Balancing act of small GTPases downstream of plexin-A4 signaling motifs promotes dendrite elaboration in mammalian cortical neurons.

The precise development of neuronal morphologies is crucial to the establishment of synaptic circuits and, ultimately, proper brain function. Signaling by the axon guidance cue semaphorin 3A (Sema3A) and its receptor complex of neuropilin-1 and plexin-A4 has multifunctional outcomes in neuronal morphogenesis. Downstream activation of the RhoGEF FARP2 through interaction with the lysine-arginine-lysine motif of plexin-A4 and consequent activation of the small GTPase Rac1 promotes dendrite arborization, but this pathway is dispensable for axon repulsion. Here, we investigated the interplay of small GTPase signaling mechanisms underlying Sema3A-mediated dendritic elaboration in mouse layer V cortical neurons in vitro and in vivo. Sema3A promoted the binding of the small GTPase Rnd1 to the amino acid motif lysine-valine-serine (LVS) in the cytoplasmic domain of plexin-A4. Rnd1 inhibited the activity of the small GTPase RhoA and the kinase ROCK, thus supporting the activity of the GTPase Rac1, which permitted the growth and branching of dendrites. Overexpression of a dominant-negative RhoA, a constitutively active Rac1, or the pharmacological inhibition of ROCK activity rescued defects in dendritic elaboration in neurons expressing a plexin-A4 mutant lacking the LVS motif. Our findings provide insights into the previously unappreciated balancing act between Rho and Rac signaling downstream of specific motifs in plexin-A4 to mediate Sema3A-dependent dendritic elaboration in mammalian cortical neuron development.

Kinesin family member 2A gates nociception.

Nociceptive axons undergo remodeling as they innervate their targets during development and in response to environmental insults and pathological conditions. How is nociceptive morphogenesis regulated? Here, we show that the microtubule destabilizer kinesin family member 2A (Kif2a) is a key regulator of nociceptive terminal structures and pain sensitivity. Ablation of Kif2a in sensory neurons causes hyperinnervation and hypersensitivity to noxious stimuli in young adult mice, whereas touch sensitivity and proprioception remain unaffected. Computational modeling predicts that structural remodeling is sufficient to explain the phenotypes. Furthermore, Kif2a deficiency triggers a transcriptional response comprising sustained upregulation of injury-related genes and homeostatic downregulation of highly specific channels and receptors at the late stage. The latter effect can be predicted to relieve the hyperexcitability of nociceptive neurons, despite persisting morphological aberrations, and indeed correlates with the resolution of pain hypersensitivity. Overall, we reveal a critical control node defining nociceptive terminal structure, which is regulating nociception.

A duplex structure of SARM1 octamers stabilized by a new inhibitor.

In recent years, there has been growing interest in SARM1 as a potential breakthrough drug target for treating various pathologies of axon degeneration. SARM1-mediated axon degeneration relies on its TIR domain NADase activity, but recent structural data suggest that the non-catalytic ARM domain could also serve as a pharmacological site as it has an allosteric inhibitory function. Here, we screened for synthetic small molecules that inhibit SARM1, and tested a selected set of these compounds in a DRG axon degeneration assay. Using cryo-EM, we found that one of the newly discovered inhibitors, a calmidazolium designated TK106, not only stabilizes the previously reported inhibited conformation of the octamer, but also a meta-stable structure: a duplex of octamers (16 protomers), which we have now determined to 4.0 Å resolution. In the duplex, each ARM domain protomer is engaged in lateral interactions with neighboring protomers, and is further stabilized by contralateral contacts with the opposing octamer ring. Mutagenesis of the duplex contact sites leads to a moderate increase in SARM1 activation in cultured cells. Based on our data we propose that the duplex assembly constitutes an additional auto-inhibition mechanism that tightly prevents pre-mature activation and axon degeneration.

Cracking the combinatorial code of neuronal wiring.

How transcription factors orchestrate the combinatorial expression of cell-surface proteins that, in turn, specify the wiring of the nervous system is an open question. In this issue of Neuron, Xie et al. reveal a new, unexpected layer of complexity.

