Impaired disassembly of the axon initial segment restricts mitochondrial entry into damaged axons.

The proteasome is essential for cellular responses to various physiological stressors. However, how proteasome function impacts the stress resilience of regenerative damaged motor neurons remains unclear. Here, we develop a unique mouse model using a regulatory element of the activating transcription factor (Atf3) gene to label mitochondria in a damage-induced manner while simultaneously genetically disrupting the proteasome. Using this model, we observed that in injury-induced proteasome-deficient mouse motor neurons, the increase of mitochondrial influx from soma into axons is inhibited because neurons fail to disassemble ankyrin G, an organizer of the axon initial segment (AIS), in a proteasome-dependent manner. Further, these motor neurons exhibit amyotrophic lateral sclerosis (ALS)-like degeneration despite having regenerative potential. Selectively vulnerable motor neurons in SOD1G93A ALS mice, which induce ATF3 in response to pathological damage, also fail to disrupt the AIS, limiting the number of axonal mitochondria at a pre-symptomatic stage. Thus, damage-induced proteasome-sensitive AIS disassembly could be a critical post-translational response for damaged motor neurons to temporarily transit to an immature state and meet energy demands for axon regeneration or preservation.

Astrocytic phagocytosis is a compensatory mechanism for microglial dysfunction.

Microglia are the principal phagocytes that clear cell debris in the central nervous system (CNS). This raises the question, which cells remove cell debris when microglial phagocytic activity is impaired. We addressed this question using Siglechdtr mice, which enable highly specific ablation of microglia. Non-microglial mononuclear phagocytes, such as CNS-associated macrophages and circulating inflammatory monocytes, did not clear microglial debris. Instead, astrocytes were activated, exhibited a pro-inflammatory gene expression profile, and extended their processes to engulf microglial debris. This astrocytic phagocytosis was also observed in Irf8-deficient mice, in which microglia were present but dysfunctional. RNA-seq demonstrated that even in a healthy CNS, astrocytes express TAM phagocytic receptors, which were the main astrocytic phagocytic receptors for cell debris in the above experiments, indicating that astrocytes stand by in case of microglial impairment. This compensatory mechanism may be important for the maintenance or prolongation of a healthy CNS.

Repeated cold stress, an animal model for fibromyalgia, elicits proprioceptor-induced chronic pain with microglial activation in mice.

BACKGROUND: Fibromyalgia is characterized by chronic pain, fatigue, and other somatic symptoms. We have recently revealed that proprioceptor hyperactivation induces chronic pain in a rat model of myalgic encephalomyelitis. The present study explores whether similar proprioceptor-induced pain is elicited in a mouse model of fibromyalgia. METHODS: Repeated cold stress (RCS) was used as a fibromyalgia model. Pain behavior was examined using the von Frey test, and neuronal activation was examined immunohistochemically as activating transcription factor (ATF)3 expression. The Atf3:BAC transgenic mouse, in which mitochondria in hyperactivated neurons are specifically labeled by green fluorescent protein, was used to trace the activated neuronal circuit. PLX3397 (pexidartinib) was used for microglial suppression. RESULTS: RCS elicited long-lasting pain in mice. ATF3, a marker of cellular hyperactivity and injury, was expressed in the lumbar dorsal root ganglion (DRG) 2 days after RCS initiation; the majority of ATF3-expressing DRG neurons were tropomyosin receptor kinase C- and/or vesicular glutamate transporter 1-positive proprioceptors. Microglial activation and increased numbers of microglia were observed in the medial part of the nucleus proprius 5 days after RCS initiation, and in the dorsal region of the ventral horn 7 days after RCS. In the ventral horn, only a subset of motor neurons was positive for ATF3; these neurons were surrounded by activated microglia. A retrograde tracer study revealed that ATF3-positive motor neurons projected to the intrinsic muscles of the foot (IMF). Using Atf3:BAC transgenic mice, we traced hyperactivated neuronal circuits along the reflex arc. Green fluorescent protein labeling was observed in proprioceptive DRG neurons and their processes originating from the IMF, as well as in motor neurons projecting to the IMF. Microglial activation was observed along this reflex arc, and PLX3397-induced microglial ablation significantly suppressed pain behavior. CONCLUSION: Proprioceptor hyperactivation leads to local microglial activation along the reflex arc; this prolonged microglial activation may be responsible for chronic pain in the present model. Proprioceptor-induced microglial activation might be the common cause of chronic pain in both the fibromyalgia and myalgic encephalomyelitis models, although the experimental models are different.

