Tomohiro Ohgomori

Minocycline selectively inhibits M1 polarization of microglia

Minocycline is commonly used to inhibit microglial activation. It is widely accepted that activated microglia exert dual functions, that is, pro-inflammatory (M1) and anti-inflammatory (M2) functions. The in vivo status of activated microglia is probably on a continuum between these two extreme states. However, the mechanisms regulating microglial polarity remain elusive. Here, we addressed this question focusing on minocycline. We used SOD1G93A mice as a model, which exhibit the motor neuron-specific neurodegenerative disease, amyotrophic lateral sclerosis. Administration of minocycline attenuated the induction of the expression of M1 microglia markers during the progressive phase, whereas it did not affect the transient enhancement of expression of M2 microglia markers during the early pathogenesis phase. This selective inhibitory effect was confirmed using primary cultured microglia stimulated by lipopolysaccharide (LPS) or interleukin (IL)-4, which induced M1 or M2 polarization, respectively. Furthermore, minocycline inhibited the upregulation of NF-κB in the LPS-stimulated primary cultured microglia and in the spinal cord of SOD1G93A mice. On the other hand, IL-4 did not induce upregulation of NF-κB. This study indicates that minocycline selectively inhibits the microglia polarization to a proinflammatory state, and provides a basis for understanding pathogeneses of many diseases accompanied by microglial activation.

Keratan Sulfate Restricts Neural Plasticity after Spinal Cord Injury

Chondroitin sulfate (CS) proteoglycans are strong inhibitors of structural rearrangement after injuries of the adult CNS. In addition to CS chains, keratan sulfate (KS) chains are also covalently attached to some proteoglycans. CS and KS sometimes share the same core protein, but exist as independent sugar chains. However, the biological significance of KS remains elusive. Here, we addressed the question of whether KS is involved in plasticity after spinal cord injury. Keratanase II (K-II) specifically degraded KS, i.e., not CS, in vivo. This enzyme digestion promoted the recovery of motor and sensory function after spinal cord injury in rats. Consistent with this, axonal regeneration/sprouting was enhanced in K-II-treated rats. K-II and the CS-degrading enzyme chondroitinase ABC exerted comparable effects in vivo and in vitro. However, these two enzymes worked neither additively nor synergistically. These data and further in vitro studies involving artificial proteoglycans (KS/CS-albumin) and heat-denatured or reduced/alkylated proteoglycans suggested that all three components of the proteoglycan moiety, i.e., the core protein, CS chains, and KS chains, were required for the inhibitory activity of proteoglycans. We conclude that KS is essential for, and has an impact comparable to that of CS on, postinjury plasticity. Our study also established that KS and CS are independent requirements for the proteoglycan-mediated inhibition of axonal regeneration/sprouting.

Perineuronal nets affect parvalbumin expression in GABAergic neurons of the mouse hippocampus.

Recent studies have suggested that the perineuronal net (PNN), a specialised extracellular matrix structure, and parvalbumin (PV), an EF‐hand calcium‐binding protein, are involved in the regulation of plasticity of neural circuits. Here, we aimed to quantitatively estimate the relationship between the two plasticity regulators, PV and PNNs, in the hippocampus of young adult mice. Dual fluorescence staining for PV and Wisteria floribunda agglutinin (a broad PNN marker) showed that a substantial population of PV‐expressing (PV+) GABAergic neurons lacked PNNs. Optical disector analysis demonstrated that there were fewer PNN+ neurons than PV+ neurons. The ratio of PNN expression in PV+ neurons was generally lower in the dendritic layers than in the principal cell layers, whereas the ratio of PV expression in PNN+ neurons was effectively 100%. The mean PV fluorescence was significantly higher in PNN+/PV+ neurons than in PNN−/PV+ neurons. Cumulative frequencies for single‐cell PV fluorescence indicated that intensely stained PV+ neurons tend to be enwrapped by PNNs, whereas weakly stained PV+ neurons are likely to lack PNNs. We digested the PNNs by a unilateral injection of chondroitinase ABC (chABC) into the dorsal CA1 region. Although the densities of PV+ neurons remained unchanged, the PV fluorescence declined 7 days after chABC injection. Quantitative real‐time polymerase chain reaction analysis demonstrated a reduction in PV mRNA expression following chABC injection. These findings indicate that the presence or absence of PNNs affects the relative PV expression in GABAergic neurons in the hippocampus.

CD44 expression in astrocytes and microglia is associated with ALS progression in a mouse model.

