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.

Basigin is released in extracellular vesicles derived from the renal tubular epithelium in response to albuminuria.

AIM: Irrespective of the cause, albumin/proteinuria induces tubulointerstitial damage and accelerates the progression of kidney diseases. Our series of studies demonstrated that proteinuria, an independent prognostic factor for chronic kidney disease (CKD), is correlated with urinary basigin/CD147 (Bsg) levels. We examined the morphology and origin of Bsg in the tubular lumen through the effects of filtered glucose and protein solutes on the tubules. METHODS: Diabetic kidney disease (DKD) patients (N = 50) were treated with spironolactone 25 mg for 4 weeks or by conservative treatment. The associations between urinary Bsg values and clinical indicators were examined. Primary-cultured proximal tubular epithelial cells (PTECs) from human adult kidneys were exposed to high glucose or bovine serum albumin (BSA). RESULTS: In patients with early phase DKD, urinary Bsg levels were closely correlated with proteinuria but not HbA1c. Full-length Bsg on extracellular vesicles (EVs) was investigated primarily in urine collected from DKD patients. EVs obtained from the urine of DKD patients included Bsg and SGLT2 proteins. Notably, spironolactone treatment concomitantly suppressed the release of Bsg-bearing EVs in correlation with decreased albuminuria. Exposure of PTECs to BSA (but not high glucose) enhanced the storage of supernatant Bsg in EVs despite the absence of exposure-specific changes in Bsg transcription. CONCLUSION: Proteinuria induces the release of Bsg-bearing EVs derived from PTECs into the tubular lumen.

Regulation of lipid synthesis in myelin modulates neural activity and is required for motor learning.

Brain function relies on both rapid electrical communication in neural circuitry and appropriate patterns or synchrony of neural activity. Rapid communication between neurons is facilitated by wrapping nerve axons with insulation by a myelin sheath composed largely of different lipids. Recent evidence has indicated that the extent of myelination of nerve axons can adapt based on neural activity levels and this adaptive myelination is associated with improved learning of motor tasks, suggesting such plasticity may enhance effective learning. In this study, we examined whether another aspect of myelin plasticity-changes in myelin lipid synthesis and composition-may also be associated with motor learning. We combined a motor learning task in mice with in vivo two-photon imaging of neural activity in the primary motor cortex (M1) to distinguish early and late stages of learning and then probed levels of some key myelin lipids using mass spectrometry analysis. Sphingomyelin levels were elevated in the early stage of motor learning while galactosylceramide levels were elevated in the middle and late stages of motor learning, and these changes were correlated across individual mice with both learning performance and neural activity changes. Targeted inhibition of oligodendrocyte-specific galactosyltransferase expression, the enzyme that synthesizes myelin galactosylceramide, impaired motor learning. Our results suggest regulation of myelin lipid composition could be a novel facet of myelin adaptations associated with learning.

Glypican-2 defines age-dependent axonal response to chondroitin sulfate.

Axons of terminally differentiated neurons in the mammalian central nervous system (CNS) are unable to regenerate after dissection. One of the mechanisms underlying this is the inhibition of axonal regeneration by chondroitin sulfate (CS) and its neuronal receptor, PTPσ. Our previous results demonstrated that the CS-PTPσ axis disrupted autophagy flux by dephosphorylating cortactin, which led to the formation of dystrophic endballs and to the inhibition of axonal regeneration. In contrast, juvenile neurons vigorously extend axons toward their targets during development and maintain regenerative activity for axons even after injury. Although several intrinsic and extrinsic mechanisms have been reported to mediate the differences, the detailed mechanisms are still elusive. Here, we report that Glypican-2, a member of heparan sulfate proteoglycans (HSPG), which are able to antagonize CS-PTPσ by competing with the receptor, is specifically expressed in the axonal tips of embryonic neurons. Glypican-2 overexpression in adult neurons rescues the dystrophic endball back to a healthy growth cone on the CSPG gradient. Consistently, Glypican-2 restored cortactin phosphorylation in the axonal tips of adult neurons on CSPG. Taken together, our results clearly demonstrated Glypican-2's pivotal role in defining the axonal response to CS and provided a new therapeutic target for axonal injury.

