Divergent signaling requirements of dSARM in injury-induced degeneration and developmental glial phagocytosis.

Elucidating signal transduction mechanisms of innate immune pathways is essential to defining how they elicit distinct cellular responses. Toll-like receptors (TLR) signal through their cytoplasmic TIR domains which bind other TIR domain-containing adaptors. dSARM/SARM1 is one such TIR domain adaptor best known for its role as the central axon degeneration trigger after injury. In degeneration, SARM1's domains have been assigned unique functions: the ARM domain is auto-inhibitory, SAM-SAM domain interactions mediate multimerization, and the TIR domain has intrinsic NAD+ hydrolase activity that precipitates axonal demise. Whether and how these distinct functions contribute to TLR signaling is unknown. Here we show divergent signaling requirements for dSARM in injury-induced axon degeneration and TLR-mediated developmental glial phagocytosis through analysis of new knock-in domain and point mutations. We demonstrate intragenic complementation between reciprocal pairs of domain mutants during development, providing evidence for separability of dSARM functional domains in TLR signaling. Surprisingly, dSARM's NAD+ hydrolase activity is strictly required for both degenerative and developmental signaling, demonstrating that TLR signal transduction requires dSARM's enzymatic activity. In contrast, while SAM domain-mediated dSARM multimerization is important for axon degeneration, it is dispensable for TLR signaling. Finally, dSARM functions in a linear genetic pathway with the MAP3K Ask1 during development but not in degenerating axons. Thus, we propose that dSARM exists in distinct signaling states in developmental and pathological contexts.

Keeping Neurons Young and Foxy: FoxOs Promote Neuronal Plasticity.

Any adult who has tried to take up the piano or learn a new language is faced with the sobering realization that acquiring such skills is more challenging as an adult than as a child. Neuronal plasticity, or the malleability of brain circuits, declines with age. Young neurons tend to be more adaptable and can alter the size and strength of their connections more readily than can old neurons. Myriad circuit- and synapse-level mechanisms that shape plasticity have been identified. Yet, molecular mechanisms setting the overall competence of young neurons for distinct forms of plasticity remain largely obscure. Recent studies indicate evolutionarily conserved roles for FoxO proteins in establishing the capacity for cell-fate, morphological, and synaptic plasticity in neurons.

FoxO regulates microtubule dynamics and polarity to promote dendrite branching in Drosophila sensory neurons.

The size and shape of dendrite arbors are defining features of neurons and critical determinants of neuronal function. The molecular mechanisms establishing arborization patterns during development are not well understood, though properly regulated microtubule (MT) dynamics and polarity are essential. We previously found that FoxO regulates axonal MTs, raising the question of whether it also regulates dendritic MTs and morphology. Here we demonstrate that FoxO promotes dendrite branching in all classes of Drosophila dendritic arborization (da) neurons. FoxO is required both for initiating growth of new branches and for maintaining existing branches. To elucidate FoxO function, we characterized MT organization in both foxO null and overexpressing neurons. We find that FoxO directs MT organization and dynamics in dendrites. Moreover, it is both necessary and sufficient for anterograde MT polymerization, which is known to promote dendrite branching. Lastly, FoxO promotes proper larval nociception, indicating a functional consequence of impaired da neuron morphology in foxO mutants. Together, our results indicate that FoxO regulates dendrite structure and function and suggest that FoxO-mediated pathways control MT dynamics and polarity.

Crimpy inhibits the BMP homolog Gbb in motoneurons to enable proper growth control at the Drosophila neuromuscular junction.

The BMP pathway is essential for scaling of the presynaptic motoneuron arbor to the postsynaptic muscle cell at the Drosophila neuromuscular junction (NMJ). Genetic analyses indicate that the muscle is the BMP-sending cell and the motoneuron is the BMP-receiving cell. Nevertheless, it is unclear how this directionality is established as Glass bottom boat (Gbb), the known BMP ligand, is active in motoneurons. We demonstrate that crimpy (cmpy) limits neuronal Gbb activity to permit appropriate regulation of NMJ growth. cmpy was identified in a screen for motoneuron-expressed genes and encodes a single-pass transmembrane protein with sequence homology to vertebrate Cysteine-rich transmembrane BMP regulator 1 (Crim1). We generated a targeted deletion of the cmpy locus and find that loss-of-function mutants exhibit excessive NMJ growth. In accordance with its expression profile, tissue-specific rescue experiments indicate that cmpy functions neuronally. The overgrowth in cmpy mutants depends on the activity of the BMP type II receptor Wishful thinking, arguing that Cmpy acts in the BMP pathway upstream of receptor activation and raising the possibility that it inhibits Gbb activity in motoneurons. Indeed, the cmpy mutant phenotype is strongly suppressed by RNAi-mediated knockdown of Gbb in motoneurons. Furthermore, Cmpy physically interacts with the Gbb precursor protein, arguing that Cmpy binds Gbb prior to the secretion of mature ligand. These studies demonstrate that Cmpy restrains Gbb activity in motoneurons. We present a model whereby this inhibition permits the muscle-derived Gbb pool to predominate at the NMJ, thus establishing the retrograde directionality of the pro-growth BMP pathway.

