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Hyperglycemia increases SCO-spondin and Wnt5a secretion into the cerebrospinal fluid to regulate ependymal cell beating and glucose sensing [1]

['Francisco Nualart', 'Laboratory Of Neurobiology', 'Stem Cells', 'Neurocellt', 'Department Of Cellular Biology', 'Faculty Of Biological Sciences', 'University Of Concepcion', 'Concepcion', 'Center For Advanced Microscopy Cma Bio Bio', 'Manuel Cifuentes']

Date: 2023-09

Hyperglycemia increases glucose concentrations in the cerebrospinal fluid (CSF), activating glucose-sensing mechanisms and feeding behavior in the hypothalamus. Here, we discuss how hyperglycemia temporarily modifies ependymal cell ciliary beating to increase hypothalamic glucose sensing. A high level of glucose in the rat CSF stimulates glucose transporter 2 (GLUT2)-positive subcommissural organ (SCO) cells to release SCO-spondin into the dorsal third ventricle. Genetic inactivation of mice GLUT2 decreases hyperglycemia-induced SCO-spondin secretion. In addition, SCO cells secrete Wnt5a-positive vesicles; thus, Wnt5a and SCO-spondin are found at the apex of dorsal ependymal cilia to regulate ciliary beating. Frizzled-2 and ROR2 receptors, as well as specific proteoglycans, such as glypican/testican (essential for the interaction of Wnt5a with its receptors) and Cx43 coupling, were also analyzed in ependymal cells. Finally, we propose that the SCO-spondin/Wnt5a/Frizzled-2/Cx43 axis in ependymal cells regulates ciliary beating, a cyclic and adaptive signaling mechanism to control glucose sensing.

Here, we report that SCO cells express glucose transporter 2 (GLUT2) with apical polarization. Since GLUT2 is a low-affinity transporter that is preferentially expressed in tissues with glucose-sensing activity, we increased the glucose concentration in CSF [ 1 ]. We demonstrated that SCO-spondin is secreted into CSF in response to hyperglycemic conditions and interacts with multiciliated ependymal cells. Thus, ependymal cells temporarily decrease ciliary beating and CSF flow. Additionally, we observed that SCO cells secrete multivesicular bodies (MVBs; CD63+ vesicles) and wingless-type MMTV integration site family member 5A (Wnt5a), which binds to ependymal cells that interact with Frizzled 2/receptor tyrosine kinase-like orphan receptor-2 (ROR2). Finally, changes in connexin distribution were also observed. We suggest the existence a hyperglycemic response system in the human brain that involves activation of a signaling pathway that includes Spondin-like proteins, Wnt5a, Frizzled-2, ROR2, and connexin-43 (Cx43) in ependymal cells.

It has been reported that fibrillary-aggregated SCO-spondin (SCO-spondin that forms RFs) plays a critical role in the maintenance of a straight body axis and spine morphogenesis in zebrafish [ 10 ]. Additionally, SCO-spondin (the soluble and/or aggregated form) has been implicated in CSF homeostasis [ 14 , 15 ], neurogenesis [ 16 – 19 ], embryonic morphogenesis [ 20 ], and hydrocephalus [ 21 ] during prenatal or early postnatal brain development; however, the function of SCO-spondin (the soluble and/or aggregated form) in the adult mammalian brain is unknown, as the SCO is an enigmatic structure inside the brain [ 12 , 22 ]. Furthermore, the mechanism by which SCO-spondin aggregation or solubilization is modulated by physiological or pathophysiological condition(s) is unknown. In vitro, it has been suggested that ATP increases SCO-spondin secretion and that 5-hydroxytryptamine (5-HT) inhibits SCO-spondin activity [ 23 ]. Additionally, in bovine SCO cells, ATP increases the [Ca 2+ ]i in approximately 85% of cells [ 24 ]. These effects are dose dependent and involve NK3 and P2Y2 receptors linked to G protein and phospholipase C activation [ 24 ].

