brain-Derived Neurotrophic Factor (BDNF) by the mammary mesenchymal cells. BDNF signals via the Tropomyosin receptor kinase B (TrkB) to promote innervation in both sexes during early embryonic development. At later embryonic stages, male sex-hormones promote expression of a truncated TrkB form (TrkB. T1) which sequesters BDNF, leading to male-specific pruning [38]. Recently, Sar-Shalom et al. showed that mammary epithelial cells, surrounded by the mesenchyme, secrete Semaphorins which have an opposite effect of BDNF. In females, reducing BDNF levels causes hypoinnervation of the gland, while decreasing Semaphorins results in hyperinnervation. In males, BDNF signaling is naturally kept low by TrkB.T1 expression; however, decreasing Semaphorins leads to reduced pruning. Overall, mammary gland innervation is determined by the relative balance between BDNF and Semaphorins [39]. Current Opinion in Neurobiology 2020, 63:1–8
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Repulsive cues of both the Semaphorin and Ephrin families are also essential for pruning in additional neural systems, reviewed elsewhere [7,40,41].
Mechanical interactions potentially shape skin innervation Many PNS neurons, especially those in the sensory system, reside in proximity to the skin, an organ which provides physical protection but is also subject to mechanical forces. In a recent study, Takahashi et al. aimed to uncover the cause of chronic itch during atopic dermatitis (AD). By inspecting the skin of both AD patients and a murine model of impaired epidermal barrier, the authors found that sensory nerve endings penetrate the keratinocyte tight junctions (TJs), leaving them exposed to noxious environmental stimuli. In healthy skin, epidermal nerve endings are confined and protected beneath TJs. Interestingly, this was shown to be maintained, at least in part, by rapid pruning of nerve fibers at sites of newly forming TJs — a process that failed to occur during AD [42]. Further mechanistic investigation is required to determine whether pruning occurs strictly due to mechanical constriction of the forming TJ around nerve fibers, or because of molecular events originating in the keratinocytes or nerves. In Zebrafish, skin innervation is dramatically remodeled from larva to adult. Embryonic peripheral sensory neurons die during post-embryonic development, and neurons of another origin re-innervate the adult skin in a fundamentally different pattern. Recently, Rasmussen et al. found that this transition coincides with scale formation during the juvenile stage, and that the innervation pattern is congruent with the scale surface tracts lined by osteoblasts known as radii. Remarkably, transplantation of denervated scales resulted in reinnervation along radii
by the regenerating host axons, while mutants lacking scales showed defective patterning, demonstrating that scales form a guidance path for sensory nerves [43]. Whether guidance is purely mechanical or also involves molecular signals from osteoblasts, remains to be determined. Together, these examples raise the potential roles of skin or bony appendages during neuronal growth or remodeling.
Neighboring cells phagocytose pruned debris During the final stages of pruning, axonal or dendritic debris must be phagocytosed by surrounding cells to avoid inflammatory responses and maintain tissue homeostasis. In the Drosophila MB, surrounding glia, specifically astrocytes, infiltrate the degenerating g-KCs lobes and engulf axon fragments for subsequent endolysosomal degradation [44–47]. This process depends on the conserved apoptotic engulfment receptor Draper [Drpr; 46–48] — which also mediates glial engulfment of axons undergoing injury-induced Wallarian degeneration [49]. Unlike g-KCs, degenerating da dendrites are cleared mainly by epidermal cells. However, similarly to g-KCs, this process is mediated, at least in part, by epithelium derived Drpr [50]. Interestingly, epidermal cells were also shown to phagocytose debris of injured sensory axons in Zebrafish [51]. While glia do not engulf da neuron dendrites, they are, nevertheless, tightly associated throughout development and localized to proximal dendrites adjacent to severing events [52], underscoring that their role during pruning warrants further investigation. Recently, Sapar et al. demonstrated that phosphatidylserine (PS), a well-known ‘eat-me’ signal for apoptotic cells, also
Figure 2
da neuron Epidermal cells
Dendrite
PS
PS-recognizing receptor (Drpr?)
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PS exposure may serve as an ‘eat-me’ signal during pruning. Severed dendrites externalize PS (red) to the cytoplasmic leaflet of the cell membrane. PS is recognized by a membranal receptor (possibly Drpr), leading to engulfment of pruned debris. Current Opinion in Neurobiology 2020, 63:1–8
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Cell–cell interactions shape neuronal remodeling Meltzer and Schuldiner 5
Figure 3
Activity
Adhesion
Secretion
Mechanical Constriction
Phagocytosis
TJ
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Cell–cell interactions during neuronal remodelling. The various ways, reviewed here, by which neurons interact with surrounding cells during neuronal remodeling. TJ; tight junction.
