Spatial and single-cell colocalisation analysis reveals MDK-mediated immunosuppressive environment with regulatory T cells in colorectal carcinogenesis.

BACKGROUND: Cell-cell interaction factors that facilitate the progression of adenoma to sporadic colorectal cancer (CRC) remain unclear, thereby hindering patient survival. METHODS: We performed spatial transcriptomics on five early CRC cases, which included adenoma and carcinoma, and one advanced CRC. To elucidate cell-cell interactions within the tumour microenvironment (TME), we investigated the colocalisation network at single-cell resolution using a deep generative model for colocalisation analysis, combined with a single-cell transcriptome, and assessed the clinical significance in CRC patients. FINDINGS: CRC cells colocalised with regulatory T cells (Tregs) at the adenoma-carcinoma interface. At early-stage carcinogenesis, cell-cell interaction inference between colocalised adenoma and cancer epithelial cells and Tregs based on the spatial distribution of single cells highlighted midkine (MDK) as a prominent signalling molecule sent from tumour epithelial cells to Tregs. Interaction between MDK-high CRC cells and SPP1+ macrophages and stromal cells proved to be the mechanism underlying immunosuppression in the TME. Additionally, we identified syndecan4 (SDC4) as a receptor for MDK associated with Treg colocalisation. Finally, clinical analysis using CRC datasets indicated that increased MDK/SDC4 levels correlated with poor overall survival in CRC patients. INTERPRETATION: MDK is involved in the immune tolerance shown by Tregs to tumour growth. MDK-mediated formation of the TME could be a potential target for early diagnosis and treatment of CRC. FUNDING: Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Science Research; OITA Cancer Research Foundation; AMED under Grant Number; Japan Science and Technology Agency (JST); Takeda Science Foundation; The Princess Takamatsu Cancer Research Fund.

Detecting and exploring kidney-derived extracellular vesicles in plasma.

BACKGROUND: Extracellular vesicles (EVs) have received considerable attention as ideal biomarkers for kidney diseases. Most reports have focused on urinary EVs, that are mainly derived from the cells in the urinary tract. However, the detection and the application of kidney-derived EVs in plasma remains uncertain. METHODS: We examined the kidney-derived small EVs (sEVs) in plasma that were supposedly released from renal mesangial and glomerular endothelial cells, using clinical samples from healthy controls and patients with kidney transplants. Plasma from healthy controls underwent ultracentrifugation, followed by on-bead flow cytometry, targeting α8 integrin, an antigen-specific to mesangial cells. To confirm the presence of kidney-derived sEVs in peripheral blood, plasma from ABO-incompatible kidney transplant recipients was ultracentrifuged, followed by western blotting for donor blood type antigens. RESULTS: Immunohistochemistry and immunoelectron microscopy confirmed α8 integrin expression in kidney mesangial cells and their sEVs. The CD9-α8 integrin double-positive sEVs were successfully detected using on-bead flow cytometry. Western blot analysis further revealed transplanted kidney-derived sEVs containing blood type B antigens in non-blood type B recipients, who had received kidneys from blood type B donors. Notably, a patient experiencing graft kidney loss exhibited diminished signals of sEVs containing donor blood type antigens. CONCLUSION: Our findings demonstrate the potential usefulness of kidney-derived sEVs in plasma in future research for kidney diseases.

Mitotic Dysregulation at Tumor Initiation Creates a Therapeutic Vulnerability to Combination Anti-Mitotic and Pro-Apoptotic Agents for MYCN-Driven Neuroblastoma.

MYCN amplification occurs in approximately 20-30% of neuroblastoma patients and correlates with poor prognosis. The TH-MYCN transgenic mouse model mimics the development of human high-risk neuroblastoma and provides strong evidence for the oncogenic function of MYCN. In this study, we identified mitotic dysregulation as a hallmark of tumor initiation in the pre-cancerous ganglia from TH-MYCN mice that persists through tumor progression. Single-cell quantitative-PCR of coeliac ganglia from 10-day-old TH-MYCN mice revealed overexpression of mitotic genes in a subpopulation of premalignant neuroblasts at a level similar to single cells derived from established tumors. Prophylactic treatment using antimitotic agents barasertib and vincristine significantly delayed the onset of tumor formation, reduced pre-malignant neuroblast hyperplasia, and prolonged survival in TH-MYCN mice. Analysis of human neuroblastoma tumor cohorts showed a strong correlation between dysregulated mitosis and features of MYCN amplification, such as MYC(N) transcriptional activity, poor overall survival, and other clinical predictors of aggressive disease. To explore the therapeutic potential of targeting mitotic dysregulation, we showed that genetic and chemical inhibition of mitosis led to selective cell death in neuroblastoma cell lines with MYCN over-expression. Moreover, combination therapy with antimitotic compounds and BCL2 inhibitors exploited mitotic stress induced by antimitotics and was synergistically toxic to neuroblastoma cell lines. These results collectively suggest that mitotic dysregulation is a key component of tumorigenesis in early neuroblasts, which can be inhibited by the combination of antimitotic compounds and pro-apoptotic compounds in MYCN-driven neuroblastoma.

