Identification of z-axis filopodia in growth cones using super-resolution microscopy.

A growth cone is a highly motile tip of an extending axon that is crucial for neural network formation. Three-dimensional-structured illumination microscopy, a type of super-resolution light microscopy with a resolution that overcomes the optical diffraction limitation (ca. 200 nm) of conventional light microscopy, is well suited for studying the molecular dynamics of intracellular events. Using this technique, we discovered a novel type of filopodia distributed along the z-axis ("z-filopodia") within the growth cone. Z-filopodia were typically oriented in the direction of axon growth, not attached to the substratum, protruded spontaneously without microtubule invasion, and had a lifetime that was considerably shorter than that of conventional filopodia. Z-filopodia formation and dynamics were regulated by actin-regulatory proteins, such as vasodilator-stimulated phosphoprotein, fascin, and cofilin. Chromophore-assisted laser inactivation of cofilin induced the rapid turnover of z-filopodia. An axon guidance receptor, neuropilin-1, was concentrated in z-filopodia and was transported together with them, whereas its ligand, semaphorin-3A, was selectively bound to them. Membrane domains associated with z-filopodia were also specialized and resembled those of lipid rafts, and their behaviors were closely related to those of neuropilin-1. The results suggest that z-filopodia have unique turnover properties, and unlike xy-filopodia, do not function as force-generating structures for axon extension.

Reduced chondroitin sulfate content prevents diabetic neuropathy through transforming growth factor-ß signaling suppression.

Diabetic neuropathy (DN) is a major complication of diabetes mellitus. Chondroitin sulfate (CS) is one of the most important extracellular matrix components and is known to interact with various diffusible factors; however, its role in DN pathology has not been examined. Therefore, we generated CSGalNAc-T1 knockout (T1KO) mice, in which CS levels were reduced. We demonstrated that diabetic T1KO mice were much more resistant to DN than diabetic wild-type (WT) mice. We also found that interactions between pericytes and vascular endothelial cells were more stable in T1KO mice. Among the RNA-seq results, we focused on the transforming growth factor ß signaling pathway and found that the phosphorylation of Smad2/3 was less upregulated in T1KO mice than in WT mice under hyperglycemic conditions. Taken together, a reduction in CS level attenuates DN progression, indicating that CS is an important factor in DN pathogenesis.

Human-Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Showed Neuronal Differentiation, Neurite Extension, and Formation of Synaptic Structures in Rodent Ischemic Stroke Brains.

Ischemic stroke is a major cerebrovascular disease with high morbidity and mortality rates; however, effective treatments for ischemic stroke-related neurological dysfunction have yet to be developed. In this study, we generated neural progenitor cells from human leukocyte antigen major loci gene-homozygous-induced pluripotent stem cells (hiPSC-NPCs) and evaluated their therapeutic effects against ischemic stroke. hiPSC-NPCs were intracerebrally transplanted into rat ischemic brains produced by transient middle cerebral artery occlusion at either the subacute or acute stage, and their in vivo survival, differentiation, and efficacy for functional improvement in neurological dysfunction were evaluated. hiPSC-NPCs were histologically identified in host brain tissues and showed neuronal differentiation into vGLUT-positive glutamatergic neurons, extended neurites into both the ipsilateral infarct and contralateral healthy hemispheres, and synaptic structures formed 12 weeks after both acute and subacute stage transplantation. They also improved neurological function when transplanted at the subacute stage with γ-secretase inhibitor pretreatment. However, their effects were modest and not significant and showed a possible risk of cells remaining in their undifferentiated and immature status in acute-stage transplantation. These results suggest that hiPSC-NPCs show cell replacement effects in ischemic stroke-damaged neural tissues, but their efficacy is insufficient for neurological functional improvement after acute or subacute transplantation. Further optimization of cell preparation methods and the timing of transplantation is required to balance the efficacy and safety of hiPSC-NPC transplantation.

Identification of the growth cone as a probe and driver of neuronal migration in the injured brain.

