Toshihiko Kadoya

Oxidized Galectin-1 Stimulates Macrophages to Promote Axonal Regeneration in Peripheral Nerves after Axotomy

Various neurotrophic factors that promote axonal regeneration have been investigated in vivo, but the signals that prompt neurons to send out processes in peripheral nerves after axotomy are not well understood. Previously, we have shown oxidized galectin-1 (GAL-1/Ox) promotes initial axonal growth after axotomy in peripheral nerves. However, the mechanism by which GAL-1/Ox promotes axonal regeneration remains unclear and is the subject of the present study. To identify possible target cells of GAL-1/Ox, a fluorescently labeled recombinant human GAL-1/Ox (rhGAL-1/Ox) was incubated with DRG neurons, Schwann cells, and intraperitoneal macrophages from adult rats. Only the cell surfaces of intraperitoneal macrophages bound the rhGAL-1/Ox, suggesting that these cells possess a receptor for GAL-1/Ox. Experiments examining tyrosine phosphorylation revealed that rhGAL-1/Ox stimulated changes in signal transduction pathways in these macrophages. These changes caused macrophages to secrete an axonal growth-promoting factor. This was demonstrated when conditioned media of macrophages stimulated with rhGAL-1/Ox in 48 hr culture strongly enhanced axonal regeneration from transected-nerve sites of DRG explants. Furthermore, activated macrophage-conditioned media also improved Schwann cell migration from the transected-nerve sites. From these results, we propose that axonal regeneration occurs in axotomized peripheral nerves as a result of cytosolic reduced galectin-1 being released from Schwann cells and injured axons, which then becomes oxidized in the extracellular space. Oxidized galectin-1 then stimulates macrophages to secrete a factor that promotes axonal growth and Schwann cell migration, thus enhancing peripheral nerve regeneration.

Galectin-1 regulates neurogenesis in the subventricular zone and promotes functional recovery after stroke

Galectin-1 (Gal-1) has recently been identified as a key molecule that plays important roles in the regulation of neural progenitor cell proliferation in two neurogenic regions: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. To test the hypothesis that Gal-1 contributes to adult neurogenesis after focal ischemia, we studied the temporal profile of endogenous Gal-1 expression and the effects of human recombinant Gal-1 on neurogenesis and neurological functions in an experimental focal ischemic model. In the normal brain, Gal-1 expression was observed only in the SVZ. In the ischemic brain, Gal-1 expression was markedly upregulated in the SVZ and the area of selective neuronal death around the infarct in the striatum. The temporal profile of Gal-1 expression was correlated with that of neural progenitor cell proliferation in the SVZ of the ischemic hemisphere. Double-labeling studies revealed that Gal-1 was localized predominantly in both reactive astrocytes and SVZ astrocytes. Administration of Gal-1, which is known to have carbohydrate-binding ability, into the lateral ventricle increased neurogenesis in the ipsilateral SVZ and improved sensorimotor dysfunction after focal ischemia. By contrast, blockade of Gal-1 in the SVZ by the administration of anti-Gal-1 neutralizing antibody strongly inhibited neurogenesis and diminished neurological function. These results suggest that Gal-1 is one of the principal regulators of adult SVZ neurogenesis through its carbohydrate-binding ability and provide evidence that Gal-1 protein has a role in the improvement of sensorimotor function after stroke.

IL-1β enhances neurite regeneration from transected-nerve terminals of adult rat DRG

WE clarified the roles of IL-β in peripheral neural regeneration after axotomy in a three-dimensional collagen gel culture system ranging from a single neurone to a dorsal root ganglion (DRG) explant with its associated nerve bundles. Application of 30 U/ml IL-1β to the culture systems clearly enhanced neural regeneration. This regeneration was evident in transected nerve terminals of DRG explants with or without associated nerve bundles, but not in dissociated single neurones. Neural survival was not affected by IL-1β in any of these culture systems. These results suggest that IL-1β stimulates surrounding non-neuronal cells to secrete neurotrophic factors, thus enhancing neurite regeneration from transected nerve terminals in cultured adult DRG explants.

Neuroprotective effect of oxidized galectin-1 in a transgenic mouse model of amyotrophic lateral sclerosis.