SMG6 regulates DNA damage and cell survival in Hippo pathway kinase LATS2-inactivated malignant mesothelioma.

Many genes responsible for Malignant mesothelioma (MM) have been identified as tumor suppressor genes and it is difficult to target these genes directly at a molecular level. We searched for the gene which showed synthetic lethal phenotype with LATS2, one of the MM causative genes and one of the kinases in the Hippo pathway. Here we showed that knockdown of SMG6 results in synthetic lethality in LATS2-inactivated cells. We found that this synthetic lethality required the nuclear translocation of YAP1 and TAZ. Both are downstream factors of the Hippo pathway. We also demonstrated that this synthetic lethality did not require SMG6 in nonsense-mediated mRNA decay (NMD) but in regulating telomerase reverse transcriptase (TERT) activity. In addition, the RNA-dependent DNA polymerase (RdDP) activity of TERT was required for this synthetic lethal phenotype. We confirmed the inhibitory effects of LATS2 and SMG6 on cell proliferation in vivo. The result suggests an interaction between the Hippo and TERT signaling pathways. We also propose that SMG6 and TERT are novel molecular target candidates for LATS2-inactivated cancers such as MM.

Close association of polarization and LC3, a marker of autophagy, in axon determination in mouse hippocampal neurons.

The autophagy-lysosome pathway is a cellular clearance system for intracellular organelles, macromolecules and microorganisms. It is indispensable for cells not only to maintain their homeostasis but also to achieve more active cellular processes such as differentiation. Therefore, impairment or disruption of the autophagy-lysosome pathway leads to a wide spectrum of human diseases, ranging from several types of neurodegenerative diseases to malignancies. In elongating axons, autophagy preferentially occurs at growth cones, and disruption of autophagy is closely associated with incapacity for axonal regeneration after injury in the central nervous system. However, the roles of autophagy in developing neurons remain elusive. In particular, whether autophagy is involved in axon-dendrite determination is largely unclear. Using primary cultured mouse embryonic hippocampal neurons, we here showed the polarized distribution of autophagosomes among minor processes of neurons at stage 2. Time-lapse observation of neurons from GFP-LC3 transgenic mice demonstrated that an "LC3 surge"-i.e., a rapid accumulation of autophagic marker LC3 that continues for several hours in one minor process-proceeded the differentiation of neurons into axons. In addition, pharmacological activation and inhibition of autophagy by trehalose and bafilomycin, respectively, accelerated and delayed axonal determination. Taken together, our findings revealed the close association between LC3, a marker of autophagy, and axon determination in developing neurons.

Screening of novel Midkine binding protein by BioID2-based proximity labeling.

Midkine (MK), a heparin-binding growth factor, is associated with the poor prognosis of the pediatric tumor, neuroblastoma. MK would be a druggable target as many studies showed inhibition of its function in various cancers suppressed tumor developments. To establish the therapy targeting MK, identification of its binding partners, and elucidation of its intracellular signaling are needed. It was reported that exogenous MK induced phosphorylation of ribosomal protein S6 (RPS6) downstream of mTOR signaling. Using RPS6 phosphorylation as a marker of MK response, we searched for MK reactive cell lines. We found that MK cell lines expressing less MK tended to respond better to MK. Next, using an MK reactive neuroblastoma cell line, MK-knocked down SH-SY5Y cells, we employed a proximity-dependent biotin identification method, which was invented to evaluate protein-protein interactions by biotinylation. We confirmed that secreted MK fused to the biotin ligase BioID2 (MK-BioID2) was able to biotinylate proteins from the cells. Biotinylated proteins were identified by liquid chromatography-mass spectrometry analyses. Twenty five proteins were found to be overlapped after three independent experiments, among which insulin-like growth binding protein 2 (IGFBP2) was further analyzed. IGFBP2 was indeed detected with immunoblotting after streptavidin pull down of MK-BioID2 labeled cell extract of MK-knocked down SH-SY5Y cells. Our study suggests that the BioID2 method is useful to identify binding partners of growth factors.

