SRF and myocardin regulate LRP-mediated amyloid-beta clearance in brain vascular cells

Abstract

Amyloid beta-peptide (Abeta) deposition in cerebral vessels contributes to cerebral amyloid angiopathy (CAA) in Alzheimer's disease (AD). Here, we report that in AD patients and two mouse models of AD, overexpression of serum response factor (SRF) and myocardin (MYOCD) in cerebral vascular smooth muscle cells (VSMCs) generates an Abeta non-clearing VSMC phenotype through transactivation of sterol regulatory element binding protein-2, which downregulates low density lipoprotein receptor-related protein-1, a key Abeta clearance receptor. Hypoxia stimulated SRF/MYOCD expression in human cerebral VSMCs and in animal models of AD. We suggest that SRF and MYOCD function as a transcriptional switch, controlling Abeta cerebrovascular clearance and progression of AD.

Figure 1

SRF and MYOCD overexpression blocks Aβ clearance by human cerebral VSMCs. (a) Multi-photon/confocal laser scanning microscopy of multi-spot glass slides coated with Cy3-labelled Aβ42 without cells (left panel) and with cerebral VSMCs (right panels) from AD individuals and age-matched controls grown for 5 days. (b) VSMCs from AD individuals transduced with Ad.shGFP or Ad.shSRF at MOI 100, plated and grown on Cy3-Aβ42-coated surfaces for 5 days and imaged by confocal microscopy. (c) VSMC from controls transduced with Ad.GFP or Ad.MYOCD plated and grown on Cy3-Aβ42-coated surfaces for 5 days and imaged by confocal microscopy. Scale bars, 50 µm (a–c). (d) Western blots of SRF and MYOCD in AD and age-matched control VSMCs incubated with vehicle and transduced with Ad.shGFP and Ad.shSRF or Ad.GFP and Ad.MYOCD, respectively. Relative levels of expression determined by scanning densitometry are shown below the blots (mean ± s.e.m. of 3–4 independent cultures). (e) Cy3-Aβ42 relative signal intensity on multi-spot slides after 5 days with control VSMCs, non-transduced or transduced with Ad.GFP or Ad.MYOCD (black bars), and with AD VSMC not transduced or transduced with Ad.shGFP or Ad.shSRF (open bars). Mean ± s.e.m. of 7–8 independent cultures are shown. (f) Cy3-Aβ40 relative signal intensity after 5 days in control VSMCs, non-transduced or transduced with Ad.GFP or Ad.MYOCD (black bars), compared with AD VSMCs (open bar). Mean ± s.e.m., from 3–4 independent cultures are shown. In e and f, the signal intensity in cell-free slides (grey bar) was arbitrarily taken as 100%. Full scans of western blot data are shown in Supplementary Information, Fig. S4.

Figure 2

LRP in cerebral VSMCs is downregulated by SRF and MYOCD. (a) Multi-photon/confocal laser scanning microscopy of Cy3-Aβ42 internalization and lysosomal colocalization in control VSMCs treated with vehicle and a LRP-specific blocking antibody. Cell Tracker, blue; Cy3-Aβ42, red; Lysotracker, green. Merged images are shown (yellow-white colour is colocalization of Aβ with lysosomes). Scale bar, 20 µm. (b) LRP levels in control and AD cerebral VSMCs, and in AD VSMCs transduced with Ad.shGFP and Ad.shSRF. (c) Real-time quantitative PCR (qPCR)for LRP mRNA in AD VSMCs alone (vehicle) and transduced with Ad.shGFP or Ad.shSRF. (d) LRP levels in control VSMC transduced with Ad.GFP or Ad.MYOCD. (e) Real-time qPCR for LRP mRNA in control VSMCs alone (vehicle) and transduced with Ad.GFP or Ad.MYOCD. Data represent mean ± s.e.m. of 3 independent cultures per group (b–e).

Figure 3

SRF and MYOCD suppress LRP through directed expression of SREBP2. (a) SREBP2 luciferase activity in PAC1 VSMCs after co-transfection with a luciferase reporter containing the promoter (1253), promoter plus intron 1 (1254) or promoter plus intron 1 with both CArG boxes mutated (1271) of human SREBP2, with pcDNA vector and EMSV vector (control) or with MYOCD and SRF overexpression plasmids. (b) Increased levels of nuclear SREBP2 (green) and low cellular expression of LRP (red) in AD VSMCs, compared with control VSMC (see d). Reduced nuclear SREBP2 levels and increased LRP expression after transduction of AD VSMCs with Ad.shSRF but not Ad.shGFP. Nuclear DAPI staining is shown in white. Scale bar, 20 µm. (c) SRF, SREBP2 (cytoplasmic long form and short nuclear form) and LRP levels determined by western blot analysis in AD VSMC transduced with Ad.shGFP and Ad.shSRF. (d) Low levels of nuclear SREBP2 (green) and increased cellular expression of LRP (red) in control VSMCs, compared with AD VSMC (see b). Increased nuclear SREBP2 and reduced LRP expression after transduction of control VSMCs with Ad.MYOCD, but not with Ad.GFP. Nuclear DAPI staining in white. Scale bars, 20 µm. (e). SRF, SREBP2 (cytoplasmic long form and short nuclear form) and LRP levels determined by western blot analysis after transduction of control VSMCs with Ad.GFP or Ad.MYOCD. Graphs in c and e show band density relative to β-actin. (f) SREBP2 and LRP levels in control VSMC transfected with control siRNA (siCTRL) or SREBP2 siRNA (siSREBP2) followed by transduction with either Ad.GFP or Ad.MYOCD. Graph shows band density for test-proteins relative to β-actin. (g) Real-time qPCR for LRP mRNA in control VSMCs transfected with control siRNA (siCTRL) or SREBP2 siRNA (siSREBP2) followed by transduction with either Ad.GFP or Ad.MYOCD. Data represent mean ± s.e.m. of 9 independent cultures; values were normalized to an internal renilla control (a), or mean ± s.e.m. of 3 independent cultures per group (c, e–g). Full scans of western blot data in panel f are shown in Supplementary Information, Fig. S4.