SCO-spondin is a glycoprotein with multiple thrombospondin type 1 repeats (TSRs) that is secreted by cells in the subcommissural organ (SCO), an epithelial structure strategically located on the roof of the third ventricle [ 9 – 11 ], and mainly forms Reissner’s fibers (RFs) [ 12 ]. Intracellularly, SCO-spondin is synthesized in elongated endoplasmic reticulum (ER) cisternae in SCO cells. Proteins are glycosylated and stored in secretory granules that accumulate in the apical zone of the cells, where SCO-spondin is released into the CSF. Extracellularly, secreted SCO-spondin forms a thin film of secretion deposited on the cilia and blebs of the SCO cells. Subsequently, this protein forms fibrillary aggregates or can also remain soluble in the CSF [ 13 ].

Under hyperglycemic conditions, glucose concentration increases proportionally in the peripheral blood and hypothalamic cerebrospinal fluid (CSF) [ 1 ]; thus, hypothalamic neurons and glial cells induce metabolic signaling to achieve satiety [ 2 ]. Glucose-sensing activity occurs in the ventral third ventricle in a specialized hypothalamic region where glial cells (tanycytes) that contact the CSF do not exhibit ciliary beating [ 3 , 4 ]. Local movement of CSF depends on ependymal cell ciliary beating in other ventricular regions (medial and dorsal). Ciliary beating and CSF flow vary inside the third ventricle and appear to be regulated by circadian clock–dependent activity [ 3 ], ethanol concentration [ 5 , 6 ], and energy balance (the ATP/ADP ratio and Ca 2+ concentration); a decrease in the glucose concentration (from 25 mM to 0.1 mM) induces a reversible increase in ciliary beating frequency [ 7 ]. Thus, increasing intracellular glucose concentrations and, consequently, ATP content should decrease fluid movement [ 7 ]. Furthermore, it has been proposed that in mice, melanin-concentrating hormone (MCH) can regulate ciliary beating in the ventral region of the third ventricle, but not in the dorsal ventricular area or at the entrance to the cerebral aqueduct (CA) [ 8 ]. Additionally, 1 report identified 3 distinct types of ependymal cells uniquely and specifically positioned within the third ventricle and classified based on their cilia beating frequency as type I (>60 Hz), type II (30 to 60 Hz), or type III (<30 Hz) [ 5 ]. In this study, alcohol had a profound effect on the beating frequency of ependymal cilia, resulting in a substantial decrease in fluid movement and volume replacement [ 5 ].