Dermatan sulphate is an activating ligand of anaplastic lymphoma kinase.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that harbours a tyrosine kinase domain in its intracellular region and is expressed in both central and peripheral nervous systems. RTKs are activated upon ligand binding and receptor clustering; however, ALK remains an orphan receptor despite its pathological significance, especially in malignancy. Recent biochemical work showed that heparan sulphate (HS), an unbranched sulphated glycan, acts as a ligand for and activates ALK. Here, we show that dermatan sulphate (DS, chondroitin sulphate B) directly interacts with the extracellular N-terminal region of ALK as well as HS. The tetrasaccharide of DS was required and was sufficient for inducing autophosphorylation of ALK at tyrosine 1604, a marker for activated ALK. Interestingly, longer oligosaccharides caused enhanced activation of ALK, as was the case for HS. Our results provide a novel example of glycans as signalling molecules and shed light on the pathophysiological roles of ALK.

CD147 Is Essential for the Development of Psoriasis via the Induction of Th17 Cell Differentiation.

Th17 cells play an important role in psoriasis. The differentiation of naïve CD4+ T cells into Th17 cells depends on glycolysis as the energy source. CD147/basigin, an integral transmembrane protein belonging to the immunoglobulin superfamily, regulates glycolysis in association with monocarboxylate transporters (MCTs)-1 and -4 in cancer cells and T cells. We examined whether CD147/basigin is involved in the pathogenesis of psoriasis in humans and psoriasis-model mice. The serum level of CD147 was increased in patients with psoriasis, and the expression of CD147 and MCT-1 was elevated in their dermal CD4+ RORγt+ T cells. In vitro, the potential of naïve CD4+ T cells to differentiate into Th17 cells was abrogated in CD147-/- T cells. Imiquimod (IMQ)-induced psoriatic dermatitis was significantly milder in CD147-/- mice and bone marrow chimeric mice lacking CD147 in the hematopoietic cells of myeloid lineage. These findings demonstrate that CD147 is essential for the development of psoriasis via the induction of Th17 cell differentiation.

Combination of tumor necrosis factor-α and epidermal growth factor induces the adrenergic-to-mesenchymal transdifferentiation in SH-SY5Y neuroblastoma cells.

Neuroblastoma, a type of cancer that is common in children, is composed of two genetically clonal but epigenetically distinct cell types: mesenchymal (MES) and adrenergic (ADRN) types, controlled by super-enhancer-associated lineage-specific transcription factor networks. Mesenchymal-type cells are more migratory, resistant to chemotherapy, and prevalent in relapse tumors. Importantly, both cell types spontaneously transdifferentiate into one another, and this interconversion can be induced by genetic manipulations. However, the mechanisms of their spontaneous transdifferentiation and extracellular factors inducing this phenomenon have not yet been elucidated. Using a unique approach involving gene set enrichment analysis, we selected six ADRN and 10 MES candidate factors, possibly inducing ADRN and MES phenotypes, respectively. Treatment with a combination of 10 MES factors clearly induced the MES gene expression profile in ADRN-type SH-SY5Y neuroblastoma cells. Considering the effects on gene expression profile, migration ability, and chemoresistance, a combination of tumor necrosis factor alpha (TNF-α) and epidermal growth factor (EGF) was sufficient to synergistically induce the ADRN-to-MES transdifferentiation in SH-SY5Y cells. In addition, human neuroblastoma cohort analysis revealed that the expression of TNF and EGF receptors was strongly associated with MES gene expression signatures, supporting their important roles in transdifferentiation in vivo. Collectively, we propose a mechanism of neuroblastoma transdifferentiation induced by extracellular growth factors, which can be controlled in clinical situations, providing a new therapeutic possibility.

Growth Factor Midkine Aggravates Pulmonary Arterial Hypertension via Surface Nucleolin.

