Expression of the ALS-causing variant hSOD1(G93A) leads to an impaired integrity and altered regulation of claudin-5 expression in an in vitro blood-spinal cord barrier model

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive paralysis due to the loss of primary and secondary motor neurons. Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene are associated with familial ALS and to date numerous hypotheses for ALS pathology exist including impairment of the blood-spinal cord barrier. In transgenic mice carrying mutated SOD1 genes, a disrupted blood-spinal cord barrier as well as decreased levels of tight junction (TJ) proteins ZO-1, occludin, and claudin-5 were detected. Here, we examined TJ protein levels and barrier function of primary blood-spinal cord barrier endothelial cells of presymptomatic hSOD1(G93A) mice and bEnd.3 cells stably expressing hSOD1(G93A). In both cellular systems, we observed reduced claudin-5 levels and a decreased transendothelial resistance (TER) as well as an increased apparent permeability. Analysis of the β-catenin/AKT/forkhead box protein O1 (FoxO1) pathway and the FoxO1-regulated activity of the claudin-5 promoter revealed a repression of the claudin-5 gene expression in hSOD1(G93A) cells, which was depended on the phosphorylation status of FoxO1. These results strongly indicate that mutated SOD1 affects the expression and localization of TJ proteins leading to impaired integrity and breakdown of the blood-spinal cord barrier.

Figure 1

Distribution of claudin-5 in spinal cords of superoxide dismutase (SOD1) transgenic mice. (A–F) Native, unfixed cryo sections from lumbar spinal cords from mice overexpression hSOD1^G93A at endstage (10 months of age) or from age-matched hSOD1^WT mice were stained with polyclonal antibodies against claudin-5 (red) and rat monoclonal antibodies against CD31 (green). Nuclei were detected with DAPI (blue). (A'–F') An enlargement of the boxed areas of the corresponding panels is shown. The arrow in (D–F) points to a blood vessel weakly stained for claudin-5 in a mutant animal. Scale bar is 20 μm. (G, H) Primary mouse spinal cord endothelial cells (pMSCECs) of 7-month-old nonsymptomatic hSOD1^G93A transgenic mice and their respective littermates were fixed with paraformaldehyde and immunostained for the tight junction (TJ) protein claudin-5, followed by incubation with AlexaFlour 546 secondary antibody. Cell nuclei were stained with DRAQ5. Scale bar is 5 μm.

Figure 2

Expression of hSOD1^G93A results in decreased claudin-5 levels in endothelial cells. (A, B) bEnd.3 cells were stably transfected via retroviral transfection. The expression of hSOD1 was analyzed by SDS-PAGE and western blot. The hSOD1 variants were fused to a C-terminal myc-tag. Blots were incubated with either mouse monoclonal 9E10 antibody recognizing the myc-tag (A) or rabbit monoclonal SOD1 antibody (B), which detects mouse and human SOD1. (C) bEnd.3 cells overexpressing hSOD1^WT or hSOD1^G93A were fixed with paraformaldehyde and immunostained for tight junction (TJ) protein claudin-5, followed by incubation with AlexaFlour 546 secondary antibody. Cell nuclei were stained with DRAQ5. Scale bar is 5 μm. (D) Surface proteins of postconfluent hSOD1^WT and hSOD1^G93A bEnd.3 cells were biotinylated using sulfo-NHS-biotin. Biotinylated samples were precipitated with NeutrAvidin agarose beads and analyzed by SDS-PAGE and western blot. All samples were analyzed on the same western blot but in a different order (PD: NeutrAvidin; left panel). As input controls, 20 μg of cell lysates was used (input; left panel). Polyclonal β-actin antibody served as a loading control to verify the absence of endomembrane contaminants in the biotinylation. (E, F) Claudin-5 levels in the lysate (E) or at the cell surface (F) were quantified by densitometric analysis of western blots. Intensities of hSOD1^WT cells were set as 100%. The data represent mean±s.e.m. of six independent experiments. *Statistically significant difference (P<0.05, t-test) between claudin-5 levels of stable hSOD1^WT and hSOD1^G93A cells is indicated. hSOD1, human SOD; mSOD1, mouse SOD1. SOD1, superoxide dismutase.