Results

Hyperglycemia induces changes in SCO cells, stimulating the release of SCO-spondin into the CSF, which interacts with ependymal cell cilia As mentioned above, under normoglycemic conditions, SCO-spondin was localized in the ER and blebs (Fig 4A). When the CSF glucose concentration was increased (5 mM), evident changes in the apical region of SCO cells were observed; SCO-spondin–positive blebs were present (B, arrowheads), and ER immunoreactivity was decreased (Fig 4B, white arrows). We also detected released SCO-spondin on ependymal cells (Fig 4B, yellow arrows and inset; S2E and S2F Fig). The most evident changes were observed when the CSF glucose concentration was increased to 10 mM CSF. In the SCO, ER cisternae exhibited a globular structure (Fig 4C, white arrows and insets). In addition, the apical region of the cells did not contain SCO-spondin–positive blebs, only disaggregated secretion (Fig 4C, yellow arrows), suggesting that many of the apical secretory granules and extracellular aggregated SCO-spondin were released into the CSF under hyperglycemic conditions. Interestingly, in the medial SCO. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Hyperglycemia increases the secretion of SCO-spondin- and CD63-positive vesicles, which is present at the apex of ependymal cells. (A-C) Immunohistochemical staining of SCO-spondin (green), vimentin (white), and acetylated α-tubulin (red) in sections of the SCO under normoglycemic conditions (A) and hyperglycemic conditions (CSF glucose concentration of 5 mM (B) or 10 mM (C)). Nuclear staining with Hoechst (blue). Scale bar: 50 μm. SCO-spondin–positive blebs, ER, BV, and cilia are depicted. The pattern of intracellular SCO-spondin immunoreactivity was altered under different glycemic conditions. (D and Dˋ) Immunohistochemical staining of SCO-spondin (green), vimentin (white), and acetylated α-tubulin (red) in frontal brain sections from hyperglycemic rats (CSF glucose concentration of 10 mM). Nuclear staining with Hoechst (blue). Scale bar: 30 μm. Most secreted SCO-spondin was detected at the apex of ependymal cells (yellow arrows). Immunofluorescence and DIC (D`) microscopy confirmed that cilia and SCO-spondin interacted. (E) TEM revealed the presence of aggregated extracellular proteins at the apex of ependymal cells (arrows) in hyperglycemia. Scale bar: 5 μm. (Eˋ) Gold immunolabeling and TEM using anti-SCO-spondin in ependymal cells, caudal area; 10 mM glucose CSF. SCO-spondin–positive reaction (black gold particles) were detected in the cilia. Scale bar: 5 μm. (F and Fˋ) Immunohistochemical staining of SCO-spondin (green), vimentin (white), and acetylated α-tubulin (red) in frontal brain sections (caudal area) from hyperglycemic rats (CSF glucose concentration of 10 mM). Nuclear staining with Hoechst (blue). Scale bar: 30 μm. Most secreted SCO-spondin was detected at the apex of ependymal cells (yellow arrows). Immunofluorescence and DIC (Fˋ) microscopy confirmed that cilia and SCO-spondin interacted. (Fˋˋ) Gold immunolabeling and TEM using anti-SCO-spondin in ependymal cells; medial area. SCO-spondin–positive reaction (black gold particles) was detected in the cilia. Scale bar: 5 μm. (G-I) Immunohistochemical staining of SCO-spondin (green) and CD63 (red) in frontal brain sections containing SCO cells under different glycemic conditions. Intensity reactivity was detected for the exosome marker CD63, mainly in ER cisternae. Scale bar: 80 μm. (J) Immunohistochemical staining of SCO-spondin (green) and CD63 (red) in sections