plays a role in mediating phagocytosis of da dendrites. The authors used genetically encoded PS-binding reporters to visualize PS externalization and found that it was exclusively externalized (i.e., flipped from the cytoplasmic-leaflet to the exoplasmic-leaflet of the cell membrane) on dendritic branches undergoing developmental pruning or Wallerian degeneration (Figure 2). PS exposure coincided with early signs of degeneration such as membrane blebbing, but preceded fragmentation and engulfment. Moreover, ectopically exposing PS on dendrites resulted in membrane vesicle shedding, suggested to derive from Drpr-mediated attack and engulfment by epidermal cells [53]. Interestingly, Shacham-Silverberg et al. have recently shown that Nerve Growth Factor-deprived Dorsal Root Ganglia explants, which serve as an in-vitro model for axon pruning, expose PS on subaxonal segments undergoing degeneration [54]. Finally, a recent study showed that coating beads by PS is sufficient to induce their engulfment by Drpr-transfected S2 cells [which are otherwise non-phagocytic; 55]. Together, these studies reinforce the relevance of PS in developmental pruning, however; whether Drpr serves as the PS receptor in the context of developmental pruning in vivo, remains to be clarified. In mammals, glia carry out similar phagocytic roles. In the PNS, Schwann cells engulf synaptic fragments following ‘axosome shedding’ during NMJ refinement [56]. In the www.sciencedirect.com
mammalian CNS, the major phagocytes responsible for debris clearance are microglia, and to a lesser extent, astrocytes [57]. Their role in synaptic pruning, mainly through engulfment of presynapses, was broadly studied in recent years, highlighting their importance during development and also in neurodegenerative and neuropsychiatric disorders, and is extensively reviewed elsewhere [58–63]. Surprisingly, it is still unknown how pruned debris is cleared during large-scale pruning in mammals, such as in the pruning of layer V corticospinal tract projections [64].
Concluding remarks Here we reviewed recent studies that have resurfaced the significance of various types of cellular interactions during neuronal remodeling (Figure 3). While substantial progress was made, a comprehensive understanding of the intercellular mechanisms that govern remodeling is still lacking. Because of recent progress in transcriptomics, genomic strategies are becoming an integral part of the genetic toolbox, enabling novel investigational approaches. A recent study [65] uncovered the transcriptional landscape of developing MB g-KCs at unprecedented temporal resolution, and more expression profiles from other model systems are likely to be uncovered. Not surprisingly, genes relating to intercellular communication, including neurotransmitter receptors and CAMs, were among the Current Opinion in Neurobiology 2020, 63:1–8
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most dynamically expressed throughout remodeling, highlighting their potential roles [65]. Advances in proteomics and methods to determine protein subcellular localization are likely to further transform the field, especially regarding spatiotemporal control. Important progress was made by identifying PS as a branch-specific ‘eat me’ signal that drives phagocytosis of specific neuronal compartments while sparing others [53] but the mechanisms remain to be further delineated. CAMs were also shown to affect pruning in a branch/compartment-specific manner, as adhesion destabilization is required for pruning of g-KCs axons, but not dendrites [26]. Therefore, uncovering the neuronal adhesome at subcellular resolution could be a pivotal step in understanding spatiotemporal regulation of remodeling. Another intriguing, unresolved aspect is the potential link between adhesion and activity during coordinated circuit remodeling [23]. An interesting insight comes from the mouse somatosensory cortex, where sensory experience was shown to drive pruning via competition between neighboring dendritic spines for limited cadherin/catenin adhesion complexes. Redistribution of these adhesion complexes from less active to more active spines results in maturation of the ‘winner’ and pruning of the ‘loser’ [66]. The molecular basis for this activity-induced redistribution of adhesion molecules is a fascinating topic for further investigation. Finally, as the function of microglia in synapse elimination during neurological disorders has been highlighted in recent years, other cell–cell interactions are also likely to play roles in aberrant pruning during disease. For example, inappropriate PS exposure might result in pathological neurite phagocytosis. Therefore, a global understanding of the mechanisms that operate within and between cells during developmental remodeling could provide insights into similar mechanisms during disease. To achieve these goals, the discovery of new neurons that undergo remodeling is critical. This should uncover entire circuits that remodel together and thereby provide excellent biological systems to address the various aspects covered in this review.
Conflict of interest statement Nothing declared.
Acknowledgements We thank Tal Bigdari for graphical assistance; Research in our lab has been funded mainly by the Israel Science Foundation (ISF), and the European Research Council (erc) consolidator (erc CoG # 615906). OS is an Incumbent of the Prof. Erwin Netter Professorial Chair of Cell Biology
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39. Sar Shalom H, Goldner R, Golan-Vaishenker Y, Yaron A: Balance between BDNF and Semaphorins gates the innervation of the mammary gland. eLife 2019, 8 Sar Shalom et al. demonstrate involvement of Semaphorin signaling during the development and male-specific pruning of mammary gland innervation, previously shown to rely on BDNF signaling. BDNF and semaphorins are secreted by mesenchimal and epithelial cells respectively, and have opposing effects, so that precise innervation is determined by their balance. This is further validated in vitro, as sensory axons become hypersensitive to Semaphorins under low BDNF levels and show increased growth cone collapse, which may represent an early step of axon pruning. This study highlights the importance of surrounding cells during remodeling, as counteracting molecular cues derived from two distinct non-neuronal cell types are crucial for determining the final innervation. 40. Vanderhaeghen P, Cheng HJ: Guidance molecules in axon pruning and cell death. Cold Spring Harb Perspect Biol 2010, 2: a001859. 41. Waimey KE, Cheng HJ: Axon pruning and synaptic development: how are they per-plexin? Neuroscientist 2006, 12:398-409. 42. Takahashi S, Ishida A, Kubo A, Kawasaki H, Ochiai S, Nakayama M, Koseki H, Amagai M, Okada T: Homeostatic pruning and activity of epidermal nerves are dysregulated in barrier-impaired skin during chronic itch development. Sci Rep 2019, 9:8625 Using intravital imaging of the epidermis, which highlights the extremely dynamic nature of the skin, the authors demonstrate that growing nerves penetrate the epidermal tight junctions in atopic dermatitis (AD). Consequently, the penetrating neurites are exposed to noxious stimuli leading to pathological itch, as reflected by aberrant neuronal calcium signaling. Interestingly, the authors suggest that this is prevented in healthy skin due to constant local pruning of nerve endings at the site of newly forming tight junctions, in a yet unknown mechanism that may involve mechanical constriction and/or molecular cues. 43. Rasmussen JP, Vo NT, Sagasti A: Fish scales dictate the pattern of adult skin innervation and vascularization. Dev Cell 2018, 46:344-359.e4. 44. Watts RJ, Schuldiner O, Perrino J, Larsen C, Luo L: Glia engulf degenerating axons during developmental axon pruning. Curr Biol 2004, 14:678-684. 45. Awasaki T, Ito K: Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis. Curr Biol 2004, 14:668-677. 46. Hakim Y, Yaniv SP, Schuldiner O: Astrocytes play a key role in Drosophila mushroom body axon pruning. PLoS One 2014, 9: e86178. 47. Tasdemir-Yilmaz OE, Freeman MR: Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons. Genes Dev 2014, 28:20-33. 48. Awasaki T, Tatsumi R, Takahashi K, Arai K, Nakanishi Y, Ueda R, Ito K: Essential role of the apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning during Drosophila metamorphosis. Neuron 2006, 50:855-867. 49. MacDonald JM, Beach MG, Porpiglia E, Sheehan AE, Watts RJ, Freeman MR: The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons. Neuron 2006, 50:869-881.