Structural basis for SARM1 inhibition and activation under energetic stress.

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1's own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.

Modular and Distinct Plexin-A4/FARP2/Rac1 Signaling Controls Dendrite Morphogenesis.

Diverse neuronal populations with distinct cellular morphologies coordinate the complex function of the nervous system. Establishment of distinct neuronal morphologies critically depends on signaling pathways that control axonal and dendritic development. The Sema3A-Nrp1/PlxnA4 signaling pathway promotes cortical neuron basal dendrite arborization but also repels axons. However, the downstream signaling components underlying these disparate functions of Sema3A signaling are unclear. Using the novel PlxnA4KRK-AAA knock-in male and female mice, generated by CRISPR/cas9, we show here that the KRK motif in the PlxnA4 cytoplasmic domain is required for Sema3A-mediated cortical neuron dendritic elaboration but is dispensable for inhibitory axon guidance. The RhoGEF FARP2, which binds to the KRK motif, shows identical functional specificity as the KRK motif in the PlxnA4 receptor. We find that Sema3A activates the small GTPase Rac1, and that Rac1 activity is required for dendrite elaboration but not axon growth cone collapse. This work identifies a novel Sema3A-Nrp1/PlxnA4/FARP2/Rac1 signaling pathway that specifically controls dendritic morphogenesis but is dispensable for repulsive guidance events. Overall, our results demonstrate that the divergent signaling output from multifunctional receptor complexes critically depends on distinct signaling motifs, highlighting the modular nature of guidance cue receptors and its potential to regulate diverse cellular responses.SIGNIFICANCE STATEMENT The proper formation of axonal and dendritic morphologies is crucial for the precise wiring of the nervous system that ultimately leads to the generation of complex functions in an organism. The Semaphorin3A-Neuropilin1/Plexin-A4 signaling pathway has been shown to have multiple key roles in neurodevelopment, from axon repulsion to dendrite elaboration. This study demonstrates that three specific amino acids, the KRK motif within the Plexin-A4 receptor cytoplasmic domain, are required to coordinate the downstream signaling molecules to promote Sema3A-mediated cortical neuron dendritic elaboration, but not inhibitory axon guidance. Our results unravel a novel Semaphorin3A-Plexin-A4 downstream signaling pathway and shed light on how the disparate functions of axon guidance and dendritic morphogenesis are accomplished by the same extracellular ligand in vivo.

Regulation of axonal morphogenesis by the mitochondrial protein Efhd1.

During development, neurons adjust their energy balance to meet the high demands of robust axonal growth and branching. The mechanisms that regulate this tuning are largely unknown. Here, we show that sensory neurons lacking liver kinase B1 (Lkb1), a master regulator of energy homeostasis, exhibit impaired axonal growth and branching. Biochemical analysis of these neurons revealed reduction in axonal ATP levels, whereas transcriptome analysis uncovered down-regulation of Efhd1 (EF-hand domain family member D1), a mitochondrial Ca2+-binding protein. Genetic ablation of Efhd1 in mice resulted in reduced axonal morphogenesis as well as enhanced neuronal death. Strikingly, this ablation causes mitochondrial dysfunction and a decrease in axonal ATP levels. Moreover, Efhd1 KO sensory neurons display shortened mitochondria at the axonal growth cones, activation of the AMP-activated protein kinase (AMPK)-Ulk (Unc-51-like autophagy-activating kinase 1) pathway and an increase in autophagic flux. Overall, this work uncovers a new mitochondrial regulator that is required for axonal morphogenesis.

Balance between BDNF and Semaphorins gates the innervation of the mammary gland.