Pulmonary arterial hypertension (PAH) is a progressive fatal disease caused by pulmonary arterial remodeling. Midkine regulates cell proliferation and migration, and it is induced by hypoxia, but its roles in pulmonary arterial remodeling remain unclear. Serum midkine levels were significantly increased in PAH patients compared with control patients. Midkine expression was increased in lungs and sera of hypoxia-induced PAH mice. Hypoxia-induced pulmonary arterial remodeling and right ventricular hypertrophy were attenuated in midkine-knockout mice. Midkine-induced proliferation and migration of pulmonary arterial smooth muscle cells (PASMC) and epidermal growth factor receptor (EGFR) signaling were significantly increased under hypoxia, which also induced cell-surface translocation of nucleolin. Nucleolin siRNA treatment suppressed midkine-induced EGFR activation in vitro, and nucleolin inhibitor AS1411 suppressed proliferation and migration of PASMC induced by midkine. Furthermore, AS1411 significantly prevented the development of PAH in Sugen hypoxia rat model. Midkine plays a crucial role in PAH development through interaction with surface nucleolin. These data define a role for midkine in PAH development and suggest midkine-nucleolin-EGFR axis as a novel therapeutic target for PAH.

Thymidylate synthase inhibitor raltitrexed can induce high levels of DNA damage in MYCN-amplified neuroblastoma cells.

MYCN gene amplification is consistently associated with poor prognosis in patients with neuroblastoma, a pediatric tumor arising from the sympathetic nervous system. Conventional anticancer drugs, such as alkylating agents and platinum compounds, have been used for the treatment of high-risk patients with MYCN-amplified neuroblastoma, whereas molecule-targeting drugs have not yet been approved. Therefore, the development of a safe and effective therapeutic approach is highly desired. Although thymidylate synthase inhibitors are widely used for colorectal and gastric cancers, their usefulness in neuroblastoma has not been well studied. Here, we investigated the efficacies of approved antifolates, methotrexate, pemetrexed, and raltitrexed (RTX), on MYCN-amplified and nonamplified neuroblastoma cell lines. Cell growth-inhibitory assay revealed that RTX showed a superior inhibitory activity against MYCN-amplified cell lines. We found no significant differences in the protein expression levels of the antifolate transporter or thymidylate synthase, a primary target of RTX, among the cell lines. Because thymidine supplementation could rescue the RTX-induced cell growth suppression, the effect of RTX was mainly due to the reduction in dTTP synthesis. Interestingly, RTX treatments induced single-stranded DNA damage response in MYCN-amplified cells to a greater extent than in the nonamplified cells. We propose that the high DNA replication stress and elevated levels of DNA damage, which are a result of deregulated expression of MYCN target genes, could be the cause of increased sensitivity to RTX.

miR-146a targeted to splenic macrophages prevents sepsis-induced multiple organ injury.

Development of a novel agent against life-threatening sepsis requires the in-depth understanding of the relevant pathophysiology and therapeutic targets. Given the function of microRNAs (miRNAs) as potent oligonucleotide therapeutics, here we investigated the pathophysiological role of exogenously applied miRNA in sepsis-induced multiple organ injury. In vitro, miR-16, miR-126, miR-146a, and miR-200b suppressed the production of pro-inflammatory cytokines in RAW264.7 macrophage cells after lipopolysaccharide (LPS) stimulation. Of these, miR-146a displayed the most highly suppressive effect, wherein the transcriptional activity of nuclear factor kappa B (NF-κB) was decreased via targeting of interleukin 1 receptor-associated kinase 1 and tumor necrosis receptor-associated factor 6. Sepsis was induced in mice via cecal ligation and puncture (CLP) and an intravenous injection of a complex of miR-146a-expressing plasmid and polyethyleneimine. Treatment with this complex significantly decreased the level of serum inflammatory cytokines, attenuated organ injury including kidney injury, and led to increased survival from polymicrobial sepsis induced by CLP. miR-146a-expressing plasmid was abundantly distributed in splenic macrophages, but not in renal parenchymal cells. CLP mice treated with miR-146a displayed significantly decreased NF-κB activation and splenocyte apoptosis. Splenectomy diminished the anti-inflammatory effects of miR-146a. The collective results support the conclusion that the induction of miR-146a expression in splenic macrophages prevents excessive inflammation and sepsis-induced multiple organ injury. This study establishes a novel and critical pathophysiological role for splenic macrophage interference in sepsis-related organ injury.

Generation and characterization of antagonistic anti-human interleukin (IL)-18 monoclonal antibodies with high affinity: Two types of monoclonal antibodies against full-length IL-18 and the neoepitope of inflammatory caspase-cleaved active IL-18.

