High endothelial venules as potential gateways for therapeutics.

High endothelial venules (HEVs) are specialized blood vessels that support the migration of lymphocytes from the bloodstream into lymph nodes (LNs). They are also formed ectopically in mammalian organs affected by chronic inflammation and cancer. The recent arrival of immunotherapy at the forefront of many cancer treatment regimens could boost a crucial role for HEVs as gateways for the treatment of cancer. In this review, we describe the microanatomical and biochemical characteristics of HEVs, mechanisms of formation of newly made HEVs, immunotherapies potentially dependent on HEV-mediated T cell homing to tumors, and finally, how HEV-targeted therapies might be used as a complementary approach to potentially shape the therapeutic landscape for the treatment of cancer and immune-mediated diseases.

Impacts of cytoplasmic p53 aggregates on the prognosis and the transcriptome in lung squamous cell carcinoma.

The tumor suppressor TP53 gene, the most frequently mutated gene in human cancers, produces the product tumor protein p53, which plays an essential role in DNA damage. p53 protein mutations may contribute to tumorigenesis by loss of tumor suppressive functions and malignancy of cancer cells via gain-of-oncogenic functions. We previously reported that mutant p53 proteins form aggregates and that cytoplasmic p53 aggregates were associated with poor prognosis in human ovarian cancer. However, the prognostic impact of p53 aggregation in other tumors including lung squamous cell carcinoma (SCC) is poorly understood. Here, we demonstrated that lung SCC cases with cytoplasmic p53 aggregates had a significantly poor clinical prognosis. Analysis via patient-derived tumor organoids (PDOs) established from lung SCC patients and possessing cytoplasmic p53 aggregates showed that eliminating cytoplasmic p53 aggregates suppressed cell proliferation. RNA sequencing and transcriptome analysis of p53 aggregate-harboring PDOs indicated multiple candidate pathways involved in p53 aggregate oncogenic functions. With lung SCC-derived cell lines, we found that cytoplasmic p53 aggregates contributed to cisplatin resistance. This study thus shows that p53 aggregates are a predictor of poor prognosis in lung SCC and suggests that detecting p53 aggregates via p53 conventional immunohistochemical analysis may aid patient selection for platinum-based therapy.

GlcNAc6ST2/CHST4 Is Essential for the Synthesis of R-10G-Reactive Keratan Sulfate/Sulfated N-Acetyllactosamine Oligosaccharides in Mouse Pleural Mesothelium.

We recently showed that 6-sulfo sialyl N-acetyllactosamine (LacNAc) in O-linked glycans recognized by the CL40 antibody is abundant in the pleural mesothelium under physiological conditions and that these glycans undergo complementary synthesis by GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5) in mice. GlcNAc6ST3 is essential for the synthesis of R-10G-positive keratan sulfate (KS) in the brain. The predicted minimum epitope of the R-10G antibody is a dimeric asialo 6-sulfo LacNAc. Whether R-10G-reactive KS/sulfated LacNAc oligosaccharides are also present in the pleural mesothelium was unknown. The question of which GlcNAc6STs are responsible for R-10G-reactive glycans was an additional issue to be clarified. Here, we show that R-10G-reactive glycans are as abundant in the pulmonary pleura as CL40-reactive glycans and that GlcNAc6ST3 is only partially involved in the synthesis of these pleural R-10G glycans, unlike in the adult brain. Unexpectedly, GlcNAc6ST2 is essential for the synthesis of R-10G-positive KS/sulfated LacNAc oligosaccharides in the lung pleura. The type of GlcNAc6ST and the magnitude of its contribution to KS glycan synthesis varied among tissues in vivo. We show that GlcNAc6ST2 is required and sufficient for R-10G-reactive KS synthesis in the lung pleura. Interestingly, R-10G immunoreactivity in KSGal6ST (encoded by Chst1) and C6ST1 (encoded by Chst3) double-deficient mouse lungs was markedly increased. MUC16, a mucin molecule, was shown to be a candidate carrier protein for pleural R-10G-reactive glycans. These results suggest that R-10G-reactive KS/sulfated LacNAc oligosaccharides may play a role in mesothelial cell proliferation and differentiation. Further elucidation of the functions of sulfated glycans synthesized by GlcNAc6ST2 and GlcNAc6ST3, such as R-10G and CL40 glycans, in pathological conditions may lead to a better understanding of the underlying mechanisms of the physiopathology of the lung mesothelium.