Axonal growth cones mediate axonal guidance and growth regulation. We show that migrating neurons in mice possess a growth cone at the tip of their leading process, similar to that of axons, in terms of the cytoskeletal dynamics and functional responsivity through protein tyrosine phosphatase receptor type sigma (PTPσ). Migrating-neuron growth cones respond to chondroitin sulfate (CS) through PTPσ and collapse, which leads to inhibition of neuronal migration. In the presence of CS, the growth cones can revert to their extended morphology when their leading filopodia interact with heparan sulfate (HS), thus re-enabling neuronal migration. Implantation of an HS-containing biomaterial in the CS-rich injured cortex promotes the extension of the growth cone and improve the migration and regeneration of neurons, thereby enabling functional recovery. Thus, the growth cone of migrating neurons is responsive to extracellular environments and acts as a primary regulator of neuronal migration.

Effects of MAP4K inhibition on neurite outgrowth.

Protein kinases are responsible for protein phosphorylation and are involved in important intracellular signal transduction pathways in various cells, including neurons; however, a considerable number of poorly characterized kinases may be involved in neuronal development. Here, we considered mitogen-activated protein kinase kinase kinase kinases (MAP4Ks), related to as candidate regulators of neurite outgrowth and synaptogenesis, by examining the effects of a selective MAP4K inhibitor PF06260933. PF06260933 treatments of the cultured neurons reduced neurite lengths, not the number of synapses, and phosphorylation of GAP43 and JNK, relative to the control. These results suggest that MAP4Ks are physiologically involved in normal neuronal development and that the resultant impaired neurite outgrowth by diminished MAP4Ks' activity, is related to psychiatric disorders.

Very-long-chain fatty acids are crucial to neuronal polarity by providing sphingolipids to lipid rafts.

Fatty acids have long been considered essential to brain development; however, the involvement of their synthesis in nervous system formation is unclear. We generate mice with knockout of GPSN2, an enzyme for synthesis of very-long-chain fatty acids (VLCFAs) and investigate the effects. Both GPSN2-/- and GPSN2+/- mice show abnormal neuronal networks as a result of impaired neuronal polarity determination. Lipidomics of GPSN2-/- embryos reveal that ceramide synthesis is specifically inhibited depending on FA length; namely, VLCFA-containing ceramide is reduced. We demonstrate that lipid rafts are highly enriched in growth cones and that GPSN2+/- neurons lose gangliosides in their membranes. Application of C24:0 ceramide, but not C16:0 ceramide or C24:0 phosphatidylcholine, to GPSN2+/- neurons rescues both neuronal polarity determination and lipid-raft density in the growth cone. Taken together, our results indicate that VLCFA synthesis contributes to physiological neuronal development in brain network formation, in particular neuronal polarity determination through the formation of lipid rafts.

Genetic manipulation resulting in decreased donor chondroitin sulfate synthesis mitigates hepatic GVHD via suppression of T cell activity.

Donor T cell activation, proliferation, differentiation, and migration are the major steps involved in graft-versus-host disease (GVHD) development following bone marrow transplantation. Chondroitin sulfate (CS) proteoglycan is a major component of the extracellular matrix and causes immune modulation by interacting with cell growth factors and inducing cell adhesion. However, its precise effects on immune function are unclear than those of other proteoglycan families. Thus, we investigated the significance of CS within donor cells in acute GVHD development utilizing CSGalNAc T1-knockout (T1KO) mice. To determine the effects of T1KO, the mice underwent allogenic bone marrow transplantation from major histocompatibility complex-mismatched donors. While transplantation resulted in hepatic GVHD with inflammatory cell infiltration of both CD4+ and CD8+ effector memory T cells, transplantation in T1KO-donors showed milder cell infiltration and improved survival with fewer splenic effector T cells. In vitro T-cell analyses showed that the ratio of effector memory T cells was significantly lower via phorbol myristate acetate/ionomycin stimulation. Moreover, quantitative PCR analyses showed significantly less production of inflammatory cytokines, such as IFN-γ and CCL-2, in splenocytes of T1KO mice. These results suggest that reduction of CS in donor blood cells may suppress the severity of acute GVHD after hematopoietic stem cell transplantation.