Interleukin-18 (IL-18) is a pro-inflammatory cytokine that evokes both innate and acquired immune responses. IL-18 is initially synthesized as an inactive precursor and the cleavage for processing into a mature, active molecule is mediated by pro-inflammatory caspases following the activation of inflammasomes. Two types of monoclonal antibodies were raised: anti-IL-1863-68 antibodies which recognize full-length1-193 and cleaved IL-18; and anti-IL-18 neoepitope antibodies which specifically recognize the new N-terminal 37YFGKLESK44 of IL-18 cleaved by pro-inflammatory caspase-1/4. These mAbs were suitable for Western blotting, capillary Western immunoassay (WES), immunofluorescence, immunoprecipitation, and function-blocking assays. WES analysis of these mAbs allowed visualization of the IL-18 bands and provided a molecular weight corresponding to the pro-inflammatory caspase-1/4 cleaved, active form IL-1837-193, and not to the inactive precursor IL-18, in the serum of patients with adult-onset Still's disease (6/14, 42%) and hemophagocytic activation syndrome (2/6, 33%). These monoclonal antibodies will be very useful in IL-18 and inflammasome biology and for diagnostic and therapeutic strategies for inflammatory diseases.

Origin and initiation mechanisms of neuroblastoma.

Neuroblastoma is an embryonal malignancy that affects normal development of the adrenal medulla and paravertebral sympathetic ganglia in early childhood. Extensive studies have revealed the molecular characteristics of human neuroblastomas, including abnormalities at genome, epigenome and transcriptome levels. However, neuroblastoma initiation mechanisms and even its origin are long-standing mysteries. In this review article, we summarize the current knowledge about normal development of putative neuroblastoma sources, namely sympathoadrenal lineage of neural crest cells and Schwann cell precursors that were recently identified as the source of adrenal chromaffin cells. A plausible origin of enigmatic stage 4S neuroblastoma is also discussed. With regard to the initiation mechanisms, we review genetic abnormalities in neuroblastomas and their possible association to initiation mechanisms. We also summarize evidences of neuroblastoma initiation observed in genetically engineered animal models, in which epigenetic alterations were involved, including transcriptomic upregulation by N-Myc and downregulation by polycomb repressive complex 2. Finally, several in vitro experimental methods are proposed that hopefully will accelerate our comprehension of neuroblastoma initiation. Thus, this review summarizes the state-of-the-art knowledge about the mechanisms of neuroblastoma initiation, which is critical for developing new strategies to cure children with neuroblastoma.

The clinical relevance of plasma CD147/basigin in biopsy-proven kidney diseases.

BACKGROUND: Precise understanding of kidney disease activity is needed to design therapeutic strategies. CD147/basigin is involved in the pathogenesis of acute kidney injury and renal fibrosis through inflammatory cell infiltration. The present study examined the clinical relevance of CD147 in biopsy-proven kidney diseases that lead to the progression of chronic kidney disease. METHODS: Kidney biopsy specimens and plasma and urine samples were obtained from patients with kidney diseases, including IgA nephropathy (IgAN), Henoch-Schönlein purpura nephritis (HSPN), diabetic kidney disease (DKD), focal segmental glomerulosclerosis (FSGS), and membranous nephropathy (MN), who underwent renal biopsy between 2011 and 2014. Plasma and urinary CD147 levels were measured and evaluated for their ability to reflect histological features. Disease activity of IgAN tissues was evaluated according to the Oxford classification and the Japanese histological grading system. RESULTS: In biopsy tissues, CD147 induction was detected in injured lesions representing renal inflammation. Plasma CD147 values correlated with eGFR in patients with inflammation-related kidney diseases such as IgAN, HSPN, and DKD. Particularly in IgAN patients, plasma CD147 levels were correlated with injured regions comprising more than 50% of glomeruli or with tubular atrophy/interstitial injury in biopsy tissues. Proteinuria showed a closer correlation with urinary values of CD147 and L-FABP. Of note, plasma and urinary CD147 levels showed a strong correlation with eGFR or proteinuria, respectively, only in DKD patients. CONCLUSION: Evaluation of plasma and urinary CD147 levels might provide key insights for the understanding of the activity of various kidney diseases.

Dual inhibition of P-glycoprotein and midkine may increase therapeutic effects of anticancer drugs.

Multidrug resistance (MDR) to chemotherapy may significantly affect the outcome of cancer treatment. ATP-dependent drug efflux pumps, including P-glycoprotein (P-gp), contribute to the resistance of various chemotherapeutic agents. Overexpression of P-gp in tumor cells induces chemoresistance via pumping the anticancer drugs out of the cells. In addition to taking part in many biological processes such as development, reproduction and repair, midkine (MK) also plays important roles in the pathogenesis of malignant diseases as well as in the regulation of MDR. Although, the mechanisms of action of P-gp and MK are different, overexpression of both proteins prevents the accumulation of many chemotherapeutics in tumor cells, leading to decreased therapeutic effects of anticancer drugs. Therefore, identification of the result of dual inhibition of P-gp and MK in overcoming chemoresistance may enhance the likelihood for a more efficient chemotherapy.