Sulfated glycosaminoglycans mediate prion-like behavior of p53 aggregates.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) are heteropolysaccharides implicated in the pathology of protein aggregation diseases including localized and systemic forms of amyloidosis. Among subdomains of sulfated GAGs, highly sulfated domains of HS, called HS S-domains, have been highlighted as being critical for HS function in amyloidoses. Recent studies suggest that the tumor suppressor p53 aggregates to form amyloid fibrils and propagates in a prion-like manner; however, molecules and mechanisms that are involved in the prion-like behavior of p53 aggregates have not been addressed. Here, we identified sulfated GAGs as molecules that mediate prion-like behavior of p53 aggregates. Sulfated GAGs at the cell surface were required for cellular uptake of recombinant and cancer cell-derived p53 aggregates and extracellular release of p53 from cancer cells. We further showed that HS S-domains accumulated within p53 deposits in human ovarian cancer tissues, and enzymatic remodeling of HS S-domains by Sulf-2 extracellular sulfatase down-regulated cellular uptake of p53 aggregates. Finally, sulfated GAG-dependent cellular uptake of p53 aggregates was critical for subsequent extracellular release of the aggregates and gain of oncogenic function in recipient cells. Our work provides a mechanism of prion-like behavior of p53 aggregates and will shed light on sulfated GAGs as a common mediator of prions.

Design and Synthesis of 6-O-Phosphorylated Heparan Sulfate Oligosaccharides to Inhibit Amyloid ß Aggregation.

Dysregulation of amyloidogenic proteins and their abnormal processing and deposition in tissues cause systemic and localized amyloidosis. Formation of amyloid ß (Aß) fibrils that deposit as amyloid plaques in Alzheimer's disease (AD) brains is an earliest pathological hallmark. The polysulfated heparan sulfate (HS)/heparin (HP) is one of the non-protein components of Aß deposits that not only modulates Aß aggregation, but also acts as a receptor for Aß fibrils to mediate their cytotoxicity. Interfering with the interaction between HS/HP and Aß could be a therapeutic strategy to arrest amyloidosis. Here we have synthesized the 6-O-phosphorylated HS/HP oligosaccharides and reported their competitive effects on the inhibition of HP-mediated Aß fibril formation in vitro using a thioflavin T fluorescence assay and a tapping mode atomic force microscopy.

Thrombospondin type 1 repeat-derived C-mannosylated peptide attenuates synaptogenesis of cortical neurons induced by primary astrocytes via TGF-ß.

C-Mannosylation is a rare type of protein glycosylation and is reportedly critical for the proper folding and secretion of parental proteins. Still, the effects of C-mannosylation on the biological functions of these modified proteins remain to be elucidated. The Trp-x-x-Trp (WxxW) sequences, whose first tryptophan (Trp) can be C-mannosylated, constitute the consensus motifs for this glycosylation modification and are commonly found in thrombospondin type 1 repeats that regulate molecular functions of thrombospondin 1 in binding and activation of transforming growth factor ß (TGF-ß). TGF-ß plays critical roles in the control of the central nervous system including synaptogenesis. Here, we investigated whether C-mannosylation of the synthetic Trp-Ser-Pro-Trp (WSPW) peptide may confer certain functions to this peptide in TGF-ß-mediated synaptogenesis. By using primary cultured rat astrocytes and cortical neurons, we found that the C-mannosylated WSPW (C-Man-WSPW) peptide, but not non-mannosylated WSPW peptide, suppressed astrocyte-conditioned medium (ACM)-stimulated synaptogenesis. C-Man-WSPW peptide inhibited both ACM- and recombinant mature TGF-ß1-induced activations of Smad 2, an important mediator in TGF-ß signaling. Interactions of recombinant mature TGF-ß with the C-Man-WSPW peptide were similar to those with non-C-mannosylated WSPW peptide. Taken together, our results reveal a novel function of C-mannosylation of the WxxW motif in signaling and synaptogenesis mediated by TGF-ß. Molecular details of how C-mannosylation affects the biological functions of WxxW motifs deserve future study for clarification.