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52. Han C, Jan LY, Jan YN: Enhancer-driven membrane markers for analysis of nonautonomous mechanisms reveal neuron-glia interactions in Drosophila. Proc Natl Acad Sci U S A 2011, 108:9673-9678. 53. Sapar ML, Ji H, Wang B, Poe AR, Dubey K, Ren X, Ni JQ, Han C: Phosphatidylserine externalization results from and causes neurite degeneration in Drosophila. Cell Rep 2018, 24:2273-2286 These two studies highlight the potential role of Phosphatidylserine (PS) as an ‘eat me’ signal in the context of neurite pruning and axon injury. Using a similar genetically encoded PS reporter, work from the Han and Yaron laboratories has demonstrated that PS externalization occurs before neurite fragmentation both in vivo in flies and in mammalian in vitro culture systems. Genetic perturbations in the fly, resulting in ectopic PS externalization, resulted in extensive degeneration and potential draper mediated engulfment by epidermal cells. Especially interesting in the context of this review, as localized PS externalization may provide spatiotemporal control of pruning. Potential mechanisms for such localized PS regulation, are however unknown. 54. Shacham-Silverberg V, Sar Shalom H, Goldner R, Golan Vaishenker Y, Gurwicz N, Gokhman I, Yaron A: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration. Cell Death Dis 2018, 9:1116 These two studies highlight the potential role of Phosphatidylserine (PS) as an ‘eat me’ signal in the context of neurite pruning and axon injury. Using a similar genetically encoded PS reporter, work from the Han and Yaron laboratories has demonstrated that PS externalization occurs before neurite fragmentation both in vivo in flies and in mammalian in vitro culture systems. Genetic perturbations in the fly, resulting in ectopic PS externalization, resulted in extensive degeneration and potential draper mediated engulfment by epidermal cells. Especially interesting in the context of this review, as localized PS externalization may provide spatiotemporal control of pruning. Potential mechanisms for such localized PS regulation, are however unknown. 55. Williamson AP, Vale RD: Spatial control of Draper receptor signaling initiates apoptotic cell engulfment. J Cell Biol 2018, 217:3977-3992.
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56. Bishop DL, Misgeld T, Walsh MK, Gan WB, Lichtman JW: Axon branch removal at developing synapses by axosome shedding. Neuron 2004, 44:651-661. 57. Jung YJ, Chung WS: Phagocytic roles of glial cells in healthy and diseased brains. Biomol Ther (Seoul) 2018, 26:350-357. 58. Hong S, Dissing-Olesen L, Stevens B: New insights on the role of microglia in synaptic pruning in health and disease. Curr Opin Neurobiol 2016, 36:128-134. 59. Presumey J, Bialas AR, Carroll MC: Complement system in neural synapse elimination in development and disease. Adv Immunol 2017, 135:53-79. 60. Lee E, Chung WS: Glial control of synapse number in healthy and diseased brain. Front Cell Neurosci 2019, 13:42. 61. Rajendran L, Paolicelli RC: Microglia-mediated synapse loss in Alzheimer’s disease. J Neurosci 2018, 38:2911-2919. 62. Luchena C, Zuazo-Ibarra J, Alberdi E, Matute C, CapetilloZarate E: Contribution of neurons and glial cells to complement-mediated synapse removal during development, aging and in Alzheimer’s disease. Mediators Inflamm 2018, 2018:2530414. 63. Wilton DK, Dissing-Olesen L, Stevens B: Neuron-glia signaling in synapse elimination. Annu Rev Neurosci 2019, 42:107-127. 64. O’Leary DD, Koester SE: Development of projection neuron types, axon pathways, and patterned connections of the mammalian cortex. Neuron 1993, 10:991-1006. 65. Alyagor I, Berkun V, Keren-Shaul H, Marmor-Kollet N, David E, Mayseless O, Issman-Zecharya N, Amit I, Schuldiner O: Combining developmental and perturbation-seq uncovers transcriptional modules orchestrating neuronal remodeling. Dev Cell 2018, 47:38-52.e6. 66. Bian WJ, Miao WY, He SJ, Qiu Z, Yu X: Coordinated spine pruning and maturation mediated by inter-spine competition for cadherin/catenin complexes. Cell 2015, 162:808-822.
