C-type natriuretic peptide preserves central neurological function by maintaining blood-brain barrier integrity

Abstract

C-type natriuretic peptide (CNP) is highly expressed in the central nervous system (CNS) and key to neuronal development; however, a broader role for CNP in the CNS remains unclear. To address this deficit, we investigated behavioral, sensory and motor abnormalities and blood-brain barrier (BBB) integrity in a unique mouse model with inducible, global deletion of CNP (gbCNP^-/-). gbCNP^-/- mice and wild-type littermates at 12 (young adult) and 65 (aged) weeks of age were investigated for changes in gait and motor coordination (CatWalk™ and rotarod tests), anxiety-like behavior (open field and elevated zero maze tests), and motor and sensory function (modified neurological severity score [mNSS] and primary SHIRPA screen). Vascular permeability was assessed in vivo (Miles assay) with complementary in vitro studies conducted in primary murine brain endothelial cells. Young adult gbCNP^-/- mice had normal gait but reduced motor coordination, increased locomotor activity in the open field and elevated zero maze, and had a higher mNSS score. Aged gbCNP^-/- animals developed recurrent spontaneous seizures and had impaired gait and wide-ranging motor and sensory dysfunction. Young adult and aged gbCNP^-/- mice exhibited increased BBB permeability, which was partially restored in vitro by CNP administration. Cultured brain endothelial cells from gbCNP^-/- mice had an abnormal ZO-1 protein distribution. These data suggest that lack of CNP in the CNS impairs tight junction protein arrangement and increases BBB permeability, which is associated with changes in locomotor activity, motor coordination and late-onset seizures.

Keywords: C-type natriuretic peptide; ZO-1; anxiety; blood brain barrier; coordination; hyperactivity; natriuretic peptide receptor C; seizure.

FIGURE 1

The effect of CNP deletion on body weight, muscle weight and the expression of CNP and its receptors. Body weight (A) and gastrocnemius weight normalized to tibia length (B) in wild-type (WT) and global CNP knockout (gbCNP^{– /–}) mice at 12 weeks of age. Densitometric quantification of CNP protein expression using diaminobenzidine (DAB) immunohistochemistry in the brain of WT and gbCNP^{– /–} mice (C) and representative images (D, white arrow indicates neuron and black arrow microglia). CNP (Nppc, E), NPR-B (Npr2, F) and NPR-C (Npr3, G) mRNA expression in brain homogenates. n = 5-7. Statistical analysis by two-tailed Student’s t-test. Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

FIGURE 2

Young adult gbCNP^{– /–} mice showed normal gait and motor coordination when walking overground but not when their balance was challenged. Rotarod latency to fall when performing in accelerating (A) and fixed speed (B) modes. Modified neurological severity score (mNSS, C). CatWalk™ analysis of gait and coordination in wild-type (WT) and global CNP knockout (gbCNP^{– /–}) mice. Stride length (D), Swing speed (E), Phase dispersion (F), Regularity index (G) and Step cycle (H). n = 6-7. Statistical analysis by two-tailed Student’s t-test, Mann–Whitney U test or two-way analysis of variance with Šídák post hoc test. Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

FIGURE 3

Young adult gbCNP^{– /–} mice were more active than their wild-type littermates in the open field and elevated zero maze. Open field analyses in wild-type (WT) and global CNP knockout (gbCNP^{– /–}) mice. Representative traces of movement of the mice in the apparatus (A), total distance traveled (B), number of entries in each zone of the open field (C), time spent in each zone of the open field (D), time spent on supported (E) and unsupported (F) rearings, and time spent grooming (G). Elevated zero maze analyses in WT and gbCNP^/ mice: Representative traces of movement of the mice in the apparatus (H), average speed (I), time spent freezing (J), average duration of the visits (K), head dips (HDIPs) (L) and stretching attend postures (SAPs, M). n = 6-7. Statistical analysis by two-tailed Student’s t-test, Mann–Whitney U test or two-way analysis of variance with Šídák post hoc test. Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

FIGURE 4

Aged gbCNP^{– /–} mice exhibited seizures. Age of seizure onset in months (M) in wild-type (WT) and global CNP knockout (gbCNP^{– /–}) mice (A). Incidence of seizures (B). Distribution of the seizure severity according to the Racine scale (0 – No seizure; 1 – Immobility, twitching; 2 – Head nodding; 3- Clonus of one forelimb; 4 – Bilateral forelimb clonus with rearing; 5 – Falling on a side, tonic-clonic seizures) (C). Representative images of a typical seizure in gbCNP^{– /–} mice (D). Limb clasping score (E) and representative images (F). n = 5-10. Statistical analysis by two-tailed Student’s t-test. Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

FIGURE 5

Abnormal gait, motor and sensory function in aged gbCNP^{– /–} mice. Primary screen of SHIRPA in aged (65 weeks) wild type (WT) and global CNP knockout (gbCNP^{– /–}) mice. Spontaneous activity (A), tremor (B), gait (C), pelvic elevation (D), tail elevation (E), touch scape (F), limb clasping (G), righting reflex (H), and representative images of WT and gbCNP^{– /–} mice (I). n = 5. Statistical analysis by two-tailed Student’s t-test. Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

FIGURE 6

Young adult and aged gbCNP^{– /–} mice had increased blood brain barrier extravasation. Representative images of wild-type (WT) and global CNP knockout (gbCNP^{– /–}) mice at 45 min following Evans blue intravenous administration (A). Evans blue extravasation in the organs of young adult (12 weeks, Y; B) and aged (65 weeks, A,C) mice, and in the brain of WT and gbCNP^{– /–} animals (D). Representative images of Evans blue extravasation in whole brains and brain sections from young adult (12 weeks, Y) and aged (65 weeks, A) mice (E). n = 5-7. Statistical analysis by two-tailed Student’s t-test. Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).

FIGURE 7

CNP prevented endothelial cell extravasation via NPR-C signaling. Transwell FITC extravasation from murine brain endothelial cells from wild-type (WT) animals induced by thrombin (5 IU/ml), in the absence and presence of CNP (100 nM) or the NPR-C selective agonist cANF^4–23 (100 nM), compared to basal unstimulated extravasation (w/o Thrombin); (A). mRNA expression of VE-cadherin (B), Zonula Occludens-1 (ZO-1); (C), claudin-5 (Cldn5); (D) and occludin (E), and representative images of C-type natriuretic peptide (CNP), CD31, VE-cadherin, ZO-1, claudin-5 and occludin inmmunohistofluorescence in murine brain endothelial cells from wild-type (WT) and global CNP knockout (gbCNP^–/–) mice (F). The white dotted line indicates a gap between the cells. n = 5-7. Statistical analysis by two-tailed Student’s t-test, Mann–Whitney U test or two-way analysis of variance with Šídák post hoc test. Each statistical comparison undertaken has an assigned P value (adjusted for multiplicity).