Dynamic changes in endoplasmic reticulum morphology and its contact with the plasma membrane in motor neurons in response to nerve injury.

The endoplasmic reticulum (ER) extends throughout a cell and plays a critical role in maintaining cellular homeostasis. Changes in ER shape could provide a clue to explore the mechanisms that underlie the fate determination of neurons after axon injury because the ER drastically changes its morphology under neuronal stress to maintain cellular homeostasis and recover from damage. Because of their tiny structures and richness in the soma, the detailed morphology of the ER and its dynamics have not been well analysed. In this study, the focused ion beam/scanning electron microscopy (FIB/SEM) analysis was performed to explore the ultra-structures of the ER in the somata of motor neuron with axon regenerative injury models. In normal motor neurons, ER in the somata is abundantly localised near the perinucleus and represents lamella-like structures. After injury, analysis of the ER volume and ER branching points indicated a collapse of the normal distribution and a transformation from lamella-like structures to mesh-like structures. Furthermore, accompanied by ER accumulation near the plasma membrane (PM), the contact between the ER and PM (ER-PM contacts) significantly increased after injury. The accumulation of extended-synaptotagmin 1 (E-Syt1), a tethering protein of the ER and PM that regulates Ca2+-dependent lipid transfer, was also identified by immunohistochemistry and quantitative Real-time PCR after injury. These morphological alterations of ER and the increase in ER-PM contacts may be crucial events that occur in motor neurons as a resilient response for the survival after axonal injury.

Brain injury triggers cell-type-specific and time-dependent endoplasmic reticulum stress responses.

The unfolded protein response (UPR) is a signal transduction network that responds to endoplasmic reticulum (ER) stress by coordinating protein homeostasis to maintain cell viability. The UPR can also trigger cell death when adaptive responses fail to improve protein homeostasis. Despite accumulating evidence suggesting that the UPR plays a role in neurodegenerative diseases and brain insults, our understanding of how ER stress is induced under neuropathological conditions is limited. Here, we investigated the cell- and time-specific patterns of the ER stress response after brain injury using ER stress-activated indicator (ERAI) mice, which enable monitoring of the UPR in vivo via increased fluorescence of a spliced XBP-1 protein fused with the green fluorescent protein (GFP) variant Venus. Following cortical stab injury of ERAI mice, the GFP signal and number of GFP+ cells increased in the ipsilateral cortex throughout the observation period (6 h to 7 days post-injury), confirming the induction of the UPR. GFP signals were observed in injured neurons early (from 6 h) after brain injury. However, non-neuronal cells, mainly endothelial cells followed by astrocytes, accounted for the majority of GFP+ cells after brain injury. Similar results were obtained in a mouse model of focal cerebral ischemia. These findings suggest that activation of the UPR in both neuronal and non-neuronal cells, especially endothelial cells and astrocytes, may play an important role in and could be a potential therapeutic target for acute brain injuries.

Phagocytic astrocytes: Emerging from the shadows of microglia.

Elimination of dead or live cells take place in both a healthy and diseased central nervous system (CNS). Dying or dead cells are quickly cleared by phagocytosis for the maintenance of a healthy CNS or for recovery after injury. Live cells or parts thereof, such as the synapses and myelin, are appropriately eliminated by phagocytosis to maintain or refine neural networks during development and adulthood. Microglia, the specific population of resident macrophages in the CNS, are classically considered as primary phagocytes; however, astrocytes have also been highlighted as phagocytes in the last decade. Phagocytic targets and receptors are reported to be mostly common between astrocytes and microglia, which raises the question of how astrocytic phagocytosis differs from microglial phagocytosis, and how these two phagocytic systems cooperate. In this review, we address the consequences of astrocytic phagocytosis, particularly focusing on these elusive points.

