Linking Notch signaling to ischemic stroke

Abstract

Vascular smooth muscle cells (SMCs) have been implicated in the pathophysiology of stroke, the third most common cause of death and the leading cause of long-term neurological disability in the world. However, there is little insight into the underlying cellular pathways that link SMC function to brain ischemia susceptibility. Using a hitherto uncharacterized knockout mouse model of Notch 3, a Notch signaling receptor paralogue highly expressed in vascular SMCs, we uncover a striking susceptibility to ischemic stroke upon challenge. Cellular and molecular analyses of vascular SMCs derived from these animals associate Notch 3 activity to the expression of specific gene targets, whereas genetic rescue experiments unambiguously link Notch 3 function in vessels to the ischemic phenotype.

Fig. 1.

Characterization of the Notch 3 knockout mice. (A) A schematic of the heterodimeric Notch 3 receptor (Upper) indicating key structural features. In the extracellular domain, the 34 EGF-like repeats (gray boxes) and the three Lin12-Notch repeats (green boxes) are indicated. The transmembrane domain (TM), and the intracellular ankyrin repeat region are also shown (red boxes). The insertional mutagenesis, which generated the knockout allele, resulted in a fusion protein (Lower) containing EGF-like repeats 1–21 of Notch 3 fused to β-gal (blue box). (B) Long-range PCR amplified intron 16–17 (≈2 kb) of the Notch 3 gene in DNA samples from WT (Notch 3^+/+) or heterozygous animals (Notch 3^+/−) but failed to amplify the larger intron containing the trapped vector in DNA from knockout mice (Notch 3^−/−). (C) The Notch 3 intracellular domain was detected by Western blot analysis of cultured aortic smooth muscle cells (SMCs) derived from WT and Notch 3^+/− but absent (also by qRT-PCR, data not shown) in those derived from Notch 3^−/− mice. (D) Notch 3 expression. In situ hybridization, using a Notch 3 antisense riboprobe labeled vessels in brain from WT mice (Upper). Likewise, X-gal staining (Lower) of Notch 3^+/− (Left) and Notch 3^−/− (Right) brain vessels. (E) Immunofluorescence of brain tissue sections demonstrated the colocalization of β-gal and Notch 3 extracellular epitopes in brain arteries from Notch 3^+/− mice. (F) Aortic SMC layers from WT mice (white) showed Notch 3 expression. (G) Low magnification electron micrographs of arterial cortical vessels (Left) and aorta (Right) from 8-week-old WT and Notch 3^−/− mice. Asterisks indicate smooth muscle cells. L, lumen. (Scale bars: Left, 5 μm; Right, 10 μm.)

Fig. 2.

Isolation of vascular smooth muscle cells from brain. (A) FACS analysis detected significant fluorescein-positive events in brain-derived cell suspensions from Notch 3^+/− (β-gal positive) but not from WT mice (β-gal negative) upon incubation with the fluorogenic β-gal substrate fluorescein di-β-d-galactopyranoside (FDG) (Center). Most fluorescein-positive cells (96.1%) were viable as demonstrated by propidium iodide (PI) exclusion but were heterogeneously distributed in the FSC-A vs. SSC-A profile (Right and data not shown). In 20 independent FACS analyses performed by using our brain digestion and FDG staining protocols (including Notch 3^+/− and Notch 3^−/− samples), the percentage of PI-positive events in the total population ranged from 0.3 to 1.5%. Fluorescein signal was only occasionally higher in brain cell suspensions derived from Notch 3^−/− mice (two copies of β-gal) compared with that of Notch 3^+/− (one copy) (Lower) consistent with a documented nonlinear relationship between fluorescence intensity and intracellular β-galactosidase activity. (B) Seventy to 80% of fluorescein-positive sorted cells in culture showed X-gal staining. (C) β-gal positive cells always expressed smooth muscle-specific alpha actin epitopes (SMC-actin).

Fig. 3.