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ScienceDirect With a little help from my friends: how intercellular communication shapes neuronal remodeling Hagar Meltzer and Oren Schuldiner Developmental neuronal remodeling shapes the mature connectivity of the nervous system in both vertebrates and invertebrates. Remodeling often combines degenerative and regenerative events, and defects in its normal progression have been linked to neurological disorders. Here we review recent progress that highlights the roles of cell–cell interactions during remodeling. We propose that these are fundamental to elucidating how spatiotemporal control of remodeling and coordinated circuit remodeling are achieved. We cover examples spanning various neuronal circuits in vertebrates and invertebrates and involving interactions between neurons and different cell types. Address Dept of Molecular Cell Biology, Weizmann Institute of Science, Israel Corresponding authors: Meltzer, Hagar ([email protected]), Schuldiner, Oren ([email protected])
Current Opinion in Neurobiology 2020, 63:xx–yy This review comes from a themed issue on Cellular neuroscience Edited by Thomas Schwarz and Holly Cline
https://doi.org/10.1016/j.conb.2020.01.018 0959-4388/ã 2020 Elsevier Ltd. All rights reserved.
instance, glia closely associate with neurons throughout the nervous system, while PNS neurons intermingle with the tissue they inhabit. So far, most studies of neuronal remodeling have focused on cell-autonomous processes, such as cytoskeletal organization and degradation pathways [2,6–8]. While the surrounding cells were never ignored, advances were relatively modest. Several recent studies began to shift the focus back towards cell–cell communication. An emerging theme from these studies suggests that a deeper understanding of circuit remodeling, and specifically its precise spatiotemporal control, must integrate both neuronal intrinsic processes and dynamic interactions with the environment. In this review we explore the roles of cell–cell interactions during neuronal remodeling and circuit maturation. We relate to various aspects of cell communication in diverse neural systems and model organisms, and raise open questions in the field. We review the existing literature based on the type of interaction between the communicating cells; however, this is mainly for convenience, as the different interaction types are not mutually exclusive, and may heavily depend on one another. Importantly, we aim to focus on recent findings, and do not cover all available literature. Topics that are extensively reviewed elsewhere were excluded from the scope of this review, as mentioned below.
Neuronal activity coordinates remodeling Introduction Precise connectivity of mature nervous systems depends on regressive and progressive events, often including elimination of exuberant connections followed by regrowth to adult-specific targets. Collectively known as developmental neuronal remodeling, such processes are used to refine neural circuits throughout evolution [1,2]. Remodeling sculpts central and peripheral nervous systems (CNS and PNS, respectively), and occurs in different scales — from pruning of single synapses or short axonal segments (normally by retraction), to elimination of long axonal stretches or entire dendritic trees (generally via local fragmentation). Defects in remodeling have been associated with neurodevelopmental disorders such as autism and schizophrenia [3–5]. A remodeling neuron is often part of a complex tissue encompassing both neurons and non-neuronal cells. For www.sciencedirect.com
Interaction via chemical synapses is probably the most unique mode of inter-neuronal communication, and not surprisingly, plays important roles during remodeling in both vertebrates and invertebrates. In mammals, classic examples of activity-dependent remodeling include the development of the neuromuscular junction (NMJ), in which motor axons compete for muscle fiber innervation, as well as the refinement of the cerebellar climbing fiber (CF) circuit, in which several CFs compete for the innervation of a single Purkinje cell (PC). In both processes [thoroughly reviewed elsewhere; 9–11] refinement involves strengthening of ‘winners’ and pruning of ‘losers’ to promote transition from poly-innervation to mono-innervation. Specifically, in the refinement of the CF-PC circuit, elimination of CF synapses requires, at different stages, both GABAergic inhibition [originating in basket cells; 12] as well as glutamatergic signaling [from parallel fibers; 13,14] onto PC somata. Notably, while this process essentially involves neuron–neuron interactions, Nakayama et al. recently suggested that microglia promote GABAergic Current Opinion in Neurobiology 2020, 63:1–8
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inhibition on PCs, in a phagocytosis-independent mechanism whose precise nature remains to be uncovered [15]. In Drosophila, olfactory sensory neurons (OSNs) synapse onto discrete glomeruli to form elaborate sensory maps [16]. Early adult sensory experience was reported to induce non-stereotypic remodeling to expand glomerulus innervation by the activated OSN [17]. However, Golovin et al. recently demonstrated an opposite effect, in which activation of a specific OSN led to volume reduction in its corresponding glomerulus due to synapse elimination [18]. This contradiction resembles the apparent conflict in the CF-PC circuit, in which both inhibitory and excitatory signals are required within the same cell to promote synapse pruning. Thus, more studies are required to dissect the precise relations between activity and pruning in different cellular contexts. Particularly fascinating is the study of neuronal networks that remodel together. In the mammalian retinogeniculate circuit, extensive remodeling refines initially overlapping projections to form segregated eye-specific domains. Retinal