TC10, a Rho family GTPase, is required for efficient axon regeneration in a neuron-autonomous manner.

Intracellular signaling pathways that promote axon regeneration are closely linked to the mechanism of neurite outgrowth. TC10, a signaling molecule that acts on neurite outgrowth through membrane transport, is a member of the Rho family G proteins. Axon injury increases the TC10 levels in motor neurons, suggesting that TC10 may be involved in axon regeneration. In this study, we tried to understand the roles of TC10 in the nervous system using TC10 knock-out mice. In cultured hippocampal neurons, TC10 ablation significantly reduced axon elongation without affecting ordinary polarization. We determined a role of TC10 in microtubule stabilization at the growth cone neck; therefore, we assume that TC10 limits axon retraction and promotes in vitro axon outgrowth. In addition, there were no notable differences in the size and structure of brains during prenatal and postnatal development between wild-type and TC10 knock-out mice. In motor neurons, axon regeneration after injury was strongly suppressed in mice lacking TC10 (both in conventional and injured nerve specific deletion). In retinal ganglion cells, TC10 ablation suppressed the axon regeneration stimulated by intraocular inflammation and cAMP after optic nerve crush. These results show that TC10 plays an important role in axon regeneration in both the peripheral and central nervous systems, and the role of TC10 in peripheral axon regeneration is neuron-intrinsic.

Morphology, localization, and postnatal development of dural macrophages.

The dura mater contains abundant macrophages whose functions remain largely elusive. Recent studies have demonstrated the origin, as well as the gene expression pattern, of dural macrophages (dMΦs). However, their histological features have not been explored yet. In this study, we performed immunohistochemistry and electron microscopy to elucidate their precise morphology, localization, and postnatal development in mice. We found that the morphology, as well as the localization, of dMΦs changed during postnatal development. In neonatal mice, dMΦ exhibited an amoeboid morphology. During postnatal development, their cell bodies elongated longitudinally and became aligned along dural blood vessels. In adulthood, nearly half of the dMΦs aligned along blood vessel networks. However, most of these cells were not directly attached to vessels; pericytes and fibroblasts interposed between dMΦs and vessels. This morphological information may provide further indications for the functional significance of dMΦs.

Hyperactivation of proprioceptors induces microglia-mediated long-lasting pain in a rat model of chronic fatigue syndrome.

BACKGROUND: Patients diagnosed with chronic fatigue syndrome (CFS) or fibromyalgia experience chronic pain. Concomitantly, the rat model of CFS exhibits microglial activation in the lumbar spinal cord and pain behavior without peripheral tissue damage and/or inflammation. The present study addressed the mechanism underlying the association between pain and chronic stress using this rat model. METHODS: Chronic or continuous stress-loading (CS) model rats, housed in a cage with a thin level of water (1.5 cm in depth), were used. The von Frey test and pressure pain test were employed to measure pain behavior. The neuronal and microglial activations were immunohistochemically demonstrated with antibodies against ATF3 and Iba1. Electromyography was used to evaluate muscle activity. RESULTS: The expression of ATF3, a marker of neuronal hyperactivity or injury, was first observed in the lumbar dorsal root ganglion (DRG) neurons 2 days after CS initiation. More than 50% of ATF3-positive neurons simultaneously expressed the proprioceptor markers TrkC or VGluT1, whereas the co-expression rates for TrkA, TrkB, IB4, and CGRP were lower than 20%. Retrograde labeling using fluorogold showed that ATF3-positive proprioceptive DRG neurons mainly projected to the soleus. Substantial microglial accumulation was observed in the medial part of the dorsal horn on the fifth CS day. Microglial accumulation was observed around a subset of motor neurons in the dorsal part of the ventral horn on the sixth CS day. The motor neurons surrounded by microglia were ATF3-positive and mainly projected to the soleus. Electromyographic activity in the soleus was two to three times higher in the CS group than in the control group. These results suggest that chronic proprioceptor activation induces the sequential activation of neurons along the spinal reflex arc, and the neuronal activation further activates microglia along the arc. Proprioceptor suppression by ankle joint immobilization significantly suppressed the accumulation of microglia in the spinal cord, as well as the pain behavior. CONCLUSION: Our results indicate that proprioceptor-induced microglial activation may be a key player in the initiation and maintenance of abnormal pain in patients with CFS.