Complementary Role of GlcNAc6ST2 and GlcNAc6ST3 in Synthesis of CL40-Reactive Sialylated and Sulfated Glycans in the Mouse Pleural Mesothelium.

Sialyl 6-sulfo Lewis X (6-sulfo sLeX) and its derivative sialyl 6-sulfo N-acetyllactosamine (LacNAc) are sialylated and sulfated glycans of sialomucins found in the high endothelial venules (HEVs) of secondary lymphoid organs. A component of 6-sulfo sLeX present in the core 1-extended O-linked glycans detected by the MECA-79 antibody was previously shown to exist in the lymphoid aggregate vasculature and bronchial mucosa of allergic and asthmatic lungs. The components of 6-sulfo sLeX in pulmonary tissues under physiological conditions remain to be analyzed. The CL40 antibody recognizes 6-sulfo sLeX and sialyl 6-sulfo LacNAc in O-linked and N-linked glycans, with absolute requirements for both GlcNAc-6-sulfation and sialylation. Immunostaining of normal mouse lungs with CL40 was performed and analyzed. The contribution of GlcNAc-6-O-sulfotransferases (GlcNAc6STs) to the synthesis of the CL40 epitope in the lungs was also elucidated. Here, we show that the expression of the CL40 epitope was specifically detected in the mesothelin-positive mesothelium of the pulmonary pleura. Moreover, GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5), but not GlcNAc6ST1 (encoded by Chst2) or GlcNAc6ST4 (encoded by Chst7), are required for the synthesis of CL40-positive glycans in the lung mesothelium. Furthermore, neither GlcNAc6ST2 nor GlcNAc6ST3 is sufficient for in vivo expression of the CL40 epitope in the lung mesothelium, as demonstrated by GlcNAc6ST1/3/4 triple-knock-out and GlcNAc6ST1/2/4 triple-knock-out mice. These results indicate that CL40-positive sialylated and sulfated glycans are abundant in the pleural mesothelium and are synthesized complementarily by GlcNAc6ST2 and GlcNAc6ST3, under physiological conditions in mice.

Glycan sulfation patterns define autophagy flux at axon tip via PTPRσ-cortactin axis.

Chondroitin sulfate (CS) and heparan sulfate (HS) are glycosaminoglycans that both bind the receptor-type protein tyrosine phosphatase PTPRσ, affecting axonal regeneration. CS inhibits axonal growth, while HS promotes it. Here, we have prepared a library of HS octasaccharides and, together with synthetic CS oligomers, we found that PTPRσ preferentially interacts with CS-E-a rare sulfation pattern in natural CS-and most HS oligomers bearing sulfate and sulfamate groups. Consequently, short and long stretches of natural CS and HS, respectively, bind to PTPRσ. CS activates PTPRσ, which dephosphorylates cortactin-herein identified as a new PTPRσ substrate-and disrupts autophagy flux at the autophagosome-lysosome fusion step. Such disruption is required and sufficient for dystrophic endball formation and inhibition of axonal regeneration. Therefore, sulfation patterns determine the length of the glycosaminoglycan segment that bind to PTPRσ and define the fate of axonal regeneration through a mechanism involving PTPRσ, cortactin and autophagy.

The Accumulation of Heparan Sulfate S-Domains in Kidney Transthyretin Deposits Accelerates Fibril Formation and Promotes Cytotoxicity.