ganglion cells (RGCs) transmit retinal input to the dorsal lateral geniculate nucleus (dLGN) in the thalamus, which is mainly comprised of relay cells that project to the visual cortex and thalamic interneurons. During early postnatal stages, RGCs locally prune their axon arbors to generate eye-specific domains within the dLGN, in an activity-dependent manner [19]. Remarkably, two recent studies demonstrated that retinal activity also drives the remodeling of relay cells and interneurons. In the absence of retinal input, relay cells demonstrate reduced dendritic surface area within the dLGN [20], and interneurons fail to branch and thus maintain a simple architecture [21]. Coordinated circuit remodeling is also evident in the Drosophila mushroom body (MB), a complex CNS neuropil, in which one neuronal type, the g-Kenyon Cells (g-KCs), undergo stereotypic remodeling during metamorphosis, including pruning of larval axons followed by regrowth of adult-specific axons [8]. g-KCs receive input from projection neurons (PNs), and modulatory signals mainly from GABAergic and dopaminergic neurons (DANs). Interestingly, embryonically born PNs and the GABAergic anterior paired lateral (APL) neuron undergo developmental remodeling in a similar timeframe as g-KCs [22,23]. In the case of the APL, Mayseless et al. used advanced genetic tools, which allow simultaneous manipulation of two distinct neuronal populations, to demonstrate that cell-autonomous inhibition of g-KC pruning inhibits APL pruning. However, once activitybased communication between unpruned g-KCs and the APL was disrupted, APL neurites pruned independently of KCs. This implies coordinated circuit remodeling by mechanisms that rely, at least in part, on g-KCs activity, which is mediated by APL nuclear calcium signaling. Current Opinion in Neurobiology 2020, 63:1–8
Taken together, these examples demonstrate that complex neuronal circuits coordinate remodeling using activitybased mechanisms that are yet to be fully explored. In this field, we anticipate that Drosophila could provide important insights, due to the existence of well-characterized circuits, combined with powerful genetic tools.
Regulation of adhesion is important for remodeling Interestingly, in the aforementioned study, Mayseless et al. also demonstrated that artificially increasing adhesion between g-KCs and the APL neuron is sufficient to inhibit pruning of both neuronal populations [23]. This finding suggests that in parallel to, or in combination with, neuronal activity, coordination of g-KC-APL remodeling may also depend on adhesion regulation. Cell adhesion molecules (CAMs) are cell surface proteins that form homophilic or heterophilic interactions and were shown to mediate different aspects of neurodevelopment [e.g. 24,25]. CAMs, and regulation of their subcellular distribution, are prime candidates to provide spatial control over remodeling. Indeed, in the Drosophila MB, in addition to their putative role in coordinating g-KC-APL remodeling, CAMs mediate pruning of g-KCs in what seems to be a compartment-specific manner. Bornstein et al. showed that defasciculation of g-KCs, mediated by reduction in membrane levels of the immunoglobulin superfamily (IgSF) CAM Fasciclin II (FasII, the NCAM ortholog) facilitates axon pruning. Overexpression of FasII, or even of other CAMs, within g-KCs is sufficient to inhibit axon pruning [26; Figure 1a]. Remarkably, pruning of g-dendrites remains unaffected by FasII overexpression, suggesting that distinct mechanisms are employed within the same cell for axon and dendrite pruning. CAMs are also important during remodeling of the Drosophila dendritic arborization (da) neurons. These sensory neurons extend highly branched dendrites that innervate the body wall. Different CAM families mediate dendrite-dendrite and dendrite-ECM interactions to ensure tiling and self-avoidance for efficient coverage of the receptive field [27–29]. During metamorphosis, da neurons of specific classes prune their entire larval dendritic tree by local fragmentation, later followed by regrowth [30]. Endocytosis-mediated downregulation of the L1-type CAM Neuroglian (Nrg) was shown to be required for proper dendrite pruning [31]. However, the binding partner of Nrg in this context, and the potential cellular interactions it mediates, remain to be further investigated (Figure 1b). Interestingly, Tenenbaum et al. recently highlighted the membrane-associated protein Coracle, the single fly 4.1 protein member, as required for dendritic enclosure within the epidermis, likely by mediating adhesion via neurexins. The authors show that this enclosure restricts dendrite branching and www.sciencedirect.com
Cell–cell interactions shape neuronal remodeling Meltzer and Schuldiner 3
Figure 1
a
IgSF protein (e.g., FasII)
b
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Adhesion regulation potentially impacts pruning. (a) Membranal downregulation of IgSF CAMs, such as FasII, results in axonal defasciculation thereby facilitating pruning. (b) Endocytosismediated CAM downregulation, promotes neurite pruning potentially by leading to de-adhesion of a neurite from its surrounding cells or from the ECM. Localized regulation of adhesion might provide a mechanism for spatiotemporal control.
growth, in order to coordinate receptive field innervation [32]. Since selective epidermal enclosure can potentially act as a spatiotemporal template for pruning, it would be interesting to further explore dendro-epidermal interactions during remodeling, whether mediated by Coracle or other molecules.
the tail tip region. LIN-44/Wnt, which originates in tail hypodermal cells, instructs PDB pruning via the LIN-17/ Frizzled receptor. Interestingly, expressing membranetethered LIN-44/Wnt in lin-44 mutants is sufficient for PDB neurite pruning, suggesting that Wnt in this context is gradient-independent and may mediate contactdependent interactions [37].