EVs move messes, not messages, at the synapse.

Extracellular vesicles are known for intercellular signaling roles but can also serve to simply dispose of unwanted cargoes. In this issue, Bostelman and Broihier discuss new work from Rodal and colleagues (https://doi.org/10.1083/jcb.202405025) that refutes prior work by showing that extracellular vesicles at Drosophila neuromuscular junctions are not required for signaling and instead likely serve a proteostasis role.

Glia control experience-dependent plasticity in an olfactory critical period.

Sensory experience during developmental critical periods has lifelong consequences for circuit function and behavior, but the molecular and cellular mechanisms through which experience causes these changes are not well understood. The Drosophila antennal lobe houses synapses between olfactory sensory neurons (OSNs) and downstream projection neurons (PNs) in stereotyped glomeruli. Many glomeruli exhibit structural plasticity in response to early-life odor exposure, indicating a general sensitivity of the fly olfactory circuitry to early sensory experience. We recently found that glia regulate the development of the antennal lobe in young adult flies, leading us to ask if glia also drive experience-dependent plasticity. Here we define a critical period for structural and functional plasticity of OSN-PN synapses in the ethyl butyrate (EB)-sensitive glomerulus VM7. EB exposure for the first two days post-eclosion drives large-scale reductions in glomerular volume, presynapse number, and post-synaptic activity. The highly conserved engulfment receptor Draper is required for this critical period plasticity. Specifically, ensheathing glia upregulate Draper expression, invade the VM7 glomerulus, and phagocytose OSN presynaptic terminals in response to critical-period EB exposure. Crucially, synapse pruning during the critical period has long-term consequences for circuit function since both OSN-PN synapse number and spontaneous activity of PNs remain persistently decreased. These data demonstrate experience-dependent pruning of synapses in olfactory circuitry and argue that the Drosophila antennal lobe will be a powerful model for defining the function of glia in critical period plasticity.

Early Draper-mediated glial refinement of neuropil architecture and synapse number in the Drosophila antennal lobe.

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.

Drosophila MMP2 regulates the matrix molecule faulty attraction (Frac) to promote motor axon targeting in Drosophila.

Matrix metalloproteinases (MMPs) are widely hypothesized to regulate signaling events through processing of extracellular matrix (ECM) molecules. We previously demonstrated that membrane-associated Mmp2 is expressed in exit glia and contributes to motor axon targeting. To identify possible substrates, we undertook a yeast interaction screen for Mmp2-binding proteins and identified the novel ECM protein faulty attraction (Frac). Frac encodes a multidomain extracellular protein rich in epidermal growth factor (EGF) and calcium-binding EGF domains, related to the vertebrate Fibrillin and Fibulin gene families. It is expressed in mesodermal domains flanking Mmp2-positive glia. The juxtaposition of Mmp2 and Frac proteins raises the possibility that Frac is a proteolytic target of Mmp2. Consistent with this hypothesis, levels of full-length Frac are increased in Mmp2 loss-of-function (LOF) and decreased in Mmp2 gain-of-function (GOF) embryos, indicating that Frac cleavage is Mmp2 dependent. To test whether frac is necessary for axon targeting, we characterized guidance in frac LOF mutants. Motor axons in frac LOF embryos are loosely associated and project ectopically, a phenotype essentially equivalent to that of Mmp2 LOF. The phenotypic similarity between enzyme and substrate mutants argues that Mmp2 activates Frac. In addition, Mmp2 overexpression pathfinding phenotypes depend on frac activity, indicating that Mmp2 is genetically upstream of frac. Last, overexpression experiments suggest that Frac is unlikely to have intrinsic signaling activity, raising the possibility that an Mmp2-generated Frac fragment acts as a guidance cue cofactor. Indeed, we present genetic evidence that Frac regulates a non-canonical LIM kinase 1-dependent bone morphogenetic protein signaling pathway in motoneurons necessary for axon pathfinding during embryogenesis.

The cytoplasmic adaptor protein Caskin mediates Lar signal transduction during Drosophila motor axon guidance.