Figure 3

Impaired integrity of hSOD1^G93A endothelial cells. (A–C) Stable hSOD1^WT and hSOD1^G93A bEnd.3 cells were cultivated on cell culture inserts in the cellZscope device. (A) Representative transendothelial electrical resistance (TER) measurement of stable bEnd.3 cells of at least three independent experiments. (B) Representative C_Cl measurement of stable bEnd.3 cells of at least three independent experiments. (C) Apparent permeability (P_app) of [¹⁴C]-inulin of hSOD1^G93A bEnd.3 cells compared with hSOD1^WT overexpressing cells. The data represent mean±s.e.m. with n⩾20 of 3 independent experiments. Statistical significance (*P<0.05, t-test) between hSOD1^G93A and hSOD1^WT overexpressing cells. (D–F) Primary mouse spinal cord endothelial cells (pMSCECs) were isolated from 7-month-old hSOD1^G93A mice and their nontransgenic littermates. Cells were cultivated on cell culture insert in the cellZscope device, which measured online the TER and C_Cl of the cells. (D) Representative TER and C_Cl measurement (E) of cultured pMSCECs of at least three independent experiments. (F) Apparent permeability (P_app) of [¹⁴C]-inulin of pMSCECs hSOD1^G93A compared with their littermates. The data represent mean±s.e.m. with n⩾9 of 3 independent experiments. Statistical significance (*P<0.05, t-test) between pMSCECs from hSOD1^G93A mice and their littermates is indicated. SOD1, superoxide dismutase; hSOD1, human SOD1.

Figure 4

Decreased levels of p[S675]β-cat, p[S473]AKT, and p[S256]FoxO1 in stable hSOD1^G93A bEnd.3 cells. The phosphorylation states of p[S675]β-cat (A, B), p[S473]AKT (C, D), and p[S256]FoxO1 (E, F) were analyzed in postconfluent hSOD1^WT and hSOD1^G93A bEnd.3 cells by SDS-PAGE and western blot (A, C, E). Levels of phosphorylated and total proteins were quantified by densitometric analysis of western blots (B, D, F). Intensities were normalized to β-tubulin and the ratio of phosphorylated to total protein was calculated. Ratios of hSOD1^WT cells were set as 100%. The data represent mean±s.e.m. of at least five independent experiments. *Statistically significant difference (P<0.05, t-test) between ratios of stable hSOD1^WT and hSOD1^G93A cells are indicated. SOD1, superoxide dismutase; hSOD1, human SOD1.

Figure 5

Binding of FoxO1 to the promoter region of claudin-5 in stable hSOD1^G93A bEnd.3 cells. The binding of FoxO1 to the promoter region of claudin-5 was analyzed by chromatin immunoprecipitation in postconfluent hSOD1^WT and hSOD1^G93A bEnd.3 cells. (A) Crosslinked chromatin was precipitated with FoxO1 antibody. As a negative control, primers for the claudin-5 CDS were used to exclude nonspecific precipitated DNA. Levels of precipitated DNA were quantified by densitometric analysis of agarose gels (B). Intensities were normalized to input and the ratio of hSOD1^WT cells was set as 100%. The data represent mean±s.e.m. of three independent experiments. *Statistically significant difference (P<0.05, t-test) between ratios of stable hSOD1^WT and hSOD1^G93A cells is indicated. SOD1, superoxide dismutase; hSOD1, human SOD1.

Figure 6

Suggested model for the regulation of the claudin-5 expression in hSOD1^G93A endothelial cells. (A) In hSOD1^WT endothelial cells, the adhesion of VE-cadherin leads downstream to an activation of the PI3K and AKT pathway. Phosphorylation of FoxO1 through AKT activation (pAKT) prevents the nuclear accumulation of β-cat and FoxO1 and consequently leads to the expression of claudin-5. (B) Binding of mutant hSOD1^G93A to Rac1 may result in a local overproduction of reactive oxygen species (ROS). Increased oxidative stress results in increased pβ-cat levels and a reduced phosphorylation of FoxO1 through the interaction of AKT. This may cause an accumulation of β-cat and FoxO1 in the nucleus leading to a complex formation of β-cat and FoxO1 with the transcription factor TCF, and this complex may bind to the claudin-5 promoter and may inhibit its transcription. This subsequently may result in the loss of claudin-5 in tight junctions (TJs), in the disruption of the endothelial barrier function and in enhanced permeability (modified from Gavard and Gutkind and Taddei et al). TCF, T-cell factor. SOD1, superoxide dismutase; hSOD1, human SOD1.