Involvement of midkine in the development of pulmonary fibrosis.

Midkine is a low-molecular-weight heparin-binding protein that is strongly expressed mainly in the midgestation period and has various physiological activities such as in development and cell migration. Midkine has been reported to be strongly expressed in cancer cells and in inflammation and repair processes, and to be involved in the pathogenesis of various diseases. However, its role in the lung is poorly understood. In this study, we analyzed the clinical characteristics of idiopathic pulmonary fibrosis patients in relation to midkine expression and used a mouse bleomycin-induced pulmonary fibrosis model to investigate the role of midkine in pulmonary fibrosis. In the idiopathic pulmonary fibrosis patients, the serum midkine level was significantly higher than in healthy subjects, and midkine levels in the serum and bronchoalveolar lavage (BAL) fluid correlated positively with the percentage of inflammatory cells in the BAL fluid. In wild-type mice, intratracheal bleomycin administration increased midkine expression in lung tissue. Additionally, compared with wild-type mice, midkine-deficient mice showed low expression of both collagen and α-smooth muscle actin, as well as a low value for the pathological lung fibrosis score after bleomycin administration. Furthermore, the total cell count and lymphocyte percentage in the BAL fluid, as well as TNF-α and transforming growth factor-ß expression in lung tissue, were significantly lower in the midkine-deficient mice compared with wild-type mice. These results suggest that midkine is involved in the development of pulmonary fibrosis by regulating inflammatory cell migration into the lung, and TNF-α and transforming growth factor-ß expression.

PRC2-Mediated Transcriptomic Alterations at the Embryonic Stage Govern Tumorigenesis and Clinical Outcome in MYCN-Driven Neuroblastoma.

Pediatric cancers such as neuroblastoma are thought to involve a dysregulation of embryonic development. However, it has been difficult to identify the critical events that trigger tumorigenesis and differentiate them from normal development. In this study, we report the establishment of a spheroid culture method that enriches early-stage tumor cells from TH-MYCN mice, a preclinical model of neuroblastoma. Using this method, we found that tumorigenic cells were evident as early as day E13.5 during embryo development, when the MYC and PRC2 transcriptomes were significantly altered. Ezh2, an essential component of PRC2, was expressed in embryonic and postnatal tumor lesions and physically associated with N-MYC and we observed that H3K27me3 was increased at PRC2 target genes. PRC2 inhibition suppressed in vitro sphere formation, derepressed its target genes, and suppressed in situ tumor growth. In clinical specimens, expression of MYC and PRC2 target genes correlated strongly and predicted survival outcomes. Together, our findings highlighted PRC2-mediated transcriptional control during embryogenesis as a critical step in the development and clinical outcome of neuroblastoma. Cancer Res; 77(19); 5259-71. ©2017 AACR.

Mechanisms of axon regeneration: The significance of proteoglycans.

BACKGROUND: Therapeutics specific to neural injury have long been anticipated but remain unavailable. Axons in the central nervous system do not readily regenerate after injury, leading to dysfunction of the nervous system. This failure of regeneration is due to both the low intrinsic capacity of axons for regeneration and the various inhibitors emerging upon injury. After many years of concerted efforts, however, these hurdles to axon regeneration have been partially overcome. SCOPE OF REVIEW: This review summarizes the mechanisms regulating axon regeneration. We highlight proteoglycans, particularly because it has become increasingly clear that these proteins serve as critical regulators for axon regeneration. MAJOR CONCLUSIONS: Studies on proteoglycans have revealed that glycans not only assist in the modulation of protein functions but also act as main players-e.g., as functional ligands mediating intracellular signaling through specific receptors on the cell surface. By regulating clustering of the receptors, glycans in the proteoglycan moiety, i.e., glycosaminoglycans, promote or inhibit axon regeneration. In addition, proteoglycans are involved in various types of neural plasticity, ranging from synaptic plasticity to experience-dependent plasticity. GENERAL SIGNIFICANCE: Although studies on proteins have progressively facilitated our understanding of the nervous system, glycans constitute a new frontier for further research and development in this field. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.