The highly sulfated domains of heparan sulfate (HS), alias HS S-domains, are made up of repeated trisulfated disaccharide units [iduronic acid (2S)-glucosamine (NS, 6S)] and are selectively remodeled by extracellular endoglucosamine 6-sulfatases (Sulfs). Although HS S-domains are critical for signal transduction of several growth factors, their roles in amyloidoses are not yet fully understood. Herein, we found HS S-domains in the kidney of a patient with transthyretin amyloidosis. In in vitro assays with cells stably expressing human Sulfs, heparin, a structural analog of HS S-domains, promoted aggregation of transthyretin in an HS S-domain-dependent manner. Interactions of cells with transthyretin fibrils and cytotoxicity of these fibrils also depended on HS S-domains at the cell surface. Furthermore, glypican-5, encoded by the susceptibility gene for nephrotic syndrome GPC5, was found to be accumulated in the transthyretin amyloidosis kidney. Our study, thus, provides a novel insight into the pathologic roles of HS S-domains in amyloidoses, and we propose that enzymatic remodeling of HS chains by Sulfs may offer an effective approach to inhibiting formation and cytotoxicity of amyloid fibrils.

Deficiency of a sulfotransferase for sialic acid-modified glycans mitigates Alzheimer's pathology.

We previously showed that microglial keratan sulfate (KS) was induced in amyotrophic lateral sclerosis. However, the functional roles of the glycan and its synthetic enzyme in neurodegenerative diseases, such as Alzheimer's disease (AD), a progressive disorder, are unclear. In our study, KS modified with sialic acids having a molecular mass of 125-220 kDa and the carbohydrate sulfotransferase GlcNAc6ST1 were up-regulated in the brains of two transgenic mouse models (J20 and Tg2576) and the brains of patients with AD. GlcNAc6ST1-deficient J20 (J20/GlcNAc6ST1-/-) mice demonstrated a complete absence of the microglial sialylated KS. J20/GlcNAc6ST1-/- primary microglia showed an increased level of amyloid-ß phagocytosis and were hyperresponsive to interleukin 4, a potent antiinflammatory cytokine. Moreover, J20/GlcNAc6ST1-/- mice manifested reduced cerebral amyloid-ß deposition. GlcNAc6ST1-synthesizing sialylated KS thus modulates AD pathology. Inhibition of KS synthesis by targeting GlcNAc6ST1 may therefore be beneficial for controlling AD pathogenesis.

Growth Factor Midkine Promotes T-Cell Activation through Nuclear Factor of Activated T Cells Signaling and Th1 Cell Differentiation in Lupus Nephritis.

Activated T cells play crucial roles in the pathogenesis of autoimmune diseases, including lupus nephritis (LN). The activation of calcineurin/nuclear factor of activated T cells (NFAT) and STAT4 signaling is essential for T cells to perform various effector functions. Here, we identified the growth factor midkine (MK; gene name, Mdk) as a novel regulator in the pathogenesis of 2,6,10,14-tetramethylpentadecane-induced LN via activation of NFAT and IL-12/STAT4 signaling. Wild-type (Mdk+/+) mice showed more severe glomerular injury than MK-deficient (Mdk-/-) mice, as demonstrated by mesangial hypercellularity and matrix expansion, and glomerular capillary loops with immune-complex deposition. Compared with Mdk-/- mice, the frequency of splenic CD69+ T cells and T helper (Th) 1 cells, but not of regulatory T cells, was augmented in Mdk+/+ mice in proportion to LN disease activity, and was accompanied by skewed cytokine production. MK expression was also enhanced in activated CD4+ T cells in vivo and in vitro. MK induced activated CD4+ T cells expressing CD69 through nuclear activation of NFAT transcription and selectively increased in vitro differentiation of naive CD4+ T cells into Th1 cells by promoting IL-12/STAT4 signaling. These results suggest that MK serves an indispensable role in the NFAT-regulated activation of CD4+ T cells and Th1 cell differentiation, eventually leading to the exacerbation of LN.

Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides.

Sulfatase 2 Modulates Fate Change from Motor Neurons to Oligodendrocyte Precursor Cells through Coordinated Regulation of Shh Signaling with Sulfatase 1.