GPR34 in spinal microglia exacerbates neuropathic pain in mice.

BACKGROUND: Neuropathic pain is caused by sensory nerve injury, but effective treatments are currently lacking. Microglia are activated in the spinal dorsal horn after sensory nerve injury and contribute to neuropathic pain. Accordingly, molecules expressed by these cells are considered potential targets for therapeutic strategies. Our previous gene screening study using a mouse model of motor nerve injury showed that the G-protein-coupled receptor 34 gene (GPR34) is induced by nerve injury. Because GPR34 is now considered a microglia-enriched gene, we explored the possibility that it might be involved in microglial activation in the dorsal horn in a mouse model of neuropathic pain. METHODS: mRNA expression of GPR34 and pro-inflammatory molecules was determined by quantitative real-time PCR in wild-type and GPR34-deficient mice with L4 spinal nerve injury. In situ hybridization was used to identify GPR34 expression in microglia, and immunohistochemistry with the microglial marker Iba1 was performed to examine microglial numbers and morphology. Mechanical sensitivity was evaluated by the von Frey hair test. Liquid chromatography-tandem mass spectrometry quantified expression of the ligand for GPR34, lysophosphatidylserine (LysoPS), in the dorsal horn, and a GPR34 antagonist was intrathecally administrated to examine the effect of inhibiting LysoPS-GPR34 signaling on mechanical sensitivity. RESULTS: GPR34 was predominantly expressed by microglia in the dorsal horn after L4 nerve injury. There were no histological differences in microglial numbers or morphology between WT and GPR34-deficient mice. However, nerve injury-induced pro-inflammatory cytokine expression levels in microglia and pain behaviors were significantly attenuated in GPR34-deficient mice. Furthermore, the intrathecal administration of the GPR34 antagonist reduced neuropathic pain. CONCLUSIONS: Inhibition of GPR34-mediated signal by GPR34 gene deletion reduced nerve injury-induced neuropathic pain by suppressing pro-inflammatory responses of microglia without affecting their morphology. Therefore, the suppression of GPR34 activity may have therapeutic potential for alleviating neuropathic pain.

New Insights of a Neuronal Peptidase DINE/ECEL1: Nerve Development, Nerve Regeneration and Neurogenic Pathogenesis.

Our understanding of the physiological relevance of unique Damage-induced neuronal endopeptidase (DINE) [also termed Endothelin-converting enzyme-like 1 (ECEL1)] has recently expanded. DINE/ECEL1 is a type II membrane-bound metalloprotease, belonging to a family including the neprilysin (NEP) and endothelin-converting enzyme (ECE). The family members degrade and/or process peptides such as amyloid ß and big-endothelins, which are closely associated with pathological conditions. Similar to NEP and ECE, DINE has been expected to play an important role in injured neurons as well as in developing neurons, because of its remarkable transcriptional response to neuronal insults and predominant neuronal expression from the embryonic stage. However, the physiological significance of DINE has long remained elusive. In the last decade, a series of genetically manipulated mice have driven research progress to elucidate the physiological aspects of DINE. The mice ablating Dine fail to arborize the embryonic motor axons in some subsets of muscles, including the respiratory muscles, and die immediately after birth. The abnormal phenotype of motor axons is also caused by one amino acid exchanges of DINE/ECEL1, which are responsible for distal arthrogryposis type 5 in a group of human congenital movement disorders. Furthermore, the mature Dine-deficient mice in which the lethality is rescued by genetic manipulation have shown the involvement of DINE in central nervous system regeneration. Here we describe recent research advances that DINE-mediated proteolytic processes are critical for nerve development, regeneration and pathogenesis, and discuss the future potential for DINE as a therapeutic target for axonal degeneration/disorder.