containing ependymal cells under hyperglycemic conditions. CD63 (large arrows) was detected at the apex of ependymal cells, which its colocalization with SCO-spondin was lower (arrowheads). Scale bar: 30 μm. (K) Gold immunostaining with anti-OSC-spondin and TEM analysis in ependymal cells. Scale bar: 2 μm. (L) Quantification of Mander’s overlap coefficient for SCO-spondin and CD63 in SCO and ependymal cells under different glycemic conditions. In normoglycemia, CD63 and SCO-spondin are not detected in ependymal cells. The graph shows data from 4 biologically independent samples. The error bars represent the SD. Data used to generate graph can be found in S1 Data. (D) SCO-spondin immunoreactivity was also observed in SCO cells and pre-RF (Fig 4D, inset and arrowheads), and positive immunoreaction was detected close to the apex of ependyma cilia (Fig 4D, yellow arrows). Using DIC analysis in ependymal cells (Dˋ), we also observed positive immunoreaction for SCO-spondin and acetylated α-tubulin on the ependymal cilia (Fig 4Dˋ, white arrows). TEM analysis showed that aggregated proteins were present in the apex of ependymal cells cilia at 10 mM glucose in CSF (Fig 4E, arrows). These secreted materials were not observed in the control ependymal cells (Fig 4E, normoglycemia). Gold immunostaining with anti-SCO-spondin and TEM analysis confirmed SCO-spondin associated with the apex of ependymal cells and MVBs (Fig 4E’and inset; S3F and S3G Fig). BV, blood vessels; DIC, differential interference contrast; d3v, dorsal third ventricle; ER, endoplasmic reticulum; MVB, multivesicular body; SCO, subcommissural organ. https://doi.org/10.1371/journal.pbio.3002308.g004 Similar results were observed in the dorsal third ventricle, where SCO-spondin was also detected intermingled with the cilia of the ependymal cells (Fig 4F, yellow arrows and inset). The interaction between cilia (white arrow) and SCO-spondin (yellow arrows) was also detected using differential interference contrast (DIC) analysis (Fig 4Eˋ, yellow arrows and inset) and by using gold immunostaining with anti-SCO-spondin and TEM analysis, which demonstrated that SCO-spondin interacts with ependymal cells cilia (Fig 4Fˋˋ, arrowheads and cilia high magnification image). We concluded that soluble SCO-spondin is gradually released into the CSF at different glucose levels and that the secreted SCO-spondin reaches the apices of ependymal cell cilia. We also observed that SCO cells were positive for the exosome marker, CD63, which was highly colocalized with SCO-spondin, mainly in basal ER cisternae (Fig 4G–4I and 4K). At a CSF glucose concentration of 10 mM, CD63 immunoreactivity was decreased, mainly in perinuclear ER cisternae (Fig 4I), suggesting increased vesicle secretion. CD63 was also detected on ependymal cells only under hyperglycemia, where poor colocalization between CD63 and SCO-spondin was observed (Fig 4J, arrowheads for SCO-spondin, white arrows for CD63, yellow arrows for colocalization). Colocalization analysis of CD63 and SCO-spondin (N = 4) showed high colocalization in SCO cells but poor colocalization in ependymal cells (Fig 4L) when the CSF glucose concentration was 5 mM or 10 mM. Only some focal areas showed colocalization between CD63 and SCO-spondin, confirming what was observed by gold immunostaining and TEM in ependymal cells, closer to the SCO (Fig 4J, yellow arrows and 4K). Then, the secretion of SCO-spondin- and CD63-positive vesicles were increased in hyperglycemia, which was detected in the apex of ependymal cells.

