Linking Notch signaling to ischemic stroke Joseph F. Arboleda-Velasquez*, Zhipeng Zhou†, Hwa Kyoung Shin†, Angeliki Louvi‡, Hyung-Hwan Kim§, Sean I. Savitz†, James K. Liao§, Salvatore Salomone†, Cenk Ayata†, Michael A. Moskowitz†, and Spyros Artavanis-Tsakonas*¶ʈ *Department of Cell Biology, Harvard Medical School, Boston, MA 02115; †Stroke and Neurovascular Regulation Laboratory, Massachusetts General Hospital, Boston, MA 02129; ‡Program on Neurogenetics and Department of Neurosurgery, Yale University School of Medicine, New Haven, CT 06520; §Vascular Medicine Research Unit, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA 02139; and ¶Colle`ge de France, 75231 Paris, France Edited by Pietro V. De Camilli, Yale University School of Medicine, New Haven, CT, and approved January 15, 2008 (received for review October 16, 2007) Vascular smooth muscle cells (SMCs) have been implicated in the from Notch 3ϩ/Ϫ or Notch 3Ϫ/Ϫ mice were isolated and shown to pathophysiology of stroke, the third most common cause of death express ␤-gal and specific markers confirming SMC identity (Fig. and the leading cause of long-term neurological disability in the 2 B and C and SI Appendix, SI Fig. 8). Availability of a highly world. However, there is little insight into the underlying cellular enriched population of SMCs allowed us to examine the impact pathways that link SMC function to brain ischemia susceptibility. of Notch 3 loss-of-function on the transcriptional profile of Using a hitherto uncharacterized knockout mouse model of Notch brain-derived SMCs (BrSMCs). Comparative analysis between 3, a Notch signaling receptor paralogue highly expressed in vas- Notch 3ϩ/Ϫ and Notch 3Ϫ/Ϫ cells revealed 662 differentially cular SMCs, we uncover a striking susceptibility to ischemic stroke regulated genes, using an arbitrary cutoff (P Յ 0.01, fold upon challenge. Cellular and molecular analyses of vascular SMCs change Ϯ 1.5). Notch 3 scores as the utmost down-regulated gene derived from these animals associate Notch 3 activity to the (Ϫ22.1-fold) in Notch 3Ϫ/Ϫ BrSMCs. Indicative of their abnor- expression of specific gene targets, whereas genetic rescue exper- mal Notch signaling capacity, the canonical Notch downstream iments unambiguously link Notch 3 function in vessels to the targets Heyl and Hes1 were down-regulated (both Ϫ1.5-fold). ischemic phenotype. Gene ontology analysis of all misregulated targets in BrSMCs showed statistical overrepresentation of genes classified under ischemia ͉ Notch3 ͉ vascular smooth muscle ͉ CADASIL four functional categories named ‘‘muscle contraction’’ (all down-regulated), ‘‘cell structure and motility,’’ ‘‘muscle devel- otch signaling defines one of the fundamental cell interac- opment,’’ and ‘‘mesoderm development’’ (SI Appendix, SI Tables Ntion mechanisms governing cell fate choices in metazoans 1 and 2), consistent with the notion that SMCs from knockout (1, 2). The central element of this signaling pathway is the Notch animals harbor significant functional differences compared with cell surface receptor, a single-pass transmembrane protein, those carrying WT Notch 3 receptors. which interacts with membrane-bound ligands expressed on Given the relevance of vascular SMCs to stroke, we examined adjacent cells linking the fate of one cell to that of its neighbor the ischemia susceptibility of mice lacking Notch 3 function in a (3, 4). In mammals, four paralogs of the Notch receptor have standard filament model of proximal middle cerebral artery Ϫ/Ϫ been identified, Notch 1–4, with overlapping but nonidentical (MCA) occlusion (11). In this assay, Notch 3 mice developed expression patterns (5). In adult tissues, Notch 3 expression is ischemic lesions approximately twice as large as those seen in ϩ/Ϫ restricted to vascular smooth muscle cells (SMCs) (5). Notwith- WT or heterozygous (Notch 3 ) 10- to 12-week-old male mice standing subtle arterial abnormalities reported in Notch 3 mu- (Fig. 3 A and B). Consistent with the severity of stroke, neuro- tant mice (6), the role of Notch 3 in vascular physiology remains logical deficits were more pronounced (Bederson neurological Ϫ/Ϫ unclear. score on day 1 median values: WT ϭ 1, Notch 3 ϭ 2, P Ͻ 0.01) and mortality higher upon MCA occlusion in Notch 3Ϫ/Ϫ mice Results compared with WT (Fig. 3C). To assess whether enlarged To explore Notch 3 function, we used a previously uncharacter- infarcts