The cyclin G-associated kinase (GAK) inhibitor SGC-GAK-1 inhibits neurite outgrowth and synapse formation.

Protein kinases are responsible for protein phosphorylation and are involved in important signal transduction pathways; however, a considerable number of poorly characterized kinases may be involved in neuronal development. Here, we considered cyclin G-associated kinase (GAK) as a candidate regulator of neurite outgrowth and synaptogenesis by examining the effects of the selective GAK inhibitor SGC-GAK-1. SGC-GAK-1 treatment of cultured neurons reduced neurite length and decreased synapse number and phosphorylation of neurofilament 200-kDa subunits relative to the control. In addition, the related kinase inhibitor erlotinib, which has distinct specificity and potency from SGC-GAK-1, had no effect on neurite growth, unlike SGC-GAK-1. These results suggest that GAK may be physiologically involved in normal neuronal development, and that decreased GAK function and the resultant impaired neurite outgrowth and synaptogenesis may be related to neurodevelopmental disorders.

JNK1-Dependent Phosphorylation of GAP-43 Serine 142 is a Novel Molecular Marker for Axonal Growth.

Mammalian axon growth has mechanistic similarities with axon regeneration. The growth cone is an important structure that is involved in both processes, and GAP-43 (growth associated protein-43 kDa) is believed to be the classical molecular marker. Previously, we used growth cone phosphoproteomics to demonstrate that S96 and T172 of GAP-43 in rodents are highly phosphorylated sites that are phosphorylated by c-jun N-terminal protein kinase (JNK). We also revealed that phosphorylated (p)S96 and pT172 antibodies recognize growing axons in the developing brain and regenerating axons in adult peripheral nerves. In rodents, S142 is another putative JNK-dependent phosphorylation site that is modified at a lower frequency than S96 and T172. Here, we characterized this site using a pS142-specific antibody. We confirmed that pS142 was detected by co-expressing mouse GAP-43 and JNK1. pS142 antibody labeled growth cones and growing axons in developing mouse neurons. pS142 was sustained until at least nine weeks after birth in mouse brains. The pS142 antibody could detect regenerating axons following sciatic nerve injury in adult mice. Comparison of amino acid sequences indicated that rodent S142 corresponds to human S151, which is predicted to be a substrate of the MAPK family, which includes JNK. Thus, we confirmed that the pS142 antibody recognized human phospho-GAP-43 using activated JNK1, and also that its immunostaining pattern in neurons differentiated from human induced pluripotent cells was similar to those observed in mice. These results indicate that the S142 residue is phosphorylated by JNK1 and that the pS142 antibody is a new candidate molecular marker for axonal growth in both rodents and human.

ChGn-2 Plays a Cardioprotective Role in Heart Failure Caused by Acute Pressure Overload.

Background Cardiac extracellular matrix is critically involved in cardiac homeostasis, and accumulation of chondroitin sulfate glycosaminoglycans (CS-GAGs) was previously shown to exacerbate heart failure by augmenting inflammation and fibrosis at the chronic phase. However, the mechanism by which CS-GAGs affect cardiac functions remains unclear, especially at the acute phase. Methods and Results We explored a role of CS-GAG in heart failure using mice with target deletion of ChGn-2 (chondroitin sulfate N-acetylgalactosaminyltransferase-2) that elongates CS chains of glycosaminoglycans. Heart failure was induced by transverse aortic constriction in mice. The role of CS-GAG derived from cardiac fibroblasts in cardiomyocyte death was analyzed. Cardiac fibroblasts were subjected to cyclic mechanical stretch that mimics increased workload in the heart. Significant CS-GAGs accumulation was detected in the heart of wild-type mice after transverse aortic constriction, which was substantially reduced in ChGn-2-/- mice. Loss of ChGn-2 deteriorated the cardiac dysfunction caused by pressure overload, accompanied by augmented cardiac hypertrophy and increased cardiomyocyte apoptosis. Cyclic mechanical stretch increased ChGn-2 expression and enhanced glycosaminoglycan production in cardiac fibroblasts. Conditioned medium derived from the stretched cardiac fibroblasts showed cardioprotective effects, which was abolished by CS-GAGs degradation. We found that CS-GAGs elicits cardioprotective effects via dual pathway; direct pathway through interaction with CD44, and indirect pathway through binding to and activating insulin-like growth factor-1. Conclusions Our data revealed the cardioprotective effects of CS-GAGs; therefore, CS-GAGs may play biphasic role in the development of heart failure; cardioprotective role at acute phase despite its possible unfavorable role in the advanced phase.