The Link between Type III Reg and STAT3-Associated Cytokines in Inflamed Colonic Tissues.

Reg (regenerating gene) family proteins are known to be overexpressed in gastrointestinal (GI) tissues under conditions of inflammation. However, the pathophysiological significance of Reg family protein overexpression and its regulation is still unclear. In the present study, we investigated the profile of Reg family gene expression in a colitis model and focused on the regulation of Reg IIIß and IIIγ, which are overexpressed in inflamed colonic mucosa. C57BL/6 mice were administered 2% dextran sulfate sodium (DSS) in drinking water for five days, and their colonic tissues were investigated histopathologically at interval for up to 12 weeks. Gene expression of the Reg family and cytokines (IL-6, IL-17, and IL-22) was evaluated by real-time RT-PCR, and Reg IIIß/γ expression was examined by immunohistochemistry. The effects of cytokines on STAT3 phosphorylation and HIP/PAP (type III REG) expression in Caco2 and HCT116 cells were examined by Western blot analysis. Among Reg family genes, Reg IIIß and IIIγ were alternatively overexpressed in the colonic tissues of mice with DSS-induced colitis. The expression of STAT3-associated cytokines (IL-6, IL-17, and IL-22) was also significantly increased in those tissues, being significantly correlated with that of Reg IIIß/γ. STAT3 phosphorylation and HIP/PAP expression were significantly enhanced in Caco2 cells upon stimulation with IL-6, IL-17, and IL-22. In HCT116 cells, those enhancements were also observed by IL-6 and IL-22 stimulations but not IL-17. The link between type III Reg and STAT3-associated cytokines appears to play a pivotal role in the pathophysiology of DSS-induced colitis.

Branched Sialylated N-glycans Are Accumulated in Brain Synaptosomes and Interact with Siglec-H.

Proper N-glycosylation of proteins is important for normal brain development and nervous system function. Identification of the localization, carrier proteins and interacting partners of N-glycans is essential for understanding the roles of glycoproteins. The present study examined the N-glycan A2G'2F (Galß1-3GlcNAcß1-2Manα1-6[Galß1-3GlcNAcß1-2Manα1-3]Manß1-4GlcNAcß1-4[Fucα1-6]GlcNAc-). A2G'2F has a branched sialic acid structural feature, and branched sialylated A2G'2F is a major N-glycan in the mouse brain. Its expression in the mouse brain increases during development, suggesting that branched sialylated N-glycans play essential roles during brain development. However, the carrier proteins, interacting partners and localization of branched sialylated N-glycans remain unknown. We previously improved our method for analyzing N-glycans from trace samples, and here we succeeded in detecting A2G'2F in small fragments excised from the two-dimensional electrophoresis gels of subcellular fractionated mouse brain proteins. A2G'2F was accumulated in mouse brain synaptosomes. We identified calreticulin as one of the candidate A2G'2F carriers and found calreticulin expression in both the endoplasmic reticulum and synaptosomal fractions. Calreticulin was observed in dendritic spines of cultured cortical neurons. Synthesized branched sialylated glycan clusters interacted with sialic acid-binding immunoglobulin-like lectin H (Siglec-H), which is known to be a microglia-specific molecule. Taken together, these results suggest that branched sialylated A2G'2F in synaptosomes plays a role in the interaction of dendritic spines with microglia.Key words: N-glycan, subcellular fractionation, calreticulin, dendritic spine, Siglec-H.

Motor Nerve Arborization Requires Proteolytic Domain of Damage-Induced Neuronal Endopeptidase (DINE) during Development.