Sulfatases (Sulfs) are a group of endosulfatases consisting of Sulf1 and Sulf2, which specifically remove sulfate from heparan sulfate proteoglycans. Although several studies have shown that Sulf1 acts as a regulator of sonic hedgehog (Shh) signaling during embryonic ventral spinal cord development, the detailed expression pattern and function of Sulf2 in the spinal cord remains to be determined. In this study, we found that Sulf2 also modulates the cell fate change from motor neurons (MNs) to oligodendrocyte precursor cells (OPCs) by regulating Shh signaling in the mouse ventral spinal cord in coordination with Sulf1. In the mouse, Sulf mRNAs colocalize with Shh mRNA and gradually expand dorsally from embryonic day (E) 10.5 to E12.5, following strong Patched1 signals (a target gene of Shh signaling). This coordinated expression pattern led us to hypothesize that in the mouse, strong Shh signaling is induced when Shh is released by Sulf1/2, and this strong Shh signaling subsequently induces the dorsal expansion of Shh and Sulf1/2 expression. Consistent with this hypothesis, in the ventral spinal cord of Sulf1 knockout (KO) or Sulf2 KO mice, the expression patterns of Shh and Patched1 differed from that in wild-type mice. Moreover, the position of the pMN and p3 domains were shifted ventrally, MN generation was prolonged, and OPC generation was delayed at E12.5 in both Sulf1 KO and Sulf2 KO mice. These results demonstrated that in addition to Sulf1, Sulf2 also plays an important and overlapping role in the MN-to-OPC fate change by regulating Shh signaling in the ventral spinal cord. However, neither Sulf1 nor Sulf2 could compensate for the loss of the other in the developing mouse spinal cord. In vitro studies showed no evidence of an interaction between Sulf1 and Sulf2 that could increase sulfatase activity. Furthermore, Sulf1/2 double heterozygote and Sulf1/2 double KO mice exhibited phenotypes similar to the Sulf1 KO and Sulf2 KO mice. These results indicate that there is a threshold for sulfatase activity (which is likely reflected in the dose of Shh) required to induce the MN-to-OPC fate change, and Shh signaling requires the coordinated activity of Sulf1 and Sulf2 in order to reach that threshold in the mouse ventral spinal cord.

Correction of spherical aberration in multi-focal multiphoton microscopy with spatial light modulator.

We demonstrate that high-quality images of the deep regions of a thick sample can be obtained from its surface by multi-focal multiphoton microscopy (MMM). The MMM system incorporates a spatial light modulator to separate the excitation beam into a multi-focal excitation beam and modulate the pre-distortion wavefront to correct spherical aberration (SA) caused by a refractive index mismatch between the immersion medium and the biological sample. When fluorescent beads in transparent epoxy resin were observed using four SA-corrected focal beams, the fluorescence signal of the obtained images was ~52 times higher than that obtained without SA correction until a depth of ~1100 µm, similar to the result for single-focal multiphoton microscopy (SMM). The MMM scanning time was four times less than that for SMM, and MMM showed an improved fluorescence intensity and depth resolution for an image of blood vessels in the brain of a mouse stained with a fluorescent dye.

Sulfated glycosaminoglycans in protein aggregation diseases.

Protein aggregation diseases are characterized by intracellular or extracellular deposition of misfolded and aggregated proteins. These aggregated deposits contain multiple proteinaceous and non-protein components that are thought to play critical roles in the etiology and pathogenesis of protein aggregation diseases in vivo. One of these components, the sulfated glycosaminoglycans (GAGs), includes heparan sulfate, chondroitin sulfate, and keratan sulfate. The sulfated GAGs are negatively charged heteropolysaccharides expressed in all mammalian tissues. Enzymatically generated structural patterns and the degree of sulfation in GAGs determine GAGs' specific interactions with their protein ligands. Here, we review the potential roles of the sulfated GAGs in the pathogenesis and progression of protein aggregation diseases from a perspective of their sulfation modification. We also discuss the possibility of sulfated GAGs as therapeutic targets for protein aggregation diseases.

Cellular interaction and cytotoxicity of the iowa mutation of apolipoprotein A-I (ApoA-IIowa) amyloid mediated by sulfate moieties of heparan sulfate.

