Dose-dependent expression of claudin-5 is a modifying factor in schizophrenia

Abstract

Schizophrenia is a neurodevelopmental disorder that affects up to 1% of the general population. Various genes show associations with schizophrenia and a very weak nominal association with the tight junction protein, claudin-5, has previously been identified. Claudin-5 is expressed in endothelial cells forming part of the blood-brain barrier (BBB). Furthermore, schizophrenia occurs in 30% of individuals with 22q11 deletion syndrome (22q11DS), a population who are haploinsufficient for the claudin-5 gene. Here, we show that a variant in the claudin-5 gene is weakly associated with schizophrenia in 22q11DS, leading to 75% less claudin-5 being expressed in endothelial cells. We also show that targeted adeno-associated virus-mediated suppression of claudin-5 in the mouse brain results in localized BBB disruption and behavioural changes. Using an inducible 'knockdown' mouse model, we further link claudin-5 suppression with psychosis through a distinct behavioural phenotype showing impairments in learning and memory, anxiety-like behaviour and sensorimotor gating. In addition, these animals develop seizures and die after 3-4 weeks of claudin-5 suppression, reinforcing the crucial role of claudin-5 in normal neurological function. Finally, we show that anti-psychotic medications dose-dependently increase claudin-5 expression in vitro and in vivo while aberrant, discontinuous expression of claudin-5 in the brains of schizophrenic patients post mortem was observed compared to age-matched controls. Together, these data suggest that BBB disruption may be a modifying factor in the development of schizophrenia and that drugs directly targeting the BBB may offer new therapeutic opportunities for treating this disorder.

Figure 1

Claudin-5 variant rs10314 causes decreased protein expression. (a) Distinctive craniofacial pattern characteristic of 22q11DS. (b) Chromosomal location of claudin-5 gene on Chr. 22q11.21. (c) Weak association of the claudin-5 variant rs10314 in a population of 67 22q11DS patients (*P=0.0388, two-sided χ ²-test). (d) Claudin-5 expression in HEK-293 cells expressing normal or rs10314 variant claudin-5. Claudin-5 protein is significantly decreased in rs10314-expressing plasmid 24 h post transfection (**P<0.01). (e) Claudin-5 expression in HEK-293 cells expressing wild-type (white arrows) or rs10314-expressing cDNA. (f) Levels of claudin-5 transcript remain unchanged 24, 48, 72 and 96 h post transfection of wild-type and rs10314-expressing cDNA. (g) Analysis of hsa-MiR-3934 and (h) hsa-MiR-125a-3p binding within 3′-untranslated region of the claudin-5 gene. The rs10314 variant shows reduced promoter activity compared to the normal variant in the control condition (*P<0.05; **P<0.01). This difference is absent following the addition of either miRNA. (i) Levels of expression of claudin-5 transcript in polysome fractions post transfection of normal (blue) or rs10314 (red)-expressing cDNA vectors. cDNA, complementary DNA; EV, empty vector; miRNA, micro RNA; UNT, untransfected.

Figure 2

Site-specific suppression of claudin-5 in the hippocampus and medial prefrontal cortex. (a) Plasmid maps of claudin-5 AAV-2/9 and non-targeting (NT) AAV-2/9. (b) eGFP-expressing AAV-2/9 injected into the dorsal hippocampus. (c) eGFP-expressing AAV-2/9 injected into the medial prefrontal cortex (mPFC). (d and e) Significant suppression of claudin-5 (red) in the microvasculature of the hippocampus (IB4: green, DAPI: blue; scale bar: 50 μm; *P<0.05). (f and g) Significant suppression of claudin-5 (red) in the microvasculature of the mPFC (IB4: green, DAPI: blue; scale bar: 50 μm; **P<0.01). (h) Biotin (red) and fibrinogen (green) extravasation in the hippocampus; scale bar: 50 μm. (i) Quantification of biotin extravasation in the hippocampus, suppression of claudin-5 significantly increases the amount of extravasation (**P<0.01). (j) Biotin (red) and fibrinogen (green) extravasation in the mPFC; scale bar: 50 μm. (k) Quantification of biotin extravasation in the mPFC, suppression of claudin-5 significantly increases the amount of biotin extravasation (**P<0.01). (l) Summary of behavioural data following suppression of claudin-5 in the hippocampus. These mice showed significantly decreased levels of grooming (*P<0.05) along with a significant impairment in the social novelty task (*P<0.05). (m) Summary of behavioural data following suppression of claudin-5 in the mPFC. These mice showed significant impairments in the social object recognition task (**P<0.01) and the T-maze (*P<0.05) along with a significant enhancement in the forced swim test (***P<0.001). Behavioural assays were performed 2 weeks post supplementation of doxycycline (2 mg/ml) to the drinking water. AAV, adeno-associated virus; eGFP, enhanced green fluorescent protein; EPM, elevated plus maze; FST, forced swimming test; LT, long-term; shRNA, short hairpin RNA; RAM, radial arm maze; OFT, open field test.