The multiprotein complexes that receive and transmit axon pathfinding cues during development are essential to circuit generation. Here, we identify and characterize the Drosophila sterile α-motif (SAM) domain-containing protein Caskin, which shares homology with vertebrate Caskin, a CASK [calcium/calmodulin-(CaM)-activated serine-threonine kinase]-interacting protein. Drosophila caskin (ckn) is necessary for embryonic motor axon pathfinding and interacts genetically and physically with the leukocyte common antigen-related (Lar) receptor protein tyrosine phosphatase. In vivo and in vitro analyses of a panel of ckn loss-of-function alleles indicate that the N-terminal SAM domain of Ckn mediates its interaction with Lar. Like Caskin, Liprin-α is a neuronal adaptor protein that interacts with Lar via a SAM domain-mediated interaction. We present evidence that Lar does not bind Caskin and Liprin-α concurrently, suggesting they may assemble functionally distinct signaling complexes on Lar. Furthermore, a vertebrate Caskin homolog interacts with LAR family members, arguing that the role of ckn in Lar signal transduction is evolutionarily conserved. Last, we characterize several ckn mutants that retain Lar binding yet display guidance defects, implying the existence of additional Ckn binding partners. Indeed, we identify the SH2/SH3 adaptor protein Dock as a second Caskin-binding protein and find that Caskin binds Lar and Dock through distinct domains. Furthermore, whereas ckn has a nonredundant function in Lar-dependent signaling during motor axon targeting, ckn and dock have overlapping roles in axon outgrowth in the CNS. Together, these studies identify caskin as a neuronal adaptor protein required for axon growth and guidance.

Pyroptosis Takes Aim at Neurodevelopment.

In a recent issue of Nature, Lammert et al. demonstrate that DNA damage drives AIM2-mediated pyroptosis during normal brain development, preventing anxiety-like behaviors acquisition in adults and revealing an important role for non-apoptotic mechanisms of cell death during neurodevelopment.

Expecto Patronin for slow and persistent minus end microtubule growth in dendrites.

Microtubule plus ends are highly dynamic in neurons, while minus ends are often capped and stable. In this issue, Feng et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201810155) demonstrate that in dendrites, free minus ends undergo slow and processive growth mediated by the minus end-binding protein Patronin.

Dying Neurons Utilize Innate Immune Signaling to Prime Glia for Phagocytosis during Development.

Glia continuously survey neuronal health during development, providing trophic support to healthy neurons while rapidly engulfing dying ones. These diametrically opposed functions necessitate a foolproof mechanism enabling glia to unambiguously identify those neurons to support versus those to engulf. To ensure specificity, glia are proposed to interact with dying neurons via a series of carefully choreographed steps. However, these crucial interactions are largely obscure. Here we show that dying neurons and glia communicate via Toll-receptor-regulated innate immune signaling. Neuronal apoptosis drives processing and activation of the Toll-6 ligand, Spätzle5. This cue activates a dSARM-mediated Toll-6 transcriptional pathway in glia, which controls the expression of the Draper engulfment receptor. Pathway loss drives early-onset neurodegeneration, underscoring its functional importance. Our results identify an upstream priming signal that prepares glia for phagocytosis. Thus, a core innate immune pathway plays an unprecedented role setting the valence of neuron-glia interactions during development.

The calcium channel subunit α2δ-3 organizes synapses via an activity-dependent and autocrine BMP signaling pathway.

Synapses are highly specialized for neurotransmitter signaling, yet activity-dependent growth factor release also plays critical roles at synapses. While efficient neurotransmitter signaling relies on precise apposition of release sites and neurotransmitter receptors, molecular mechanisms enabling high-fidelity growth factor signaling within the synaptic microenvironment remain obscure. Here we show that the auxiliary calcium channel subunit α2δ-3 promotes the function of an activity-dependent autocrine Bone Morphogenetic Protein (BMP) signaling pathway at the Drosophila neuromuscular junction (NMJ). α2δ proteins have conserved synaptogenic activity, although how they execute this function has remained elusive. We find that α2δ-3 provides an extracellular scaffold for an autocrine BMP signal, suggesting a mechanistic framework for understanding α2δ's conserved role in synapse organization. We further establish a transcriptional requirement for activity-dependent, autocrine BMP signaling in determining synapse density, structure, and function. We propose that activity-dependent, autocrine signals provide neurons with continuous feedback on their activity state for modulating both synapse structure and function.

Drosophila homeodomain protein dHb9 directs neuronal fate via crossrepressive and cell-nonautonomous mechanisms.

Here we present the identification and characterization of dHb9, the Drosophila homolog of vertebrate Hb9, which encodes a factor central to motorneuron (MN) development. We show that dHb9 regulates neuronal fate by restricting expression of Lim3 and Even-skipped (Eve), two homeodomain (HD) proteins required for development of distinct neuronal classes. Also, dHb9 and Lim3 are activated independently of each other in a virtually identical population of ventrally and laterally projecting MNs. Surprisingly, dHb9 represses Lim3 cell nonautonomously in a subset of dorsally projecting MNs, revealing a novel role for intercellular signaling in the establishment of neuronal fate in Drosophila. Lastly, we provide evidence that dHb9 and Eve regulate each other's expression through Groucho-dependent crossrepression. This mutually antagonistic relationship bears similarity to the crossrepressive relationships between pairs of HD proteins that pattern the vertebrate neural tube.