The single amino acid mutation G26R in human apolipoprotein A-I (apoA-I) is associated with familial amyloid polyneuropathy III. ApoA-I carrying this mutation (apoA-IIowa) forms amyloid fibrils in vitro. Heparan sulfate (HS) is a glycosaminoglycan that is abundant at the cell surface and in the extracellular matrix. Although HS and its highly sulfated domains are involved in aggregation of amyloid-ß and accumulate in cerebral amyloid plaques of patients with Alzheimer disease and mouse models of this disease, the role of HS in familial amyloid polyneuropathy III has never been addressed. Here, we used cell models to investigate the possible role of HS in the cytotoxicity of apoA-IIowa amyloid. Wild-type CHO cells, but not pgsD-677 cells, an HS-deficient CHO mutant, demonstrated uptake of apoA-IIowa amyloid after incubation with the amyloid. Addition of sulfated glycosaminoglycans to culture media prevented interaction with and cytotoxicity of apoA-IIowa amyloid to CHO cells. Elimination of cell surface HS or inhibition of HS sulfation with chemical reagents interfered with interaction of apoA-IIowa amyloid with CHO cells. We also found that cellular interaction and cytotoxicity of apoA-IIowa amyloid were significantly attenuated in CHO cells that stably expressed the human extracellular endoglucosamine 6-sulfatases HSulf-1 and HSulf-2. Our results thus suggest that cell surface HS mediates cytotoxicity of apoA-IIowa amyloid and that enzymatic remodeling of HS mitigates the cytotoxicity.

Apical membrane expression of distinct sulfated glycans represents a novel marker of cholangiolocellular carcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver neoplasm, followed by hepatocellular carcinoma. ICC can be further subclassified as (i) perihilar and (ii) peripheral types, the latter histologically resembling small-sized intrahepatic bile ducts, such as interlobular bile ducts, cholangioles/ductules and the canals of Hering. Cholangiolocellular carcinoma (CoCC), now classified by the World Health Organization as a subtype of combined hepatocellular-cholangiocarcinoma, is currently regarded as a subtype of peripheral-type ICC. The present study was undertaken to determine whether sulfated glycans recognized by the MECA-79 monoclonal antibody could serve as a CoCC marker. Using immunohistochemistry, we show that MECA-79 sulfated glycans are preferentially expressed at the apical membrane of cholangiocytes found in small-sized intrahepatic bile ducts in normal liver and in canalicular structures formed in CoCC. We also report that apical membrane MECA-79 sulfated glycan expression colocalizes with that of mucin 1 (MUC1) core proteins. We also present immunoblotting of Chinese hamster ovary cells overexpressing FLAG-tagged MUC1 to show that MUC1 serves as a MECA-79 scaffold. Furthermore, we report that SSP-25 human ICC cells overexpressing N-acetylglucosamine-6-O-sulfotransferase 2 (GlcNAc6ST-2), but not GlcNAc6ST-1, exhibit membrane expression of MECA-79 sulfated glycans, suggesting that GlcNAc6ST-2 catalyzes MECA-79 epitope biosynthesis in cholangiocytes. Moreover, both wild-type and GlcNAc6ST-1 knockout mice exhibit apical membrane MECA-79 expression in small-sized intrahepatic bile ducts, namely interlobular bile ducts, whereas MECA-79 expression was completely absent in comparable tissues from GlcNAc6ST-1 and GlcNAc6ST-2 double knockout mice. These data collectively indicate that apical membrane localization of MUC1 proteins decorated with GlcNAc6ST-2-dependent MECA-79 sulfated glycans may mark cholangiocytes with cholangiolar/ductular differentiation and could serve as a useful CoCC marker.

Microglial keratan sulfate epitope elicits in central nervous tissues of transgenic model mice and patients with amyotrophic lateral sclerosis.