Amyotrophic lateral sclerosis (ALS) is a motor neuron-specific neurodegenerative disease. An increasing body of evidence suggests that, in addition to cell autonomous regulation, i.e., pathological changes in motor neurons, non-cell autonomous mechanisms involving glial cells play critical roles in the pathogenesis of ALS. CD44 functions as a receptor for osteopontin and hyaluronan, and has been implicated in inflammation associated with neuronal injuries. However, this membrane glycoprotein has been poorly studied in ALS. Here we investigated its expression during ALS progression using SOD1(G93A) mice. CD44 expression increased around the onset of disease and then increased continuously. Astrocytes and microglia expressed CD44 in vivo. Consistent with these findings, primary cultured microglia began to express CD44 upon activation with LPS and interferon-γ. CD44 expression in primary cultured astrocytes was also enhanced by activation with interferon-γ+TNF-α or bFGF alone. As CD44 was detected in cell lysate, but not in culture media of astrocytes and microglia, it was likely that these glial cells expressed a membrane-bound form of CD44. Our study demonstrates that CD44 expression in astrocytes and microglia is closely associated with the pathogenesis of ALS, and suggests that inflammatory responses involving CD44 may play a role in this disease.

Ablation of keratan sulfate accelerates early phase pathogenesis of ALS.

Biopolymers consist of three major classes, i.e., polynucleotides (DNA, RNA), polypeptides (proteins) and polysaccharides (sugar chains). It is widely accepted that polynucleotides and polypeptides play fundamental roles in the pathogenesis of neurodegenerative diseases. But, sugar chains have been poorly studied in this process, and their biological/clinical significance remains largely unexplored. Amyotrophic lateral sclerosis (ALS) is a motoneuron-degenerative disease, the pathogenesis of which requires both cell autonomous and non-cell autonomous processes. Here, we investigated the role of keratan sulfate (KS), a sulfated long sugar chain of proteoglycan, in ALS pathogenesis. We employed ALS model SOD1G93A mice and GlcNAc6ST-1−/− mice, which are KS-deficient in the central nervous system. Unexpectedly, SOD1G93AGlcNAc6ST-1−/− mice exhibited a significantly shorter lifespan than SOD1G93A mice and an accelerated appearance of clinical symptoms (body weight loss and decreased rotarod performance). KS expression was induced exclusively in a subpopulation of microglia in SOD1G93A mice, and became detectable around motoneurons in the ventral horn during the early disease phase before body weight loss. During this phase, the expression of M2 microglia markers was transiently enhanced in SOD1G93A mice, while this enhancement was attenuated in SOD1G93AGlcNAc6ST-1−/− mice. Consistent with this, M2 microglia were markedly less during the early disease phase in SOD1G93AGlcNAc6ST-1−/− mice. Moreover, KS expression in microglia was also detected in some human ALS cases. This study suggests that KS plays an indispensable, suppressive role in the early phase pathogenesis of ALS and may represent a new target for therapeutic intervention.

ADAMTS-13 is produced by glial cells and upregulated after spinal cord injury

ADAMTS-13, a member of the family of disintegrins and metalloproteinases with thrombospondin motifs, is produced primarily in the liver, particularly by hepatic stellate cells. This metalloproteinase cleaves von Willebrand factor multimers and thereby regulates blood coagulation. Here, we investigated the expression of ADAMTS-13 in the central nervous system. ADAMTS-13 mRNA was expressed in cultured astrocytes and microglia but not in neurons. The protein production of ADAMTS-13 was also detected in these cultured glial cells. Furthermore, we found that the expression of ADAMTS-13 was significantly increased in the rat spinal cord after injury. Supporting the in vivo data, ADAMTS-13 protein was detected in GFAP- and CD11b-positive glial cells in injured spinal cord. Consistent with this, the proteolytic activity of ADAMTS-13 was increased after spinal cord injury. Our data suggest that ADAMTS-13 may have a critical role in the central nervous system, particularly after neuronal injuries.

Keratan sulfate expression in microglia is diminished in the spinal cord in experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN) is an animal model of Guillain–Barré syndrome, an inflammatory demyelination disease of the peripheral nervous system. Although this disease has been extensively studied on peripheral nerves, the pathology of the central nervous system has not been fully understood. Previous studies demonstrate that expression of keratan sulfate (KS), the sugar chain of proteoglycan, is associated with activated microglia/macrophages accumulated after neuronal injuries. Unexpectedly, we found here that KS is rather diminished in rat EAN. KS was restrictively expressed in microglia in the spinal cord of normal rats. KS was positive in 50% microglia in the ventral horn and 20% in the dorsal horn. In EAN, microglia increased in number and expressed the activation marker CD68, but KS expression was abolished. Concomitantly, pro-inflammatory cytokines, i.e., interferon (IFN)-γ, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α, were increased in the spinal cord of EAN rats, whereas anti-inflammatory cytokines, such as IL-4 and IL-10, were decreased. In addition, silencing of KSGal6ST attenuated KS expression on the primary cultured microglia and upregulated expression of some activation markers (TNF-α, IL-1β, and iNOS) under the stimulation with lipopolysaccharide and IFN-γ. This study demonstrates for the first time a close association of EAN and disappearance of KS on microglia. KS expression could be a useful marker to evaluate the status of polyneuropathy.