UNLABELLED: Damage-induced neuronal endopeptidase (DINE)/endothelin-converting enzyme-like 1 (ECEL1) is a membrane-bound metalloprotease, which we originally identified as a nerve regeneration-associated molecule. Abundant expression of DINE is observed in regenerating neurons, as well as in developing spinal motor neurons. In line with this, DINE-deficient (DINE KO) embryos fail to arborize phrenic motor nerves in the diaphragm and to form proper neuromuscular junctions (NMJ), which lead to death shortly after birth. However, it is unclear whether protease activity of DINE is involved in motor nerve terminal arborization and how DINE participates in the process. To address these issues, we performed an in vivo rescue experiment in which three types of motor-neuron specific DINE transgenic mice were crossed with DINE KO mice. The DINE KO mice, which overexpressed wild-type DINE in motor neurons, succeeded in rescuing the aberrant nerve terminal arborization and lethality after birth, while those overexpressing two types of protease domain-mutated DINE failed. Further histochemical analysis showed abnormal behavior of immature Schwann cells along the DINE-deficient axons. Coculture experiments of motor neurons and Schwann cells ensured that the protease domain of neuronal DINE was required for proper alignment of immature Schwann cells along the axon. These findings suggest that protease activity of DINE is crucial for intramuscular innervation of motor nerves and subsequent NMJ formation, as well as proper control of interactions between axons and immature Schwann cells. SIGNIFICANCE STATEMENT: Damage-induced neuronal endopeptidase (DINE) is a membrane-bound metalloprotease; expression is abundant in developing spinal motor neurons, as well as in nerve-injured neurons. DINE-deficient (KO) embryos fail to arborize phrenic motor nerves in the diaphragm and to form a neuromuscular junction, leading to death immediately after birth. To address whether proteolytic activity of DINE is involved in this process, we performed in vivo rescue experiments with DINE KO mice. Transgenic rescue of DINE KO mice was accomplished by overexpression of wild-type DINE, but not by protease domain-mutated DINE. Immature Schwann cells were abnormally aligned along the DINE protease-deficient axons. Thus, the protease activity of DINE is crucial for motor axon arborization, as well as the interaction between axons and immature Schwann cells.

TREM2/DAP12 Signal Elicits Proinflammatory Response in Microglia and Exacerbates Neuropathic Pain.

Neuropathic pain afflicts millions of people, and the development of an effective treatment for this intractable pain is an urgent issue. Recent evidence has implicated microglia in neuropathic pain. The present study showed that the DNAX-activating protein of 12 kDa (DAP12) and its associated "triggering receptor expressed on myeloid cells 2" (TREM2) were predominantly expressed by microglia in the dorsal horn after spinal nerve injury, revealing a role for TREM2/DAP12 signaling in neuropathic pain. Nerve injury-induced proinflammatory cytokine expression in microglia and pain behaviors were significantly suppressed in Dap12-deficient mice. Furthermore, intrathecal administration of TREM2 agonistic antibody induced proinflammatory cytokine expression, as well as neuropathic pain, in mice without nerve injury. The agonistic antibody induced proinflammatory responses and neuropathic pain was not observed in Dap12-deficient mice. Together, these results suggest that TREM2/DAP12-mediated signals in microglia exacerbate nerve injury-induced neuropathic pain by inducing proinflammatory cytokine secretion from microglia. Suppression of DAP12-mediated signals could be a therapeutic target for neuropathic pain. SIGNIFICANCE STATEMENT: Recent studies have revealed that activated microglia in the spinal dorsal horn exacerbate neuropathic pain, which has suggested that suppression of microglial activity should be considered as a therapeutic target. However, only a few molecules have been identified as regulators of microglial activity. In this study, we focused on a receptor complex of TREM2 and DAP12, both of which are expressed by microglia and have been implicated in the pathogenesis of Alzheimer's disease, and demonstrated that TREM2/DAP12 signaling promoted proinflammatory responses in microglia and exacerbates neuropathic pain. The present results revealed the functional significance of TREM2/DAP12 signaling in microglial activation after neuronal injury, and could help in the development of treatments for neuropathic pain and neurodegenerative diseases.