Towards the in vivo identification of protein-protein interactions.

Protein-protein interactions (PPIs) play crucial roles in biological processes. The conventional methods based on affinity purification of a protein of interest (POI) have been widely used to identify unknown PPIs. Recently, proximity-dependent biotin identification (BioID) has been used increasingly to investigate PPIs. BioID utilizes the proximity-dependent biotinylation, in the presence of biotin, of endogenous proteins that are located within a certain distance of POI-fused biotin ligase, which enables us to reveal the more physiologically relevant PPIs in vivo compared to the conventional methods. However, there is little information on an appropriate way to administer biotin in vivo. Recent studies reported some biotin supplementations for in vivo BioID. In this commentary, we review the BioID technique as a tool to examine PPIs, and we introduce a potential method to achieve efficient proximity labelling for in vivo BioID.

Single-cell Glycogenomics Deciphers Links Between Altered Transcriptional Regulation and Aberrant Glycosylation in Alzheimer's Disease.

Glycosylation is increasingly recognized as a potential new therapeutic target in Alzheimer's disease. In recent years, evidence for Alzheimer's disease-specific glycoproteins has been established. However, the mechanisms of their dysregulation, including tissue and cell type specificity, are not fully understood. We aimed to explore upstream regulators of aberrant glycosylation by integrating multiple data sources and using a glycogenomics approach. We identified dysregulation by the glycosyltransferase PLOD3 in oligodendrocytes as an upstream regulator in cerebral vessels, and found that it is involved in COL4A5 synthesis, which is strongly correlated with amyloid fiber formation. Furthermore, COL4A5 was suggested to interact with astrocytes via ECM receptors as a ligand. This study suggests directions for new therapeutic strategies for Alzheimer's disease targeting glycosyltransferases.

Systematic characterization of seed overlap microRNA cotargeting associated with lupus pathogenesis.

BACKGROUND: Combinatorial gene regulation by multiple microRNAs (miRNAs) is widespread and closely spaced target sites often act cooperatively to achieve stronger repression ("neighborhood" miRNA cotargeting). While miRNA cotarget sites are suggested to be more conserved and implicated in developmental control, the pathological significance of miRNA cotargeting remains elusive. RESULTS: Here, we report the pathogenic impacts of combinatorial miRNA regulation on inflammation in systemic lupus erythematosus (SLE). In the SLE mouse model, we identified the downregulation of two miRNAs, miR-128 and miR-148a, by TLR7 stimulation in plasmacytoid dendritic cells. Functional analyses using human cell lines demonstrated that miR-128 and miR-148a additively target KLF4 via extensively overlapping target sites ("seed overlap" miRNA cotargeting) and suppress the inflammatory responses. At the transcriptome level, "seed overlap" miRNA cotargeting increases susceptibility to downregulation by two miRNAs, consistent with additive but not cooperative recruitment of two miRNAs. Systematic characterization further revealed that extensive "seed overlap" is a prevalent feature among broadly conserved miRNAs. Highly conserved target sites of broadly conserved miRNAs are largely divided into two classes-those conserved among eutherian mammals and from human to Coelacanth, and the latter, including KLF4-cotargeting sites, has a stronger association with both "seed overlap" and "neighborhood" miRNA cotargeting. Furthermore, a deeply conserved miRNA target class has a higher probability of haplo-insufficient genes. CONCLUSIONS: Our study collectively suggests the complexity of distinct modes of miRNA cotargeting and the importance of their perturbations in human diseases.

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.

Metabolome and transcriptome analysis on muscle of sporadic inclusion body myositis.