The functional role of 5D4 antibody-reactive keratan sulfate (KS) in the pathogenesis of neurodegenerative diseases is unknown. We therefore studied the expression of 5D4-reactive KS in amyotrophic lateral sclerosis (ALS), a motor neuron-degenerative disease, with the use of SOD1(G93A) ALS model mice and patients with ALS. Histochemical and immunoelectron microscopic characterizations showed that the 5D4-reactive KS is expressed in Mac2/galectin-3-positive activated or proliferating microglia of SOD1(G93A) ALS model mice at disease end stage and that the KS is an O-linked glycan modified with sialic acid and fucose, which was thus far shown to exist in cartilage. Intriguingly, microglial KS was detected in the spinal cord and brainstem but not in the cerebral cortex of SOD1(G93A) mice. We found that KSGal6ST, a galactose-6-sulfotransferase, is required for biosynthesis of the microglial 5D4-reactive KS by generating SOD1(G93A)/KSGal6ST(-/-) mice. The requirement of GlcNAc6ST1 for this synthesis was corroborated by analyzing SOD1(G93A)/GlcNAc6ST1(-/-) mice. These results indicate that both galactose-6- and N acteylglucosamine-6-sulfated KS elicited in the spinal cord and brainstem are associated with the degeneration of spinal and bulbar lower motor neurons in ALS pathology and may play a role in disease progression via microglial activation and proliferation.

Keratan Sulfate Regulates the Switch from Motor Neuron to Oligodendrocyte Generation During Development of the Mouse Spinal Cord.

Keratan sulfate (KS) is a sulfated glycosaminoglycan and has been shown to bind to sonic hedgehog (Shh), which act as a morphogen to regulate the embryonic spinal cord development. We found highly sulfated KS was present in the floor plate (including lateral floor plate) and the notochord . This expression colocalized with Shh expression. To understand the roles of KS, we analyzed the embryonic spinal cord of GlcNAc6ST-1, KS chain synthesizing enzyme, knock-out (KO) mice. At E12.5, the pMN domain, whose formation is controlled by Shh signaling, became shifted ventrally in GlcNAc6ST-1 KO mice. In addition, the expression patterns of Patched1 and Gli1, two Shh signaling reporter genes, differed between wild type (WT) and GlcNAc6ST-1 KO mice at E12.5. Next, we focused on cell types generated from the pMN domain; namely, motor neurons and subsequently oligodendrocytes. The number of PDGFRα(+) [a marker for oligodendrocyte precursor cells (OPCs)] cells was low in the E12.5 mutant spinal cord, while motor neuron production was increased. Thus the switch from motor neuron generation to OPC generation was delayed in the pMN domain. Furthermore, we investigated the cause for this delayed switch in the pMN domain. The number of Olig2, Nkx2.2 double-positive cells was less in GlcNAc6ST-1 KO mice than in WT mice. In contrast, the number of Olig2, Neurogenin2 (Ngn2) double-positive cells related to the motor neuron specification was significantly greater in the KO mice. These results indicate that KS is important for the late phase Shh signaling and contributes to motor neuron to OPC generation switch.

Reduced molecular size and altered disaccharide composition of cerebral chondroitin sulfate upon Alzheimer's pathogenesis in mice.

Alzheimer's disease (AD) is a progressive disorder leading to cognitive impairment and neuronal loss. Cerebral extracellular accumulation and deposition of amyloid ß plaques is a pathological hallmark of AD. Chondroitin sulfate (CS) is an extracellular component abundant in the brain. CS is a sulfated glycosaminoglycan covalently attached to a core protein, forming chondroitin sulfate proteoglycan. The structure of CS is heterogeneous with sulfation modification and elongation of the chain. The structural diversity of CS allows it to play various roles in the brain. Increasing evidence has shown that CS promotes aggregation of amyloid ß peptides into higher-order species such as insoluble amyloid ß fibrils. Difficulties in the structural analysis of brain CS, as well as its heterogeneity, limit the study of potential roles of CS in AD pathology. Here we established a microanalysis method with reversed-phase ion-pair high performance liquid chromatography and found that CS in the brains of Tg2576 AD model mice show a lower molecular size and an increased ratio of CS-B motif di-sulfated disaccharide. Our findings provide insight into the structural changes of cerebral CS upon Alzheimer's pathogenesis.