Siglec-H is a microglia-specific marker that discriminates microglia from CNS-associated macrophages and CNS-infiltrating monocytes.

Several types of myeloid cell are resident in the CNS. In the steady state, microglia are present in the CNS parenchyma, whereas macrophages reside in boundary regions of the CNS, such as perivascular spaces, the meninges and choroid plexus. In addition, monocytes infiltrate into the CNS parenchyma from circulation upon blood-brain barrier breakdown after CNS injury and inflammation. Although several markers, such as CD11b and ionized calcium-binding adapter molecule 1 (Iba1), are frequently used as microglial markers, they are also expressed by other types of myeloid cell and microglia-specific markers were not defined until recently. Previous transcriptome analyses of isolated microglia identified a transmembrane lectin, sialic acid-binding immunoglobulin-like lectin H (Siglec-H), as a molecular signature for microglia; however, this was not confirmed by histological studies in the nervous system and the reliability of Siglec-H as a microglial marker remained unclear. Here, we demonstrate that Siglec-H is an authentic marker for microglia in mice by immunohistochemistry using a Siglec-H-specific antibody. Siglec-H was expressed by parenchymal microglia from developmental stages to adulthood, and the expression was maintained in activated microglia under injury or inflammatory condition. However, Siglec-H expression was absent from CNS-associated macrophages and CNS-infiltrating monocytes, except for a minor subset of cells. We also show that the Siglech gene locus is a feasible site for specific targeting of microglia in the nervous system. In conclusion, Siglec-H is a reliable marker for microglia that will allow histological identification of microglia and microglia-specific gene manipulation in the nervous system.

Phospholipid localization implies microglial morphology and function via Cdc42 in vitro.

Under a quiescent state, microglia exhibit a ramified shape, rather than the amoeboid-like morphology following injury or inflammation. The manipulation of microglial morphology in vitro has not been very successful, which has impeded the progress of microglial studies. We demonstrate that lysophosphatidylserine (LysoPS), a kind of lysophospholipids, rapidly and substantially alters the morphology of primary cultured microglia to an in vivo-like ramified shape in a receptor independent manner. This mechanism is mediated by Cdc42 activity. LysoPS is incorporated into the plasma membrane and converted to phosphatidylserine (PS) via the Lands' cycle. The accumulated PS on the membrane recruits Cdc42. Both Cdc42 and PS colocalize predominantly in primary and secondary processes, but not in peripheral branches or tips of microglia. Along with the morphological changes LysoPS suppresses inflammatory cytokine production and NF-kB activity. The present study provides a tool to manipulate a microglial phenotype from an amoeboid to a fully ramified in vitro, which certainly contributes to studies exploring microglial physiology and pathology.

Agonists for G-protein-coupled receptor 84 (GPR84) alter cellular morphology and motility but do not induce pro-inflammatory responses in microglia.

BACKGROUND: Several G-protein-coupled receptors (GPCRs) have been shown to be important signaling mediators between neurons and glia. In our previous screening for identification of nerve injury-associated GPCRs, G-protein-coupled receptor 84 (GPR84) mRNA showed the highest up-regulation by microglia after nerve injury. GPR84 is a pro-inflammatory receptor of macrophages in a neuropathic pain mouse model, yet its function in resident microglia in the central nervous system is poorly understood. METHODS: We used endogenous, natural, and surrogate agonists for GPR84 (capric acid, embelin, and 6-OAU, respectively) and examined their effect on mouse primary cultured microglia in vitro. RESULTS: 6-n-Octylaminouracil (6-OAU), embelin, and capric acid rapidly induced membrane ruffling and motility in cultured microglia obtained from C57BL/6 mice, although these agonists failed to promote microglial pro-inflammatory cytokine expression. Concomitantly, 6-OAU suppressed forskolin-induced increase of cAMP in cultured microglia. Pertussis toxin, an inhibitor of Gi-coupled signaling, completely suppressed 6-OAU-induced microglial membrane ruffling and motility. In contrast, no 6-OAU-induced microglial membrane ruffling and motility was observed in microglia from DBA/2 mice, a mouse strain that does not express functional GPR84 protein due to endogenous nonsense mutation of the GPR84 gene. CONCLUSIONS: GPR84 mediated signaling causes microglial motility and membrane ruffling but does not promote pro-inflammatory responses. As GPR84 is a known receptor for medium-chain fatty acids, those released from damaged brain cells may be involved in the enhancement of microglial motility through GPR84 after neuronal injury.