The innervation of the mammary gland is controlled by brain-derived neurotrophic factor (BDNF), and sexually dimorphic sequestering of BDNF by the truncated form of TrkB (TrkB.T1) directs male-specific axonal pruning in mice. It is unknown whether other cues modulate these processes. We detected specific, non-dimorphic, expression of Semaphorin family members in the mouse mammary gland, which signal through PlexinA4. PlexinA4 deletion in both female and male embryos caused developmental hyperinnervation of the gland, which could be reduced by genetic co-reduction of BDNF. Moreover, in males, PlexinA4 ablation delayed axonal pruning, independently of the initial levels of innervation. In support of this, in vitro reduction of BDNF induced axonal hypersensitivity to PlexinA4 signaling. Overall, our study shows that precise sensory innervation of the mammary gland is regulated by the balance between trophic and repulsive signaling. Upon inhibition of trophic signaling, these repulsive factors may promote axonal pruning.

Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration.

Apoptotic cells expose Phosphatidylserine (PS), that serves as an "eat me" signal for engulfing cells. Previous studies have shown that PS also marks degenerating axonsduring developmental pruning or in response to insults (Wallerian degeneration), but the pathways that control PS exposure on degenerating axons are largely unknown. Here, we used a series of in vitro assays to systematically explore the regulation of PS exposure during axonal degeneration. Our results show that PS exposure is regulated by the upstream activators of axonal pruning and Wallerian degeneration. However, our investigation of signaling further downstream revealed divergence between axon degeneration and PS exposure. Importantly, elevation of the axonal energetic status hindered PS exposure, while inhibition of mitochondrial activity caused PS exposure, without degeneration. Overall, our results suggest that the levels of PS on the outer axonal membrane can be dissociated from the degeneration process and that the axonal energetic status plays a key role in the regulation of PS exposure.

Brown-adipose-tissue macrophages control tissue innervation and homeostatic energy expenditure.

Tissue macrophages provide immunological defense and contribute to the establishment and maintenance of tissue homeostasis. Here we used constitutive and inducible mutagenesis to delete the nuclear transcription regulator Mecp2 in macrophages. Mice that lacked the gene encoding Mecp2, which is associated with Rett syndrome, in macrophages did not show signs of neurodevelopmental disorder but displayed spontaneous obesity, which was linked to impaired function of brown adipose tissue (BAT). Specifically, mutagenesis of a BAT-resident Cx3Cr1+ macrophage subpopulation compromised homeostatic thermogenesis but not acute, cold-induced thermogenesis. Mechanistically, malfunction of BAT in pre-obese mice with mutant macrophages was associated with diminished sympathetic innervation and local titers of norepinephrine, which resulted in lower expression of thermogenic factors by adipocytes. Mutant macrophages overexpressed the signaling receptor and ligand PlexinA4, which might contribute to the phenotype by repulsion of sympathetic axons expressing the transmembrane semaphorin Sema6A. Collectively, we report a previously unappreciated homeostatic role for macrophages in the control of tissue innervation. Disruption of this circuit in BAT resulted in metabolic imbalance.

TIR Axons Apart: Unpredicted NADase Controls Axonal Degeneration.

SARM1 is a key regulator of axonal degeneration. However, SARM1 mechanism of action is not clear. In this issue of Neuron, Essuman et al. (2017) reveal an intrinsic NADase activity in the SARM1-TIR domain that is required for axonal degeneration.

Harnessing the natural inhibitory domain to control TNFα Converting Enzyme (TACE) activity in vivo.

Dysregulated activity of A Disintegrin And Metalloproteinase 17 (ADAM17)/TNFα Converting Enzyme (TACE) is associated with inflammatory disorders and cancer progression by releasing regulatory membrane-tethered proteins like TNFα, IL6R and EGFR ligands. Although specific inhibition of TACE is thought to be a viable strategy for inflammatory disorders and for malignancies treatment, the generation of effective inhibitors in vivo has been proven to be challenging. Here we report on the development of a protein inhibitor that leverages the endogenous modulator of TACE. We have generated a stable form of the auto-inhibitory TACE prodomain (TPD), which specifically inhibits in vitro and cell-surface TACE, but not the related ADAM10, and effectively modulated TNFα secretion in cells. TPD significantly attenuated TACE-mediated disease models of sepsis, rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), and reduced TNFα in synovial fluids from RA patients. Our results demonstrate that intervening with endogenous ADAM sheddase modulatory mechanisms holds potential as a general strategy for the design of ADAM inhibitors.