Stroke susceptibility of Notch 3 knockout mice. (A and B) Infarct volume (indirect method) and infarct areas in individual coronal slices in WT, Notch 3^+/− (N3^+/−), and Notch 3^−/− (N3^−/−) mice analyzed 22 h after a 1-h transient filament middle cerebral artery occlusion (fMCAO). Both infarct area and volume were substantially larger in Notch 3^−/− mice compared with those of WT and Notch 3^+/− mice (10- to 12-week-old male mice, n = 9 per group; P < 0.01). (C) A separate cohort of WT and Notch 3^−/− mice (10- to 12-week-old male mice, n = 5 per group) underwent 1 h transient fMCAO. Notch 3^−/− mice had 60% mortality over 7 days, compared with no mortality in WT mice. (D) Representative laser speckle contrast images taken 1 h after distal MCA occlusion (dMCAO) are shown from WT and Notch 3^−/− mice. Distal MCA was clipped through a small temporal craniotomy (arrows). Superimposed areas (blue) indicate regions with severe cerebral blood flow (CBF) deficit (i.e., <20% residual CBF). Notch 3^−/− mice developed significantly larger area of severe CBF deficit compared with WT. The imaging field (5.24 × 7 mm) was positioned over the entire right hemisphere as shown in Left Inset. (E) Composite bar graph showing the areas of severe (residual CBF ≤20%), moderate (21–30%), and mild (31–40%) CBF deficit in WT and Notch 3^−/− mice 60 min after dMCAO. The area of severe CBF deficit was significantly larger in Notch 3^−/− animals compared with WT (P < 0.01), whereas the areas of moderate or mild CBF deficit did not differ between the two genotypes (P > 0.05; two way ANOVA for repeated measures). Error bars indicate standard deviations.

Fig. 4.

Abnormal CBF changes upon ischemic challenge in Notch 3 knockout mice. Representative tracings showing the time-course of CBF changes after dMCAO (at time 0) in severe (black), moderate (green), or mildly ischemic cortex (red) in WT and Notch 3^−/− (N3^−/−) mice. Black dots indicate spontaneous peri-infarct depolarizations (PIDs). Notch 3^−/− mice developed more frequent PIDs; however, the characteristic transient hypoperfusion response observed in WT during the PIDs (arrows) was absent in Notch 3^−/− mice.

Fig. 5.

Rescuing stroke susceptibility with human NOTCH 3. (A) Schematic representation of the targeting construct used to generate mice carrying a WT human NOTCH 3 transgene that can be conditionally expressed by Cre-mediated recombination. The vector contains ROSA genomic sequences allowing for homologous recombination in the ROSA locus, an adenovirus splice acceptor site (SA), a PGK-neo-tpA “stop” cassette flanked by LoxP sites (black triangles), the coding region for human NOTCH 3, an internal ribosomal entry sequence (IRES), nuclear EGFP, and the bovine growth hormone polyadenylation sequence (bpA). (B) The EcoRV restriction sites allowed identification of WT vs. targeted alleles by Southern blot analysis of DNA from ES cell clones (clone 76 generated chimeric mice capable of germ line transmission). (C and D) Upon Cre expression, the PGK-Neo-tpA cassette is excised, thereby allowing expression of the NOTCH 3 transgene detected here by RT-PCR in aorta tissue (C) and in brain arterioles from a Notch 3^−/−; ROSA NOTCH 3^+/−; SM22-Cre^+/− mouse (Cre under the control of the smooth muscle-specific transgelin promoter), using an antibody specific for intracellular epitopes of the receptor (D). (E and F) Graphics depict indirect infarct volume and infarct area of genetically rescued (Notch 3^−/−; ROSA NOTCH 3^+/−; SM22-Cre^+/−) and control (Notch 3^−/−; ROSA NOTCH 3^+/−) mice after 1 h of MCAO and 22 h of reperfusion (10- to 12-week-old male mice, n = 5 per group; P < 0.01). Error bars represent standard deviations.