PI4P/PS countertransport by ORP10 at ER-endosome membrane contact sites regulates endosome fission.

Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER-endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER-endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER-endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.

Coactosin Promotes F-Actin Protrusion in Growth Cones Under Cofilin-Related Signaling Pathway.

During brain development, axon outgrowth and its subsequent pathfinding are reliant on a highly motile growth cone located at the tip of the axon. Actin polymerization that is regulated by actin-depolymerizing factors homology (ADF-H) domain-containing family drives the formation of lamellipodia and filopodia at the leading edge of growth cones for axon guidance. However, the precise localization and function of ADF-H domain-containing proteins involved in axon extension and retraction remain unclear. We have previously shown that transcripts and proteins of coactosin-like protein 1 (COTL1), an ADF-H domain-containing protein, are observed in neurites and axons in chick embryos. Coactosin overexpression analysis revealed that this protein was localized to axonal growth cones and involved in axon extension in the midbrain. We further examined the specific distribution of coactosin and cofilin within the growth cone using superresolution microscopy, structured illumination microscopy, which overcomes the optical diffraction limitation and is suitable to the analysis of cellular dynamic movements. We found that coactosin was tightly associated with F-actin bundles at the growth cones and that coactosin overexpression promoted the expansion of lamellipodia and extension of growth cones. Coactosin knockdown in oculomotor neurons resulted in an increase in the levels of the inactive, phosphorylated form of cofilin and dysregulation of actin polymerization and axonal elongation, which suggests that coactosin promoted axonal growth in a cofilin-dependent manner. Indeed, the application of a dominant-negative form of LIMK1, a downstream effector of GTPases, reversed the effect of coactosin knockdown on axonal growth by enhancing cofilin activity. Combined, our results indicate that coactosin functions promote the assembly of protrusive actin filament arrays at the leading edge for growth cone motility.

Phosphorylation of GAP-43 T172 is a molecular marker of growing axons in a wide range of mammals including primates.

GAP-43 is a vertebrate neuron-specific protein and that is strongly related to axon growth and regeneration; thus, this protein has been utilized as a classical molecular marker of these events and growth cones. Although GAP-43 was biochemically characterized more than a quarter century ago, how this protein is related to these events is still not clear. Recently, we identified many phosphorylation sites in the growth cone membrane proteins of rodent brains. Two phosphorylation sites of GAP-43, S96 and T172, were found within the top 10 hit sites among all proteins. S96 has already been characterized (Kawasaki et al., 2018), and here, phosphorylation of T172 was characterized. In vitro (cultured neurons) and in vivo, an antibody specific to phosphorylated T172 (pT172 antibody) specifically recognized cultured growth cones and growing axons in developing mouse neurons, respectively. Immunoblotting showed that pT172 antigens were more rapidly downregulated throughout development than those of pS96 antibody. From the primary structure, this phosphorylation site was predicted to be conserved in a wide range of animals including primates. In the developing marmoset brainstem and in differentiated neurons derived from human induced pluripotent stem cells, immunoreactivity with pT172 antibody revealed patterns similar to those in mice. pT172 antibody also labeled regenerating axons following sciatic nerve injury. Taken together, the T172 residue is widely conserved in a wide range of mammals including primates, and pT172 is a new candidate molecular marker for growing axons.

Distinct effects of chondroitin sulfate on hematopoietic cells and the stromal microenvironment in bone marrow hematopoiesis.