Axonal Degeneration Is Regulated by a Transcriptional Program that Coordinates Expression of Pro- and Anti-degenerative Factors.

Developmental neuronal cell death and axonal elimination are controlled by transcriptional programs, of which their nature and the function of their components remain elusive. Here, we identified the dual specificity phosphatase Dusp16 as part of trophic deprivation-induced transcriptome in sensory neurons. Ablation of Dusp16 enhanced axonal degeneration in response to trophic withdrawal, suggesting that it has a protective function. Moreover, axonal skin innervation was severely reduced while neuronal elimination was increased in the Dusp16 knockout. Mechanistically, Dusp16 negatively regulates the transcription factor p53 and antagonizes the expression of the pro-degenerative factor, Puma (p53 upregulated modulator of apoptosis). Co-ablation of Puma with Dusp16 protected axons from rapid degeneration and specifically reversed axonal innervation loss early in development with no effect on neuronal deficits. Overall, these results reveal that physiological axonal elimination is regulated by a transcriptional program that integrates regressive and progressive elements and identify Dusp16 as a new axonal preserving factor.

Nucleolin-Mediated RNA Localization Regulates Neuron Growth and Cycling Cell Size.

How can cells sense their own size to coordinate biosynthesis and metabolism with their growth needs? We recently proposed a motor-dependent bidirectional transport mechanism for axon length and cell size sensing, but the nature of the motor-transported size signals remained elusive. Here, we show that motor-dependent mRNA localization regulates neuronal growth and cycling cell size. We found that the RNA-binding protein nucleolin is associated with importin ß1 mRNA in axons. Perturbation of nucleolin association with kinesins reduces its levels in axons, with a concomitant reduction in axonal importin ß1 mRNA and protein levels. Strikingly, subcellular sequestration of nucleolin or importin ß1 enhances axonal growth and causes a subcellular shift in protein synthesis. Similar findings were obtained in fibroblasts. Thus, subcellular mRNA localization regulates size and growth in both neurons and cycling cells.

Common and Divergent Mechanisms in Developmental Neuronal Remodeling and Dying Back Neurodegeneration.

Cell death is an inherent process that is required for the proper wiring of the nervous system. Studies over the last four decades have shown that, in a parallel developmental pathway, axons and dendrites are eliminated without the death of the neuron. This developmentally regulated 'axonal death' results in neuronal remodeling, which is an essential mechanism to sculpt neuronal networks in both vertebrates and invertebrates. Studies across various organisms have demonstrated that a conserved strategy in the formation of adult neuronal circuitry often involves generating too many connections, most of which are later eliminated with high temporal and spatial resolution. Can neuronal remodeling be regarded as developmentally and spatially regulated neurodegeneration? It has been previously speculated that injury-induced degeneration (Wallerian degeneration) shares some molecular features with 'dying back' neurodegenerative diseases. In this opinion piece, we examine the similarities and differences between the mechanisms regulating neuronal remodeling and those being perturbed in dying back neurodegenerative diseases. We focus primarily on amyotrophic lateral sclerosis and peripheral neuropathies and highlight possible shared pathways and mechanisms. While mechanistic data are only just beginning to emerge, and despite the inherent differences between disease-oriented and developmental processes, we believe that some of the similarities between these developmental and disease-initiated degeneration processes warrant closer collaborations and crosstalk between these different fields.

Mechanisms of developmental neurite pruning.

The precise wiring of the nervous system is a combined outcome of progressive and regressive events during development. Axon guidance and synapse formation intertwined with cell death and neurite pruning sculpt the mature circuitry. It is now well recognized that pruning of dendrites and axons as means to refine neuronal networks, is a wide spread phenomena required for the normal development of vertebrate and invertebrate nervous systems. Here we will review the arising principles of cellular and molecular mechanisms of neurite pruning. We will discuss these principles in light of studies in multiple neuronal systems, and speculate on potential explanations for the emergence of neurite pruning as a mechanism to sculpt the nervous system.