What neurons tell themselves: autocrine signals play essential roles in neuronal development and function.

Although retrograde neurotrophin signaling has provided an immensely influential paradigm for understanding growth factor signaling in the nervous system, recent studies indicate that growth factors also signal via cell-autonomous, or autocrine, mechanisms. Autocrine signals have been discovered in many neuronal contexts, providing insights into their regulation and function. The growing realization of the importance of cell-autonomous signaling stems from advances in both conditional genetic approaches and in sophisticated analyses of growth factor dynamics, which combine to enable rigorous in vivo dissection of signaling pathways. Here we review recent studies defining autocrine roles for growth factors such as BDNF, and classical morphogens, including Wnts and BMPs, in regulating neuronal development and plasticity. Collectively, these studies highlight an intimate relationship between activity-dependent autocrine signaling and synaptic plasticity, and further suggest a common principle for coordinating paracrine and autocrine signaling in the nervous system.

MYRFs on the Move to Rewire Circuits.

Synaptic plasticity occurs in response to intrinsic and extrinsic cues and is a key step in the formation of mature neuronal circuits. In this issue of Developmental Cell, Meng et al. (2017) find that two conserved Myrf transcription factors coexist in the same complex to promote developmental circuit remodeling.

BMP signaling turns up in fragile X syndrome: FMRP represses BMPR2.

Fragile X syndrome is the most common inherited form of intellectual disability and results from a loss of function of the translational repressor FMRP. In this issue of Science Signaling, Kashima et al find that FMRP binds to and represses a specific isoform of BMPR2, a type II bone morphogenetic protein (BMP) receptor. Reducing signaling through this BMP pathway reverses neuroanatomical defects observed in fragile X models.

A Toll receptor-FoxO pathway represses Pavarotti/MKLP1 to promote microtubule dynamics in motoneurons.

FoxO proteins are evolutionarily conserved regulators of neuronal structure and function, yet the neuron-specific pathways within which they act are poorly understood. To elucidate neuronal FoxO function in Drosophila melanogaster, we first screened for FoxO's upstream regulators and downstream effectors. On the upstream side, we present genetic and molecular pathway analyses indicating that the Toll-6 receptor, the Toll/interleukin-1 receptor domain adaptor dSARM, and FoxO function in a linear pathway. On the downstream side, we find that Toll-6-FoxO signaling represses the mitotic kinesin Pavarotti/MKLP1 (Pav-KLP), which itself attenuates microtubule (MT) dynamics. We next probed in vivo functions for this novel pathway and found that it is essential for axon transport and structural plasticity in motoneurons. We demonstrate that elevated expression of Pav-KLP underlies transport and plasticity phenotypes in pathway mutants, indicating that Toll-6-FoxO signaling promotes MT dynamics by limiting Pav-KLP expression. In addition to uncovering a novel molecular pathway, our work reveals an unexpected function for dynamic MTs in enabling rapid activity-dependent structural plasticity.

Crimpy enables discrimination of presynaptic and postsynaptic pools of a BMP at the Drosophila neuromuscular junction.

Distinct pools of the bone morphogenetic protein (BMP) Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, whereas muscle-derived Gbb regulates neuromuscular junction growth. Yet how cells differentiate between these ligand pools is not known. Here we present evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre- and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbb's proneurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, we propose that Gbb/Cmpy corelease from presynaptic terminals defines a neuronal protransmission signal.

FoxO limits microtubule stability and is itself negatively regulated by microtubule disruption.

Transcription factors are essential for regulating neuronal microtubules (MTs) during development and after axon damage. In this paper, we identify a novel neuronal function for Drosophila melanogaster FoxO in limiting MT stability at the neuromuscular junction (NMJ). foxO loss-of-function NMJs displayed augmented MT stability. In contrast, motor neuronal overexpression of wild-type FoxO moderately destabilized MTs, whereas overexpression of constitutively nuclear FoxO severely destabilized MTs. Thus, FoxO negatively regulates synaptic MT stability. FoxO family members are well-established components of stress-activated feedback loops. We hypothesized that FoxO might also be regulated by cytoskeletal stress because it was well situated to shape neuronal MT organization after cytoskeletal damage. Indeed, levels of neuronal FoxO were strongly reduced after acute pharmacological MT disruption as well as sustained genetic disruption of the neuronal cytoskeleton. This decrease was independent of the dual leucine zipper kinase-Wallenda pathway and required function of Akt kinase. We present a model wherein FoxO degradation is a component of a stabilizing, protective response to cytoskeletal insult.