Increased a-series gangliosides positively regulate leptin/Ob receptor-mediated signals in hypothalamus of GD3 synthase-deficient mice.

Gangliosides are widely involved in the regulation of cells and organs. However, little is known about their roles in adipose tissues and hypothalamus. In GD3 synthase-knockout (GD3S KO) mice, deletion of b-series gangliosides resulted in the reduction of serum leptin due to disturbed secretion from adipocytes. To examine whether leptin signals altered, leptin/leptin receptor (ObR)-mediated signaling in hypothalamus was analyzed. Hypothalamus of GD3S KO mouse showed increased expression of GM1 and GD1a, and increased activation of ObR-mediated signals such as pSTAT3 and c-Fos. Leptin stimulation of hypothalamus-derived N-41 cells and their transfectants with GD3S cDNA showed that a-series gangliosides positively regulate leptin/ObR-mediated signals. Co-precipitation analysis revealed that ObR interacts with a-series gangliosides with increased association by leptin stimulation. In brown adipose tissues (BAT) of GD3S KO mice, their weights and adipocyte numbers were increased, and BAT markers such as PGC1α and UCP-1 were also up-regulated. These results suggested that leptin/ObRb-mediated signals were enhanced in hypothalamus of GD3S KO mice due to increased a-series gangliosides, leading to the apparently similar features of energy expenditure between the KO and wild type mice.

ECEL1 mutation implicates impaired axonal arborization of motor nerves in the pathogenesis of distal arthrogryposis.

The membrane-bound metalloprotease endothelin-converting enzyme-like 1 (ECEL1) has been newly identified as a causal gene of a specific type of distal arthrogryposis (DA). In contrast to most causal genes of DA, ECEL1 is predominantly expressed in neuronal cells, suggesting a unique neurogenic pathogenesis in a subset of DA patients with ECEL1 mutation. The present study analyzed developmental motor innervation and neuromuscular junction formation in limbs of the rodent homologue damage-induced neuronal endopeptidase (DINE)-deficient mouse. Whole-mount immunostaining was performed in DINE-deficient limbs expressing motoneuron-specific GFP to visualize motor innervation throughout the limb. Although DINE-deficient motor nerves displayed normal trajectory patterns from the spinal cord to skeletal muscles, they indicated impaired axonal arborization in skeletal muscles in the forelimbs and hindlimbs. Systematic examination of motor innervation in over 10 different hindlimb muscles provided evidence that DINE gene disruption leads to insufficient arborization of motor nerves after arriving at the skeletal muscle. Interestingly, the axonal arborization defect in foot muscles appeared more severe than in other hindlimb muscles, which was partially consistent with the proximal-distal phenotypic discordance observed in DA patients. Additionally, the number of innervated neuromuscular junction was significantly reduced in the severely affected DINE-deficient muscle. Furthermore, we generated a DINE knock-in (KI) mouse model with a pathogenic mutation, which was recently identified in DA patients. Axonal arborization defects were clearly detected in motor nerves of the DINE KI limb, which was identical to the DINE-deficient limb. Given that the encoded sequences, as well as ECEL1 and DINE expression profiles, are highly conserved between mouse and human, abnormal arborization of motor axons and subsequent failure of NMJ formation could be a primary cause of DA with ECEL1 mutation.