The bone marrow (BM) microenvironment, known as the BM niche, regulates hematopoiesis but is also affected by interactions with hematopoietic cells. Recent evidence indicates that extracellular matrix components are involved in these interactions. Chondroitin sulfate (CS), a glycosaminoglycan, is a major component of the extracellular matrix; however, it is not known whether CS has a physiological role in hematopoiesis. Here, we analyzed the functions of CS in hematopoietic and niche cells. CSGalNAcT1, which encodes CS N-acetylgalactosaminyltransferase-1 (T1), a key enzyme in CS biosynthesis, was highly expressed in hematopoietic stem and progenitor cells (HSPCs) and endothelial cells (ECs), but not in mesenchymal stromal cells (MSCs) in BM. In T1 knockout (T1KO) mice, a greater number of HSPCs existed compared with the wild-type (WT), but HSPCs from T1KO mice showed significantly impaired repopulation in WT recipient mice on serial transplantation. RNA sequence analysis revealed the activation of IFN-α/ß signaling and endoplasmic reticulum stress in T1KO HSPCs. In contrast, the number of WT HSPCs repopulated in T1KO recipient mice was larger than that in WT recipient mice after serial transplantation, indicating that the T1KO niche supports repopulation of HSPCs better than the WT niche. There was no obvious difference in the distribution of vasculature and MSCs between WT and T1KO BM, suggesting that CS loss alters vascular niche functions without affecting its structure. Our results revealed distinct roles of CS in hematopoietic cells and BM niche, indicating that crosstalk between these components is important to maintain homeostasis in BM.

Chondroitin Sulfate N-acetylgalactosaminyltransferase-2 Impacts Foam Cell Formation and Atherosclerosis by Altering Macrophage Glycosaminoglycan Chain.

OBJECTIVE: Chondroitin sulfate proteoglycans are the primary constituents of the macrophage glycosaminoglycan and extracellular microenvironment. To examine their potential role in atherogenesis, we investigated the biological importance of one of the chondroitin sulfate glycosaminoglycan biosynthesis gene, ChGn-2 (chondroitin sulfate N-acetylgalactosaminyltransferase-2), in macrophage foam cell formation. Approach and Results: ChGn-2-deficient mice showed decreased and shortened glycosaminoglycans. ChGn-2-/-/LDLr-/- (low-density lipoprotein receptor) mice generated less atherosclerotic plaque after being fed with Western diet despite exhibiting a metabolic phenotype similar to that of the ChGn-2+/+/LDLr-/- littermates. We demonstrated that in macrophages, ChGn-2 expression was upregulated in the presence of oxLDL (oxidized LDL), and glycosaminoglycan was substantially increased. Foam cell formation was significantly altered by ChGn-2 in both mouse peritoneal macrophages and the RAW264.7 macrophage cell line. Mechanistically, ChGn-2 enhanced oxLDL binding on the cell surface, and as a consequence, CD36-an important macrophage membrane scavenger receptor-was differentially regulated. CONCLUSIONS: ChGn-2 alteration on macrophages conceivably influences LDL accumulation and subsequently accelerates plaque formation. These results collectively suggest that ChGn-2 is a novel therapeutic target amenable to clinical translation in the future. Graphic Abstract: A graphic abstract is available for this article.

Chondroitin sulfate N-acetylgalactosyltransferase-1 knockout shows milder phenotype in experimental autoimmune encephalomyelitis than in wild type.