The speed of ependymal flow is significantly slower when the CSF glucose concentration is 10 mM and upon coincubation with SCO-spondin Abnormalities in cilia beating frequently result in reduced ependymal flow speed [28–31]. Continuous beating of cilia on the apical surface of ependymal cells generates unidirectional fluid flow [32], which can be visualized and quantified by placing polystyrene latex fluorescent microbeads on live preparations of the dorsal/lateral wall of the third ventricle [30,33–35]. We next investigated ependymal flow in whole-mount preparations (Fig 6A), positive for acetylate α-tubulin (Fig 6Aˋ), and incubated with 3 mM glucose-artificial CSF (aCSF) as basal condition, or with 10 mM glucose-aCSF for 15 min (Fig 6B). When a small number of microbeads were placed in the ventral region of the third ventricle (3 mM glucose-aCSF, control), a strong ventro-dorsal to anteroposterior current was observed (Fig 6B). The overall directionality of flow in whole-mount preparations incubated with 10 mM glucose-aCSF was similar to that in control preparations (Fig 6B), but the speed of the fluorescent beads was significantly slower than that in control preparations (Fig 6B). The speed of the fluorescent beads was 261.4 ± 14.1 μm/s after incubation with 3 mM glucose-aCSF for 15 min (control) and was reduced to 78.22 ± 10.8 μm/s after incubation with 10 mM glucose-aCSF after 15 min (Fig 6C). When the samples were reincubated with 3 mM glucose-aCSF, substantial recovery of fluid flow was observed after a short time, with the flow speed reaching 159.67 ± 26.68 μm/s after 15 min (Fig 6C). Additionally, we incubated the samples with 10 mM glucose-aCSF containing 20 μg/mL SCO-spondin for 15 min, and the speed of the beads slowly recovered following incubation with 3 mM glucose-aCSF (Fig 6D). After incubation for 10 to 15 min, no recovery of flow speed was observed; however, after incubation with 3 mM glucose-aCSF for 30 min, the speed of the beads was 140.48 ± 12.72 μm/s (Fig 6D). This finding suggested that SCO-spondin retards the recovery of ciliary movement. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Ependymal flow is significantly slower at high glucose concentrations. (A, B) Analysis of the migration speed of fluorescent beads placed on whole-mount preparations of the dorsal wall of the third ventricle (A and inset; A, anterior; D, dorsal; P, posterior; V, ventral.). The fluorescent beads were taken up with a glass needle and placed on whole-mount preparations in the dorsal area (asterisk). Immunofluorescence analysis using anti-acetylated α-tubulin to localize the cilia of ependymal cells (blue signal), in the area used for flow analyses (Aˋ). The speed of fluorescent beads was determined with Imaris software (B). (C) The migration speed of the fluorescent beads in aCSF containing 3 mM glucose (control), aCSF containing 10 mM glucose, and aCSF containing 3 mM glucose (recovery). The data are expressed as the mean ± SD. Graph of representative data from 4 independent experiments (***P < 0.001, one-way ANOVA with Tukey’s multiple comparison test). (D) Recovery of the speed of the fluorescent beads placed on whole-mount preparations of the dorsal wall of the third ventricle in aCSF containing 3 mM glucose after incubation with aCSF containing 10 mM glucose + bovine SCO-spondin (20 μg/mL) for 15 min. The data are expressed as the mean ± SD. Graph of representative data from 4 independent experiments (***P < 0.001, n.s. = not significant, one-way ANOVA with Tukey’s multiple comparison test). (E) Migration speed of the fluorescent beads in aCSF containing 3 mM glucose (same data showed in C), aCSF containing 10 mM glucose (same data showed in C), aCSF containing 3 mM glucose with bovine SCO-spondin (20 μg/mL), or aCSF containing 10 mM glucose with bovine SCO-spondin (20 μg/mL). The data are expressed as the mean ± SD. Graph of representative data from 3 independent experiments (*P < 0.05, ***P < 0.001, n.s. = not significant, one-way ANOVA with Tukey’s multiple comparison test). (F, G) Immunohistochemical analysis of whole-mount preparations in 3 mM glucose (control) or 10 mM glucose. CellMask staining (white) and staining of fluorescent microbeads (green), acetylated α-tubulin (1:2,000, blue), and SCO-spondin (1:1,000, red). Scale bar: 10 μm. (H) The migration speed of the fluorescent beads that were coincubated or not with aCSF containing 10 mM glucose + BAY876 (GLUT1 inhibitor) after incubation with aCSF containing 3 mM glucose in aCSF containing 10 mM glucose. The data are expressed as the mean ± SD. Graph of representative data from 4independent experiments (***P < 0.001, unpaired t test with Welch’s correction). Data used to generate graphs can be found in S1 Data. aCSF, artificial CSF; d3v, dorsal third ventricle; SCO, subcommissural organ. https://doi.org/10.1371/journal.pbio.3002308.g006 We further compared the data showed in C (3 mM and 10 mM glucose in aCSF), with samples treated additionally with SCO-spondin. In these conditions, the effect was increased when the samples were incubated with 10 mM glucose-aCSF + SCO-spondin, with the flow speed reaching 33.19 ± 5.7 μm/s (Fig 6E, pink analysis). These data demonstrated that SCO-spondin affects ciliary movement under hyperglycemic conditions. To confirm the interaction between SCO-spondin and the ependymal wall in the whole-mount preparations, the ventricular wall was fixed with paraformaldehyde and incubated with CellMask (plasma membrane stain for in vivo analysis), following treatment with glucose-aCSF to identify the ependymal cell membrane (Fig 6F and 6G, white staining). We detected SCO-spondin (red staining) on the surface of ependymal cells in contact with cilia (blue staining) and fluorescent microbeads (green staining) (Fig 6F and 6G). Finally, to determine whether this effect is mediated by the entry of glucose into ependymal cells, we incubated whole-mount preparations in 3 mM glucose for 15 min and subsequently followed by 10 mM glucose (control) or 10 mM glucose containing BAY876, a specific inhibitor of GLUT1 (Fig 6H). No decrease in flow speed was observed within 9 min during this treatment protocol; however, in the control group, the flow speed decreased rapidly from 225.1 ± 16.3 μm/s to 54.72 ± 10.9 μm/s within 9 min. These results indicated that an increase in the glucose concentration decreases ependymal flow and that this effect is enhanced by SCO-spondin.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002308

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