OBJECTIVE: Sporadic inclusion body myositis (sIBM) is the most common acquired myopathy in patients older than 50 years of age. sIBM is hardly responds to any immunosuppressing theraphies, and its pathophysiology remains elusive. This study aims to explore pathogenic pathways underlying sIBM and identify novel therapeutic targets using metabolomic and transcriptomic analyses. METHODS: In this retrospective observational study, we analyzed biopsied muscle samples from 14 sIBM patients and six non-diseased subjects to identify metabolic profiles. Frozen muscle samples were used to measure metabolites with cation and anion modes of capillary electrophoresis time of flight mass spectrometry. We validated the metabolic pathway altered in muscles of sIBM patients through RNA sequencing and histopathological studies. RESULTS: A total of 198 metabolites were identified. Metabolomic and transcriptomic analyses identified specific metabolite changes in sIBM muscle samples. The pathways of histamine biosynthesis and certain glycosaminoglycan biosynthesis were upregulated in sIBM patients, whereas those of carnitine metabolism and creatine metabolism were downregulated. Histopathological examination showed infiltration of mast cells and deposition of chondroitin sulfate in skeletal muscle samples, supporting the results of metabolomic and transcriptomic analyses. INTERPRETATION: We identified alterations of several metabolic pathways in muscle samples of sIBM patients. These results suggest that mast cells, chondroitin sulfate biosynthesis, carnitine, and creatine play roles in sIBM pathophysiology.

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.

Basigin deficiency prevents anaplerosis and ameliorates insulin resistance and hepatosteatosis.

Monocarboxylates, such as lactate and pyruvate, are precursors for biosynthetic pathways, including those for glucose, lipids, and amino acids via the tricarboxylic acid (TCA) cycle and adjacent metabolic networks. The transportation of monocarboxylates across the cellular membrane is performed primarily by monocarboxylate transporters (MCTs), the membrane localization and stabilization of which are facilitated by the transmembrane protein basigin (BSG). Here, we demonstrate that the MCT/BSG axis sits at a crucial intersection of cellular metabolism. Abolishment of MCT1 in the plasma membrane was achieved by Bsg depletion, which led to gluconeogenesis impairment via preventing the influx of lactate and pyruvate into the cell, consequently suppressing the TCA cycle. This net anaplerosis suppression was compensated in part by the increased utilization of glycogenic amino acids (e.g., alanine and glutamine) into the TCA cycle and by activated ketogenesis through fatty acid ß-oxidation. Complementary to these observations, hyperglycemia and hepatic steatosis induced by a high-fat diet were ameliorated in Bsg-deficient mice. Furthermore, Bsg deficiency significantly improved insulin resistance induced by a high-fat diet. Taken together, the plasma membrane-selective modulation of lactate and pyruvate transport through BSG inhibition could potentiate metabolic flexibility to treat metabolic diseases.

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.

Axonal Regeneration by Glycosaminoglycan.

Like other biomolecules including nucleic acid and protein, glycan plays pivotal roles in various cellular processes. For instance, it modulates protein folding and stability, organizes extracellular matrix and tissue elasticity, and regulates membrane trafficking. In addition, cell-surface glycans are often utilized as entry receptors for viruses, including SARS-CoV-2. Nevertheless, its roles as ligands to specific surface receptors have not been well understood with a few exceptions such as selectins and siglecs. Recent reports have demonstrated that chondroitin sulfate and heparan sulfate, both of which are glycosaminoglycans, work as physiological ligands on their shared receptor, protein tyrosine phosphatase sigma (PTPσ). These two glycans differentially determine the fates of neuronal axons after injury in our central nervous system. That is, heparan sulfate promotes axonal regeneration while chondroitin sulfate inhibits it, inducing dystrophic endbulbs at the axon tips. In our recent study, we demonstrated that the chondroitin sulfate (CS)-PTPσ axis disrupted autophagy flux at the axon tips by dephosphorylating cortactin. In this minireview, we introduce how glycans work as physiological ligands and regulate their intracellular signaling, especially focusing on chondroitin sulfate.