Proteoglycans (PGs) are one of the main components in the extracellular matrix of the central nervous system. Chondroitin sulfate (CS) is a glycosaminoglycan (GAG), which is composed of major PGs. Similar to keratin sulfate (KS), another GAG, CS inhibits axon regeneration. However, the influence of these GAGs on the pathogenicity of neuroimmunological diseases is unclear. Here, we induced experimental autoimmune encephalomyelitis (EAE) in mice lacking CS N-acetylgalactosaminyltransferase-1 (CSGalNAcT1-KO), an important enzyme for CS synthesis. In our study, CSGalNAcT1-KO mice showed milder EAE symptoms than those in wild-type (WT) mice. The recall response of antigen-specific lymphocytes showed that CSGalNAcT1-KO-derived lymphocytes had a milder cell proliferation response than that in WT-derived lymphocytes. These results suggest that CS contributes toward the induction phase of EAE. We previously performed EAE experiments in GlcNAc-6-O-sulfotransferase KO (GlcNAc6ST-KO) and C6ST1-KO mice, which had reduced KS and reduced CS-C, respectively. EAE in CSGalNAcT1-KO mice was more similar to that in GlcNAc6ST-KO mice than in C6ST1-KO mice. In conclusion, the distinct GAG sugar chains are associated with severe or mild phenotypes of EAE and are therefore potential new therapeutic targets for neuroimmunological diseases, including multiple sclerosis.

Neuronal Signaling Involved in Neuronal Polarization and Growth: Lipid Rafts and Phosphorylation.

Neuronal polarization and growth are developmental processes that occur during neuronal cell differentiation. The molecular signaling mechanisms involved in these events in in vivo mammalian brain remain unclear. Also, cellular events of the neuronal polarization process within a given neuron are thought to be constituted of many independent intracellular signal transduction pathways (the "tug-of-war" model). However, in vivo results suggest that such pathways should be cooperative with one another among a given group of neurons in a region of the brain. Lipid rafts, specific membrane domains with low fluidity, are candidates for the hotspots of such intracellular signaling. Among the signals reported to be involved in polarization, a number are thought to be present or translocated to the lipid rafts in response to extracellular signals. As part of our analysis, we discuss how such novel molecular mechanisms are combined for effective regulation of neuronal polarization and growth, focusing on the significance of the lipid rafts, including results based on recently introduced methods.

Microtubule elongation along actin filaments induced by microtubule-associated protein 4 contributes to the formation of cellular protrusions.

Actin-microtubule crosstalk is implicated in the formation of cellular protrusions, but the mechanism remains unclear. In this study, we examined the regulation of cell protrusion involving a ubiquitously expressed microtubule-associated protein (MAP) 4, and its superfamily proteins, neuronal MAP2 and tau. Fluorescence microscopy revealed that these MAPs bound to F-actin and microtubules simultaneously, and formed F-actin/microtubule hybrid bundles. The hybrid bundle-forming activity was in the order of MAP2 > MAP4 ≫ tau. Interestingly, the microtubule assembly-promoting activity of MAP4 and MAP2, but not of tau, was upregulated by their interaction with F-actin. When MAP4 was overexpressed in NG108-15 cells, the number of cell processes and maximum process length of each cell increased significantly by 28% and 30%, respectively. Super-resolution microscopy revealed that 95% of microtubules in cell processes colocalized with F-actin, and MAP4 was always found in their vicinity. These results suggest that microtubule elongation along F-actin induced by MAP4 contributes to the formation of cellular protrusions. Since MAP4, MAP2 and tau had different crosstalk activity between F-actin and microtubules, it is likely that the functional differentiation of these MAPs is a driving force for neural evolution, causing significant changes in cell morphology.

Phosphoproteomic and bioinformatic methods for analyzing signaling in vertebrate axon growth and regeneration.

Phosphorylation is the most important post-translational modification of proteins in many cells, including neurons. Phosphoproteomics is a relatively new technique for comprehensively identifying phosphorylation sites in the whole proteome of a given system. We applied this method to developmental neurobiology research to understand the signaling pathways that regulate the mammalian growth cone, which is formed at the tips of developing neurites to ensure accurate neuronal network formation. Using this powerful technique, we identified at least four phosphorylation sites tightly associated with axon growth. Because phosphoproteomic results include relatively large numbers of phosphopeptides, the data are typically analyzed using bioinformatics. We utilized three bioinformatics tools to identify the responsible protein kinases, the putative functions of the phosphorylated protein groups, and the evolutional aspects of the phosphorylated proteins. Collectively, these data indicate phosphoproteomics is a cutting-edge tool for neuroscience research.