Cells secrete pruning-regulating factors An additional type of cell–cell communication is factor secretion, such as growth factors or axon guidance molecules, which function in short or long range. In Drosophila, glia surrounding MB g-KCs secrete the Transforming Growth Factor (TGF)-b ligand Myoglianin (Myo), whose signaling is mediated by the TGF-b receptor Baboon and facilitated by the IgSF protein Plum [33,34]. Recently, glial-derived Myo and neuronal Plum were shown to additionally mediate axon midline stopping of another KC type, the ɑ/b-KCs [35]. Interestingly, astrocyte-derived TGF-b plays a role in the retinogeniculate refinement in mammals [36], implying a conserved function. Lu and Mizumoto recently demonstrated that in Caenorhabditis elegans, Wnt signaling promotes the stereotypic pruning of the cholinergic motor neuron called PDB. During larval development, PDB undergoes asymmetrical pruning to achieve its typical V-shaped turn at www.sciencedirect.com
In mammals, innervation of the mammary gland undergoes sexually dimorphic remodeling, controlled by secretion of Brain-Derived Neurotrophic Factor (BDNF) by the mammary mesenchymal cells. BDNF signals via the Tropomyosin receptor kinase B (TrkB) to promote innervation in both sexes during early embryonic development. At later embryonic stages, male sex-hormones promote expression of a truncated TrkB form (TrkB. T1) which sequesters BDNF, leading to male-specific pruning [38]. Recently, Sar-Shalom et al. showed that mammary epithelial cells, surrounded by the mesenchyme, secrete Semaphorins which have an opposite effect of BDNF. In females, reducing BDNF levels causes hypoinnervation of the gland, while decreasing Semaphorins results in hyperinnervation. In males, BDNF signaling is naturally kept low by TrkB.T1 expression; however, decreasing Semaphorins leads to reduced pruning. Overall, mammary gland innervation is determined by the relative balance between BDNF and Semaphorins [39]. Current Opinion in Neurobiology 2020, 63:1–8
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Repulsive cues of both the Semaphorin and Ephrin families are also essential for pruning in additional neural systems, reviewed elsewhere [7,40,41].
Mechanical interactions potentially shape skin innervation Many PNS neurons, especially those in the sensory system, reside in proximity to the skin, an organ which provides physical protection but is also subject to mechanical forces. In a recent study, Takahashi et al. aimed to uncover the cause of chronic itch during atopic dermatitis (AD). By inspecting the skin of both AD patients and a murine model of impaired epidermal barrier, the authors found that sensory nerve endings penetrate the keratinocyte tight junctions (TJs), leaving them exposed to noxious environmental stimuli. In healthy skin, epidermal nerve endings are confined and protected beneath TJs. Interestingly, this was shown to be maintained, at least in part, by rapid pruning of nerve fibers at sites of newly forming TJs — a process that failed to occur during AD [42]. Further mechanistic investigation is required to determine whether pruning occurs strictly due to mechanical constriction of the forming TJ around nerve fibers, or because of molecular events originating in the keratinocytes or nerves. In Zebrafish, skin innervation is dramatically remodeled from larva to adult. Embryonic peripheral sensory neurons die during post-embryonic development, and neurons of another origin re-innervate the adult skin in a fundamentally different pattern. Recently, Rasmussen et al. found that this transition coincides with scale formation during the juvenile stage, and that the innervation pattern is congruent with the scale surface tracts lined by osteoblasts known as radii. Remarkably, transplantation of denervated scales resulted in reinnervation along radii
by the regenerating host axons, while mutants lacking scales showed defective patterning, demonstrating that scales form a guidance path for sensory nerves [43]. Whether guidance is purely mechanical or also involves molecular signals from osteoblasts, remains to be determined. Together, these examples raise the potential roles of skin or bony appendages during neuronal growth or remodeling.
Neighboring cells phagocytose pruned debris During the final stages of pruning, axonal or dendritic debris must be phagocytosed by surrounding cells to avoid inflammatory responses and maintain tissue homeostasis. In the Drosophila MB, surrounding glia, specifically astrocytes, infiltrate the degenerating g-KCs lobes and engulf axon fragments for subsequent endolysosomal degradation [44–47]. This process depends on the conserved apoptotic engulfment receptor Draper [Drpr; 46–48] — which also mediates glial engulfment of axons undergoing injury-induced Wallarian degeneration [49]. Unlike g-KCs, degenerating da dendrites are cleared mainly by epidermal cells. However, similarly to g-KCs, this process is mediated, at least in part, by epithelium derived Drpr [50]. Interestingly, epidermal cells were also shown to phagocytose debris of injured sensory axons in Zebrafish [51]. While glia do not engulf da neuron dendrites, they are, nevertheless, tightly associated throughout development and localized to proximal dendrites adjacent to severing events [52], underscoring that their role during pruning warrants further investigation. Recently, Sapar et al. demonstrated that phosphatidylserine (PS), a well-known ‘eat-me’ signal for apoptotic cells, also
Figure 2
da neuron Epidermal cells
Dendrite
PS
PS-recognizing receptor (Drpr?)