Midkine Interaction with Chondroitin Sulfate Model Synthetic Tetrasaccharides and Their Mimetics: The Role of Aromatic Interactions.

Midkine (MK) is a neurotrophic factor that participates in the embryonic central nervous system (CNS) development and neural stem cell regulation, interacting with sulfated glycosaminoglycans (GAGs). Chondroitin sulfate (CS) is the natural ligand in the CNS. In this work, we describe the interactions between a library of synthetic models of CS-types and mimics. We did a structural study of this library by NMR and MD (Molecular Dynamics), concluding that the basic shape is controlled by similar geometry of the glycosidic linkages. Their 3D structures are a helix with four residues per turn, almost linear. We have studied the tetrasaccharide-midkine complexes by ligand observed NMR techniques and concluded that the shape of the ligands does not change upon binding. The ligand orientation into the complex is very variable. It is placed inside the central cavity of MK formed by the two structured beta-sheets domains linked by an intrinsically disordered region (IDR). Docking analysis confirmed the participation of aromatics residues from MK completed with electrostatic interactions. Finally, we test the biological activity by increasing the MK expression using CS tetrasaccharides and their capacity in enhancing the growth stimulation effect of MK in NIH3T3 cells.

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.

Type IIa RPTPs and Glycans: Roles in Axon Regeneration and Synaptogenesis.

Type IIa receptor tyrosine phosphatases (RPTPs) play pivotal roles in neuronal network formation. It is emerging that the interactions of RPTPs with glycans, i.e., chondroitin sulfate (CS) and heparan sulfate (HS), are critical for their functions. We highlight here the significance of these interactions in axon regeneration and synaptogenesis. For example, PTPσ, a member of type IIa RPTPs, on axon terminals is monomerized and activated by the extracellular CS deposited in neural injuries, dephosphorylates cortactin, disrupts autophagy flux, and consequently inhibits axon regeneration. In contrast, HS induces PTPσ oligomerization, suppresses PTPσ phosphatase activity, and promotes axon regeneration. PTPσ also serves as an organizer of excitatory synapses. PTPσ and neurexin bind one another on presynapses and further bind to postsynaptic leucine-rich repeat transmembrane protein 4 (LRRTM4). Neurexin is now known as a heparan sulfate proteoglycan (HSPG), and its HS is essential for the binding between these three molecules. Another HSPG, glypican 4, binds to presynaptic PTPσ and postsynaptic LRRTM4 in an HS-dependent manner. Type IIa RPTPs are also involved in the formation of excitatory and inhibitory synapses by heterophilic binding to a variety of postsynaptic partners. We also discuss the important issue of possible mechanisms coordinating axon extension and synapse formation.

Enoxaparin promotes functional recovery after spinal cord injury by antagonizing PTPRσ.

The receptor-type protein tyrosine phosphatase sigma (PTPRσ) regulates axonal regeneration/sprouting as a molecular switch in response to glycan ligands. Cell surface heparan sulfate oligomerizes PTPRσ and inactivates its enzymatic activity, which in turn promotes axonal growth. In contrast, matrix-associated chondroitin sulfate monomerizes PTPRσ and activates it. This leads to dephosphorylation of its specific substrates, such as cortactin, resulting in a failure of axonal regeneration after injury. However, this molecular switch model has never been challenged in a clinical situation. In this study, we demonstrated that enoxaparin, a globally approved anticoagulant consisting of heparin oligosaccharides with an average molecular weight of 45 kDa, induced clustering and inactivated PTPRσ in vitro. Enoxaparin induced PTPRσ clustering, and counteracted PTPRσ-mediated dephosphorylation of cortactin, which was shown to be important for inhibition of axonal regeneration. Systemic administration of enoxaparin promoted anatomical recovery after both optic nerve and spinal cord injuries in rats at clinically tolerated doses. Moreover, enoxaparin promoted recovery of motor function without obvious hemorrhage. Collectively, our data provide a new strategy for the treatment of traumatic axonal injury.