were the result of more severe cerebral blood flow (CBF) ized Notch 3 knockout mouse model provided by W. C. Skarnes deficits, we used laser speckle flowmetry (LSF) during distal and M. Tessier-Lavigne (7). Like Notch 3 knockouts studied in MCA occlusion (12). This two-dimensional optical imaging refs. 8 and 9, this mutant mouse was viable and fertile. The null technique measures cortical blood flow with high spatial reso- allele was generated by insertional mutagenesis with a lacZ lution, quantifies the ischemic area, and allows for monitoring of carrying vector so that ␤-galactosidase (␤-gal) expression par- spontaneous periinfarct depolarizations (PIDs) triggered by anoxic release of Kϩ and excitatory amino acids from the infarct allels that of Notch 3 (Fig. 1). Our analysis generally agrees with Ϫ Ϫ reported Notch 3 expression studies in the vasculature (10) but core (13, 14). Using this method, we found that Notch 3 / mice did reveal a broader distribution, including the neuronal pro- developed a 60% larger area of severe CBF deficit than WT mice genitor-containing ventricular zone of the developing neural (P Ͻ 0.01) (Fig. 3 D and E). Thus, using two distinct approaches, tube between embryonic day 12.5 (E12.5) and E15.5 and the we find complete loss of Notch 3 function to be associated with neonatal brain [supporting information (SI) Appendix, SI Fig. 6, significant ischemic abnormalities. Interestingly, the frequency and data not shown]. Relevant to this study, in situ hybridization, of spontaneous PIDs, which are known to aggravate stroke (15), X-gal staining, and immunofluorescence confirmed expression of Notch 3 in SMCs from brain vessels and aorta (Fig. 1 D–F and Author contributions: J.F.A.-V. and Z.Z. contributed equally to this work; J.F.A.-V., Z.Z., A.L., data not shown). A morphological study involving immunostain- S.S., C.A., M.A.M., and S.A.-T. designed research; J.F.A.-V., Z.Z., H.K.S., A.L., H.-H.K., S.I.S., ing with SMC-specific antibodies and electron microscopy did S.S., and C.A. performed research; J.F.A.-V., Z.Z., H.K.S., A.L., H.-H.K., S.I.S., J.K.L., S.S., C.A., not reveal any abnormalities in either brain vessels or the aorta M.A.M., and S.A.-T. analyzed data; and J.F.A.-V., A.L., C.A., M.A.M., and S.A.-T. wrote the of knockout mice (Fig. 1G; SI Appendix, SI Fig. 7; and data not paper. shown). The authors declare no conflict of interest. To investigate further the properties of SMCs lacking Notch This article is a PNAS Direct Submission. 3 function, we developed a FACS-based cell purification proto- ʈTo whom correspondence should be addressed. E-mail: [email protected]. col, taking advantage of the ␤-gal expression associated with the This article contains supporting information online at www.pnas.org/cgi/content/full/ Notch 3 knockout allele in SMCs, virtually the only cells in the 0709867105/DC1. adult brain to express Notch 3 (Figs. 1 and 2). Brain-derived cells © 2008 by The National Academy of Sciences of the USA 4856–4861 ͉ PNAS ͉ March 25, 2008 ͉ vol. 105 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0709867105 Downloaded by guest on September 23, 2021 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 ␤-gal (blue box). (B) Long-range PCR amplified intron 16–17 (Ϸ2 kb) of the Notch 3 gene in DNA samples from WT (Notch 3ϩ/ϩ) or heterozygous animals (Notch 3ϩ/Ϫ) but failed to amplify the larger intron containing the trapped vector in DNA from knockout mice (Notch 3Ϫ/Ϫ). (C) The Notch 3 intracellular domain was detected by Western blot analysis of cultured aortic smooth muscle cells (SMCs) derived from WT and Notch 3ϩ/Ϫ but absent (also by qRT-PCR, data not shown) in those derived from Notch 3Ϫ/Ϫ 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)ofNotch 3ϩ/Ϫ (Left) and Notch 3Ϫ/Ϫ (Right) brain vessels. (E) Immunofluorescence of brain tissue sections demonstrated the colocalization of ␤-gal and Notch 3 extracellular epitopes in brain arteries from Notch 3ϩ/Ϫ 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 3Ϫ/Ϫ mice. Asterisks indicate smooth muscle cells. L, lumen. (Scale bars: Left,5␮m; Right,10␮m.) was more than doubled in Notch 3Ϫ/Ϫ compared with WT mice Discussion (6.0 Ϯ 2.5 vs. 2.9 Ϯ 2.5 PIDs per h, P Ͻ 0.05) (Fig. 4). However, Stroke burden is a key factor in determining short- and long-term Ϫ Ϫ Notch 3 / mice did not exhibit the characteristic transient neurological disability.