A combination of keratan sulfate digestion and rehabilitation promotes anatomical plasticity after rat spinal cord injury

Functional recovery after neuronal injuries relies on neuronal network reconstruction which involves many repair processes, such as sealing of injured axon ends, axon regeneration/sprouting, and construction and refinement of synaptic connections. Chondroitin sulfate (CS) is a major inhibitor of axon regeneration/sprouting. It has been reported that the combination of task-specific rehabilitation and CS-digestion is much more effective than either treatment alone with regard to the promotion of functional and anatomical plasticity for dexterity in acute and chronic spinal cord injury models. We previously reported that keratan sulfate (KS) is another inhibitor and has a potency equal to CS. Here, we compared the effects of KS- or CS-digestion plus rehabilitation on recovery from spinal cord injury. Keratanase II or chondroitinase ABC was locally administered at the lesion after spinal cord injury at C3/4. Task-specific rehabilitation training, i.e., a single pellet reaching task using a Whishaw apparatus, was done for 3 weeks before injury, and then again at 1-6 weeks after injury. The combination of KS-digestion and rehabilitation yielded a better rate of pellet removal than either KS-digestion alone or rehabilitation alone, although these differences were not statistically significant. The combination of CS-digestion and rehabilitation showed similar results. Strikingly, both KS-digestion/rehabilitation and CS-digestion/rehabilitation showed significant increases in neurite growth in vivo as estimated by 5-hydroxytryptamine and GAP43 staining. Thus, KS-digestion and rehabilitation exerted a synergistic effect on anatomical plasticity, and this effect was comparable with that of CS-digestion/rehabilitation. KS-digestion might widen the therapeutic window of spinal cord injury if combined with rehabilitation.

Comparative morphometric analysis of microglia in the spinal cord of SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis

It has long been recognized that reactive microglia undergo a series of phenotypic changes accompanying morphological transformation. However, the morphological classification of microglia has not yet been achieved. To address this issue, here we morphometrically analysed three‐dimensionally reconstructed ionized calcium binding adaptor molecule 1‐immunoreactive (Iba1+) microglia in the ventral horn of the lumbar spinal cord of SOD1G93A transgenic mice, a model of amyotrophic lateral sclerosis. The hierarchical cluster analysis revealed that microglia were objectively divided into four groups: type S (named after surveillant microglia) and types R1, R2 and R3 (named after reactive microglia). For the purpose of comparative morphometry, we also analysed two pharmacological disease models using wild‐type mice: 3,3′‐iminodipropionitrile (IDPN)‐induced axonopathy and lipopolysaccharide (LPS)‐induced neuroinflammation. Type S microglia showed a typical ramified morphology of surveillant microglia, and were mostly observed in wild‐type controls. Type R1 microglia were seen at the early stage of disease in SOD1G93A mice, and also frequently occurred in IDPN‐treated mice. They exhibited small cell bodies with shorter and simple processes. Type R2 microglia were morphologically similar to type R1 microglia, but only transiently occurred in the middle stage of disease in SOD1G93A mice and in IDPN‐treated mice. Type R3 microglia exhibited a bushy shape, and were observed in the end stage of disease in SOD1G93A mice and in LPS‐treated mice. These findings indicate that microglia of SOD1G93A mice can be classified into four types, and also suggest that the phenotypic changes may be induced by the events related to axonopathy and neuroinflammation.

Structural study of the N-glycans of intercellular adhesion molecule-5 (telencephalin)

Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized membrane glycoprotein expressed in tissues distinct from those expressing other ICAMs. Here, we determined the N-glycan structure of ICAM-5 purified from adult rat brain and compared it with that of other ICAMs. N-glycans were released by N-glycosidase F digestion and labeled with p-amino benzoic octylester (ABOE). ABOE-labeled glycans were analyzed by high performance liquid chromatography (HPLC) and mass spectrometry. The N-glycans obtained from rat brain ICAM-5 consisted of approximately 85% neutral, 10.2% sialylated-only, 2.8% sulfated-only, and 1.2% sialylated and sulfated glycans. Compared with the N-glycan structures of human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells, rat brain ICAM-5 had less highly branched glycans, sialylated glycans, and N-acetyllactosamine structures. In contrast, high-mannose-type N-glycans and Lewis X were more commonly found in rat brain ICAM-5 than in human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells. In addition, sulfated glycans contained GlcNAc 6-O-sulfate on the non-reducing terminal side. Our data will be important for the elucidation of the roles of the N-glycans expressed in neural cells, including those present on ICAM-5.