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PS exposure may serve as an ‘eat-me’ signal during pruning. Severed dendrites externalize PS (red) to the cytoplasmic leaflet of the cell membrane. PS is recognized by a membranal receptor (possibly Drpr), leading to engulfment of pruned debris. Current Opinion in Neurobiology 2020, 63:1–8
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Figure 3
Activity
Adhesion
Secretion
Mechanical Constriction
Phagocytosis
TJ
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Cell–cell interactions during neuronal remodelling. The various ways, reviewed here, by which neurons interact with surrounding cells during neuronal remodeling. TJ; tight junction.
plays a role in mediating phagocytosis of da dendrites. The authors used genetically encoded PS-binding reporters to visualize PS externalization and found that it was exclusively externalized (i.e., flipped from the cytoplasmic-leaflet to the exoplasmic-leaflet of the cell membrane) on dendritic branches undergoing developmental pruning or Wallerian degeneration (Figure 2). PS exposure coincided with early signs of degeneration such as membrane blebbing, but preceded fragmentation and engulfment. Moreover, ectopically exposing PS on dendrites resulted in membrane vesicle shedding, suggested to derive from Drpr-mediated attack and engulfment by epidermal cells [53]. Interestingly, Shacham-Silverberg et al. have recently shown that Nerve Growth Factor-deprived Dorsal Root Ganglia explants, which serve as an in-vitro model for axon pruning, expose PS on subaxonal segments undergoing degeneration [54]. Finally, a recent study showed that coating beads by PS is sufficient to induce their engulfment by Drpr-transfected S2 cells [which are otherwise non-phagocytic; 55]. Together, these studies reinforce the relevance of PS in developmental pruning, however; whether Drpr serves as the PS receptor in the context of developmental pruning in vivo, remains to be clarified. In mammals, glia carry out similar phagocytic roles. In the PNS, Schwann cells engulf synaptic fragments following ‘axosome shedding’ during NMJ refinement [56]. In the www.sciencedirect.com
mammalian CNS, the major phagocytes responsible for debris clearance are microglia, and to a lesser extent, astrocytes [57]. Their role in synaptic pruning, mainly through engulfment of presynapses, was broadly studied in recent years, highlighting their importance during development and also in neurodegenerative and neuropsychiatric disorders, and is extensively reviewed elsewhere [58–63]. Surprisingly, it is still unknown how pruned debris is cleared during large-scale pruning in mammals, such as in the pruning of layer V corticospinal tract projections [64].
Concluding remarks Here we reviewed recent studies that have resurfaced the significance of various types of cellular interactions during neuronal remodeling (Figure 3). While substantial progress was made, a comprehensive understanding of the intercellular mechanisms that govern remodeling is still lacking. Because of recent progress in transcriptomics, genomic strategies are becoming an integral part of the genetic toolbox, enabling novel investigational approaches. A recent study [65] uncovered the transcriptional landscape of developing MB g-KCs at unprecedented temporal resolution, and more expression profiles from other model systems are likely to be uncovered. Not surprisingly, genes relating to intercellular communication, including neurotransmitter receptors and CAMs, were among the Current Opinion in Neurobiology 2020, 63:1–8
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most dynamically expressed throughout remodeling, highlighting their potential roles [65]. Advances in proteomics and methods to determine protein subcellular localization are likely to further transform the field, especially regarding spatiotemporal control. Important progress was made by identifying PS as a branch-specific ‘eat me’ signal that drives phagocytosis of specific neuronal compartments while sparing others [53] but the mechanisms remain to be further delineated. CAMs were also shown to affect pruning in a branch/compartment-specific manner, as adhesion destabilization is required for pruning of g-KCs axons, but not dendrites [26]. Therefore, uncovering the neuronal adhesome at subcellular resolution could be a pivotal step in understanding spatiotemporal regulation of remodeling. Another intriguing, unresolved aspect is the potential link between adhesion and activity during coordinated circuit remodeling [23]. An interesting insight comes from the mouse somatosensory cortex, where sensory experience was shown to drive pruning via competition between neighboring dendritic spines for limited cadherin/catenin adhesion complexes. Redistribution of these adhesion complexes from less active to more active spines results in maturation of the ‘winner’ and pruning of the ‘loser’ [66]. The molecular basis for this activity-induced redistribution of adhesion molecules is a fascinating topic for further investigation. Finally, as the function of microglia in synapse elimination during neurological disorders has been highlighted in recent years, other cell–cell interactions are also likely to play roles in aberrant pruning during disease. For example, inappropriate PS exposure might result in pathological neurite phagocytosis. Therefore, a global understanding of the mechanisms that operate within and between cells during developmental remodeling could provide insights into similar mechanisms during disease. To achieve these goals, the discovery of new neurons that undergo remodeling is critical. This should uncover entire circuits that remodel together and thereby provide excellent biological systems to address the various aspects covered in this review.
Conflict of interest statement Nothing declared.