Abnormal accumulation of neurofilaments in motor neurons is a characteristic pathological finding in amyotrophic lateral sclerosis (ALS). Recently, we revealed that galectin-1, whose oxidized form has axonal regeneration-enhancing activity, accumulates in the neurofilamentous lesions in ALS. To investigate whether oxidized galectin-1 has a beneficial effect on ALS, oxidized recombinant human galectin-1 (rhGAL-1/ox) or physiological saline was injected into the left gastrocnemius muscle of the transgenic mice over-expressing a mutant copper/zinc superoxide dismutase (SOD1) with a substitution of histidine to arginine at position 46 (H46R SOD1). The H46R SOD1 transgenic mice, which represented a new animal model of familial ALS, were subsequently assessed for their disease onset, life span, duration of illness, and motor function. Furthermore, the number of remaining large anterior horn cells of spinal cords was also compared between the two groups. The results showed that administration of rhGAL-1/ox to the mice delayed the onset of their disease and prolonged the life of the mice and the duration of their illness. Motor function, as evaluated by a Rotarod performance, was improved in rhGAL-1/ox-treated mice. Significantly more anterior horn neurons of the lumbar and cervical cords were preserved in the mice injected with rhGAL-1/ox than in those injected with physiological saline. The study suggests that rhGAL-1/ox administration could be a new therapeutic strategy for ALS.

Identification of oxidized galectin-1 as an initial repair regulatory factor after axotomy in peripheral nerves.

Various neurotrophic factors that promote axonal regeneration have been investigated in vivo, but the signals that prompt the axons to send out processes in peripheral nerves after axotomy are not well understood. We have shown using two specific strategies that galectin-1 can play an important role in this initial stage. One used an in vitro nerve regeneration model that allowed us to monitor the initial axon and support cell outgrowth from the proximal nerve stump comparable to the initial stages of nerve repair. The other strategy was to clarify the axonal regeneration-promoting factor from kidney-derived cells. Using these strategies, we discovered that oxidized galectin-1 from the cell (COS1 cell) conditioned media acts as an axonal regeneration-promoting factor without the lectin activity. Oxidized recombinant human galectin-1 (rhGAL-1/Ox) showed the same activity at low concentrations (pg/ml range). A similarly low concentration also effectively promoted axonal regeneration in both transection and crush experiments in vivo. Moreover, the application of functional anti-galectin-1 antibody strongly inhibited the regeneration in vivo. Since galectin-1was shown to be secreted and localized in the regenerating sciatic nerve, this suggests that secreted galectin-1 may be oxidized and change its molecular structure to regulate initial repair after axotomy as a kind of cytokine.

Galectin-1 Is Part of Human Trophoblast Invasion Machinery - A Functional Study In Vitro

Background Interactions of glycoconjugates with endogenous galectins, have been long proposed to participate in several reproductive processes including implantation. In human placenta gal-1, gal-3, gal-8, and gal-13 proteins are known to be present. Each of them has been proposed to play multiple functions, but so far no clear picture has emerged. We hypothesized that gal-1 participates in trophoblast invasion, and conducted Matrigel invasion assay using isolated cytotrophoblast from first trimester placenta and HTR-8/SVneo cell line to test it. Methods and Findings Function blocking anti-gal-1 antibody was employed to assess participation of endogenous gal-1 in cell adhesion, cell invasion of HTR-8/SVneo cells. When gal-1 was blocked in isolated trophoblast cell invasion was reduced to 75% of control (SEM±6.3, P<0.001) and to 66% of control (SEM±1.7, P<0.001) in HTR-8/SVneo cell line. Increased availability of gal-1, as two molecular forms of recombinant human gal-1 (CS-gal-1 and Ox-gal-1), resulted in increased cell invasion by cytotrophoblast to 151% (SEM±16, P<0.01) with 1 ng/ml of CS-gal-1, and to 192% (SEM±51, P<0.05) with 1 µg/ml of Ox-gal-1. Stimulation was also observed in HTR-8/SVneo cells, to 317% (SEM±58, P<0.001) by CS-gal-1, and to 200% (SEM±24, P<0.001) by Ox-gal-1 at 1 µg/ml. Both sets of results confirmed involvement of gal-1 in trophoblast invasion. Galectin profile of isolated cytotrophoblast and HTR-8/SVneo cells was established using RT-PCR and real-time PCR and found to consist of gal-1, gal-3 and gal-8 for both cell types. Only gal-1 was located at the trophoblast cell membrane, as determined by FACS analysis, which is consistent with the results of the functional tests. Conclusion and Significance These findings qualify gal-1 as a member of human trophoblast cell invasion machinery.

Structural and functional studies of galectin-1: a novel axonal regeneration-promoting activity for oxidized galectin-1.