Figure 3

Generation and characterization of inducible claudin-5 knockdown mice. (a) Significant suppression of claudin-5 expression 24 h post transfection of two different shRNAs targeting claudin-5 in vitro (*P<0.05; **P<0.01). (b) Levels of expression of claudin-5 at the tight junction 24 h post transfection of claudin-5 shRNA in a monolayer of mouse brain endothelial cells; scale bar: 50 μm. (c) Schematic representation of inducible claudin-5 knockdown mouse model. (d) Inducible suppression of claudin-5 protein using shRNA in the vasculature of the mouse brain (***P<0.001) 72 h following i.p. injection of 40 mg/kg doxycycline in 0.9% saline. No significant changes in the protein expression levels of the other tight junction proteins, (e) occludin, and ZO-1 and (f) tricellulin. (g) Inducible suppression of claudin-5 mRNA using shRNA in the vasculature of the mouse brain (***P<0.001). No changes in the transcript levels of (h) occludin, (i) ZO-1 or (j) tricellulin. (k) Expression of rtTA3 (red, mKate) and claudin-5 shRNA (green, turbo green fluorescent protein) confirmed in the vasculature of the mouse brain 72 h following i.p. injection of doxycycline; scale bar: 50 μm. (l) Survival chart for inducible claudin-5 knockdown mice. Inducible claudin-5 knockdown mice supplemented with doxycycline (2 mg/ml) all die compared to inducible claudin-5 mice fed water only, a scrambled control mouse (NT) and Cre-negative littermates. mRNA, messenger RNA; NT, non-targeting; shRNA, short hairpin RNA.

Figure 4

Phenotype of inducible claudin-5 knockdown mice. (a) Reduced spontaneous alternation in the T-maze in claudin-5 knockdown mice (*P<0.05). (b) Reduced discrimination index in claudin-5 knockdown mice in the object recognition task (*P<0.05). (c) Reduced open arm entries observed in the elevated plus maze in the claudin-5 knockdown mice (*P<0.05). (d) Increased side bias in inducible claudin-5 knockdown mice in a spontaneous alternation task in the Y-maze (*P<0.05). (e) Decreased acoustic prepulse inhibition (PPI) in inducible claudin-5 knockdown mice with a 77 dB prepulse at 110 dB (**P<0.01) and 120 dB (*P<0.05). (f) Summary of behavioural data following suppression of claudin-5 in the inducible claudin-5 knockdown mouse model. (g) Contrast-enhanced magnetic resonance imaging (MRI) showing significant extravasation of contrast agent in the brain of inducible claudin-5 knockdown mice (right) compared to non-targeting control mice (left; **P<0.01). All assays were performed 2–4 weeks post supplementation of doxycycline (2 mg/ml) to the drinking water. RQ, relative quantity.

Figure 5

Regulation of claudin-5 levels by anti-psychotic drugs and examples of aberrant claudin-5 expression in schizophrenia. (a) Levels of expression of claudin-5 in primary mouse brain endothelial cells exposed for 24 h to lithium (LiCl), haloperidol (HAL) or chlorpromazine (CPZ). (b) Immunocytochemical analysis of claudin-5 (red)-staining pattern in primary mouse brain endothelial cells treated with anti-psychotic drugs; scale bar: 50 μm. (c) Levels of expression of claudin-5 in capillary fractions from mouse brains 24 h following administration (i.v.) of LiCl, HAL or CPZ. (d) Claudin-5 expression was significantly higher in mice treated with antipsychotics (*P<0.05). (e) Claudin-5 mRNA levels were significantly higher in mice treated with CPZ (*P<0.05). (f) Claudin-5 levels in normal control or schizophrenia donor brain tissues from the parietal lobe. Sixty-two per cent of schizophrenia patients showed aberrant claudin-5 staining in the parietal lobe. (g) There are significantly lower levels of claudin-5 in the parietal lobe in individuals who have a diagnosis of schizophrenia and the presence of the rs10314 allele compared to controls who have the rs10314 allele (*P<0.05).