Acknowledgements We thank Tal Bigdari for graphical assistance; Research in our lab has been funded mainly by the Israel Science Foundation (ISF), and the European Research Council (erc) consolidator (erc CoG # 615906). OS is an Incumbent of the Prof. Erwin Netter Professorial Chair of Cell Biology
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39. Sar Shalom H, Goldner R, Golan-Vaishenker Y, Yaron A: Balance between BDNF and Semaphorins gates the innervation of the mammary gland. eLife 2019, 8 Sar Shalom et al. demonstrate involvement of Semaphorin signaling during the development and male-specific pruning of mammary gland innervation, previously shown to rely on BDNF signaling. BDNF and semaphorins are secreted by mesenchimal and epithelial cells respectively, and have opposing effects, so that precise innervation is determined by their balance. This is further validated in vitro, as sensory axons become hypersensitive to Semaphorins under low BDNF levels and show increased growth cone collapse, which may represent an early step of axon pruning. This study highlights the importance of surrounding cells during remodeling, as counteracting molecular cues derived from two distinct non-neuronal cell types are crucial for determining the final innervation. 40. Vanderhaeghen P, Cheng HJ: Guidance molecules in axon pruning and cell death. Cold Spring Harb Perspect Biol 2010, 2: a001859. 41. Waimey KE, Cheng HJ: Axon pruning and synaptic development: how are they per-plexin? Neuroscientist 2006, 12:398-409. 42. Takahashi S, Ishida A, Kubo A, Kawasaki H, Ochiai S, Nakayama M, Koseki H, Amagai M, Okada T: Homeostatic pruning and activity of epidermal nerves are dysregulated in barrier-impaired skin during chronic itch development. Sci Rep 2019, 9:8625 Using intravital imaging of the epidermis, which highlights the extremely dynamic nature of the skin, the authors demonstrate that growing nerves penetrate the epidermal tight junctions in atopic dermatitis (AD). Consequently, the penetrating neurites are exposed to noxious stimuli leading to pathological itch, as reflected by aberrant neuronal calcium signaling. Interestingly, the authors suggest that this is prevented in healthy skin due to constant local pruning of nerve endings at the site of newly forming tight junctions, in a yet unknown mechanism that may involve mechanical constriction and/or molecular cues. 43. Rasmussen JP, Vo NT, Sagasti A: Fish scales dictate the pattern of adult skin innervation and vascularization. Dev Cell 2018, 46:344-359.e4. 44. Watts RJ, Schuldiner O, Perrino J, Larsen C, Luo L: Glia engulf degenerating axons during developmental axon pruning. Curr Biol 2004, 14:678-684. 45. Awasaki T, Ito K: Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis. Curr Biol 2004, 14:668-677. 46. Hakim Y, Yaniv SP, Schuldiner O: Astrocytes play a key role in Drosophila mushroom body axon pruning. PLoS One 2014, 9: e86178. 47. Tasdemir-Yilmaz OE, Freeman MR: Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons. Genes Dev 2014, 28:20-33. 48. Awasaki T, Tatsumi R, Takahashi K, Arai K, Nakanishi Y, Ueda R, Ito K: Essential role of the apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning during Drosophila metamorphosis. Neuron 2006, 50:855-867. 49. MacDonald JM, Beach MG, Porpiglia E, Sheehan AE, Watts RJ, Freeman MR: The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons. Neuron 2006, 50:869-881.
35. Marmor-Kollet N, Gutman I, Issman-Zecharya N, Schuldiner O: Glial derived TGF-beta instructs axon midline stopping. Front Mol Neurosci 2019, 12:232.
50. Han C, Song Y, Xiao H, Wang D, Franc NC, Jan LY, Jan YN: Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila. Neuron 2014, 81:544-560.
36. Bialas AR, Stevens B: TGF-beta signaling regulates neuronal C1q expression and developmental synaptic refinement. Nat Neurosci 2013, 16:1773-1782.
51. Rasmussen JP, Sack GS, Martin SM, Sagasti A: Vertebrate epidermal cells are broad-specificity phagocytes that clear sensory axon debris. J Neurosci 2015, 35:559-570.
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52. Han C, Jan LY, Jan YN: Enhancer-driven membrane markers for analysis of nonautonomous mechanisms reveal neuron-glia interactions in Drosophila. Proc Natl Acad Sci U S A 2011, 108:9673-9678. 53. Sapar ML, Ji H, Wang B, Poe AR, Dubey K, Ren X, Ni JQ, Han C: Phosphatidylserine externalization results from and causes neurite degeneration in Drosophila. Cell Rep 2018, 24:2273-2286 These two studies highlight the potential role of Phosphatidylserine (PS) as an ‘eat me’ signal in the context of neurite pruning and axon injury. Using a similar genetically encoded PS reporter, work from the Han and Yaron laboratories has demonstrated that PS externalization occurs before neurite fragmentation both in vivo in flies and in mammalian in vitro culture systems. Genetic perturbations in the fly, resulting in ectopic PS externalization, resulted in extensive degeneration and potential draper mediated engulfment by epidermal cells. Especially interesting in the context of this review, as localized PS externalization may provide spatiotemporal control of pruning. Potential mechanisms for such localized PS regulation, are however unknown. 54. Shacham-Silverberg V, Sar Shalom H, Goldner R, Golan Vaishenker Y, Gurwicz N, Gokhman I, Yaron A: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration. Cell Death Dis 2018, 9:1116 These two studies highlight the potential role of Phosphatidylserine (PS) as an ‘eat me’ signal in the context of neurite pruning and axon injury. Using a similar genetically encoded PS reporter, work from the Han and Yaron laboratories has demonstrated that PS externalization occurs before neurite fragmentation both in vivo in flies and in mammalian in vitro culture systems. Genetic perturbations in the fly, resulting in ectopic PS externalization, resulted in extensive degeneration and potential draper mediated engulfment by epidermal cells. Especially interesting in the context of this review, as localized PS externalization may provide spatiotemporal control of pruning. Potential mechanisms for such localized PS regulation, are however unknown. 55. Williamson AP, Vale RD: Spatial control of Draper receptor signaling initiates apoptotic cell engulfment. J Cell Biol 2018, 217:3977-3992.
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