Recently, we discovered oxidized galectin-1 as a factor that regulates initial axonal growth in the peripheral nerve after axotomy. Galectin-1 is a member of the galectins, a family of animal lectins ranging from Caenorhabditis elegans to humans, which is defined by their affinity for beta-galactosides and by significant sequence similarity in the carbohydrate-binding site. Galectin-1 is a homodimer with a subunit molecular mass of 14.5 kDa, which contains six cysteine residues per subunit. The cysteine residues should be in a free state in order to maintain a molecular structure that is capable of showing lectin activity. However, our structural analysis revealed that the axonal regeneration-promoting factor exists as an oxidized form of galectin-1, containing three intramolecular disulfide bonds. The oxidized galectin-1 exhibited marked peripheral nerve regeneration-promoting activity, although it showed no lectin activity. It was also revealed that oxidized galectin-1 exists as a monomer in a physiological solution. Galectin-1 seems to have a variety of biological functions. These functions could vary according to the time at which a biological function is taking place, as well as the site in which a biological function is taking place. In addition, these functions could vary according to the structure of galectin-1 by which a particular biological function is taking place. Disulfide bond formation alters the structure of galectin-1, so as to confer the novel ability to promote axonal regeneration. Oxidized galectin-1 likely acts as an autocrine or paracrine factor to promote axonal regeneration, functioning more like a cytokine than as a lectin.

Synthesis, localization and externalization of galectin-1 in mature dorsal root ganglion neurons and Schwann cells

We recently confirmed that oxidized galectin‐1 is a novel factor enhancing axonal growth in peripheral nerves after axotomy, but the process of extracellular release and oxidization of endogenous galectin‐1 in the injured nervous tissue remains unknown. In the present study, we examined the distribution of galectin‐1 in adult rat dorsal root ganglia (DRG) in vivo and in vitro. By RT‐PCR analysis and in situ hybridization histochemistry, galectin‐1 mRNA was detected in both DRG neurons and non‐neuronal cells. Immunohistochemical analyses revealed that galectin‐1 was distributed diffusely throughout the cytoplasm in smaller diameter neurons and Schwann cells in DRG sections. In contrast, the immunoreactivity for galectin‐1 was detected in almost all DRG neurons from an early stage in culture (3 h after seeding) and was restricted to the surface and/or extracellular region of neurons and Schwann cells at later stages in culture. In a manner similar to the primary cultured cells, we also observed the surface and extracellular expression of this molecule in immortalized adult mouse Schwann cells (IMS32). Western blot analysis has revealed that both reduced and oxidized forms of galectin‐1 were detected in culture media of DRG neurons and IMS32. These findings suggest that galectin‐1 is externalized from DRG neurons and Schwann cells upon axonal injury. Some of the molecules in the extracellular milieu may be converted to the oxidized form, which lacks lectin activity but could act on neural tissue as a cytokine.

Galectin-1 promotes basal and kainate-induced proliferation of neural progenitors in the dentate gyrus of adult mouse hippocampus

We examined the expression of galectin-1, an endogenous lectin with one carbohydrate-binding domain, in the adult mouse hippocampus after systemic kainate administration. We found that the expression of galectin-1 was remarkably increased in activated astrocytes of the CA3 subregion and dentate gyrus of the hippocampus, and in nestin-positive neural progenitors in the dentate gyrus. Quantitative reverse transcription PCR (RT-PCR) analysis revealed that the galectin-1 mRNA level in hippocampus began to increase 1 day after kainate administration and that a 13-fold increase was attained within 3 days. Western blotting analysis confirmed that the level of galectin-1 protein increased to more than three-fold a week after the exposure. We showed that isolated astrocytes express and secrete galectin-1. To clarify the significance of the increased expression of galectin-1 in hippocampus, we compared the levels of hippocampal cell proliferation in galectin-1 knockout and wild-type mice after saline or kainate administration. The number of 5-bromo-2′-deoxyuridine (BrdU)-positive cells detected in the subgranular zone (SGZ) of galectin-1 knockout mice decreased to 62% with saline, and to 52% with kainate, as compared with the number seen in the wild-type mice. Most of the BrdU-positive cells in SGZ expressed doublecortin and neuron-specific nuclear protein, indicating that they are immature neurons. We therefore concluded that galectin-1 promotes basal and kainate-induced proliferation of neural progenitors in the hippocampus.

Proteomic approach to the identification of cell membrane proteins

The expression of plasma membrane proteins in human monocyte‐derived U937 cells was examined by cell disruption and isolation of microsomal fractions. Two alternative procedures for cell disruption, Dounce homogenization and nitrogen cavitation, were compared. Cell homogenization and sequential centrifugation resulted in an approximately fivefold enrichment of plasma membrane proteins in the microsomal fraction. However, identification of 30 such apparently enriched proteins by two‐dimensional (2‐D) electrophoresis, proteolytic digestion, and mass spectrometry revealed that only eight were plasma membrane proteins, the remaining 22 being contaminants. In contrast, nitrogen cavitation followed by sequential centrifugation and solubilization of proteins with sodium dodecyl sulfate (SDS) and 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propane‐sulfonate (CHAPS) detergent yielded subcellular fractions, including microsomes, that showed little overlap in constituent proteins as revealed by 2‐D electrophoresis. These results highlight the importance of obtaining pure plasma mem branes and complete solubilization of membrane proteins for proteomic analysis.