Figure 4

MYOCD overexpression in pial arteries suppresses focal LRP-mediated Aβ clearance in mice. (a, b) MYOCD, SRF, smooth muscle α-actin, smooth muscle calponin, SREBP2 and LRP levels determined by western blotting in cerebral pial vessels isolated after in vivo transduction with Ad.GFP or Ad.MYOCD in 5-month-old C57Bl6 mice (a). Graph shows band density of test-proteins relative to β-actin (mean ± s.e.m. from 5 animals per group, b). (c) Immunocytochemical staining for nuclear SREBP2 and cellular LRP in pial vessels after MYOCD gene transfer. LRP, red; SREBP2, green; TO-PRO3, grey. Scale bars, 20 µm. (d, e) Effects of MYOCD overexpression on endogenous Aβ42 levels in mouse transduced pial vessels (d) and focally in brain (e). Each point represents an individual animal value. Full scans of western blot data are shown in Supplementary Information, Fig. S4.

Figure 5

SRF gene transfer to pial arteries reduces Aβ pathology in AD models. (a) Aβ immunostaining in transduced pial vessels (upper panels) and focally in brain tissue (lower panels) after expression of a control vector (Ad.shGFP) or Ad.shSRF in 24-month-old Dutch/Iowa APP mice. Scale bars,50 µm. (b, c) Aβ load in transduced pial vessels (b) and focally in brain (c) after adenoviral-mediated transfer of shGFP or shSRF in 24-month-old Dutch/Iowa APP mice. (d, e) Amyloid load in transduced pial vessels (d) and focal Aβ40 and Aβ42 levels in brain (e) after adenoviral-mediated transfer of shGFP and shSRF in 24-month-old Dutch/Iowa APP mice. (f) The levels of SREBP2, immature LRP (LRP 600 band) and mature LRP (LRP 85 band) in pial vessels after overexpression of shGFP and shSRF in 24-month-old Dutch/Iowa APP mice. Graphs show relative band density, compared with β-actin. (g) 2-photon in vivo longitudinal imaging of amyloid with methoxy-XO4 and Texas Red dextran angiography in the cortical lamina I in 16-month-old APPsw^± mice transduced locally in the pial vessels with either Ad.shGFP or Ad.shSRF. Scale bars, 100 µm. Graphs show XO4 relative signal intensity. (h) Aβ40 and Aβ42 focal brain levels in the areas containing pial vessels transduced with Ad.shGFP or Ad.shSRF in 16-month-old APPsw^± mice. Data represent mean ± s.e.m. of 5 animals per group (b–d, f–h).

Figure 6

MYOCD overexpression in pial arteries aggravates Aβ pathology in the Dutch/Iowa APP mice. (a) Aβ40 and Aβ42 levels in the pial vessels after transfer of Ad.GFP and Ad.MYOCD. Each point represents an individual value. (b) Aβ immunostaining in transduced pial vessels (upper panels) and focally in brain tissue (lower panels) after expression of a control vector (Ad.LacZ) or Ad.MYOCD. Scale bars, 50 µm. (c) Aβ load in transduced pial vessels and focally in brain after transfer of Ad.LacZ or Ad.MYOCD. (d) Amyloid load in pial vessels after transduction of Ad.LacZ or Ad.MYOCD. (e) The levels of SREBP2 long and short forms, immature LRP (LRP 600 band) and mature LRP (LRP 85 band) in pial vessels after overexpression of GFP and MYOCD. Graph shows relative band density, compared with β-actin. Data represent mean ± s.e.m. of 5 animals per group (c–e). MYOCD and GFP gene transfer was performed in 15-month-old Dutch/Iowa APP mice.

Figure 7

Hypoxia increases MYOCD and SRF expression in human cerebral VSMC and pial vessels in APPsw^± mice. (a) MYOCD luciferase activity in PAC1 VSMCs after transfection with a luciferase reporter, with or without the hypoxia response element (+HRE or −HRE) in the promoter region of human MYOCD gene. Cells were cultured for 48 h under normoxic or hypoxic (1% O₂) conditions. (b) MYOCD luciferase activity in PAC1 cells after transfection with a luciferase reporter with or without the HRE in the promoter region of human MYOCD gene and co-transfected with pcDNA vector or HIF-1α overexpression plasmid. Cells were cultured for 48 h under normoxic condition. (c) Real-time qPCR for MYOCD mRNA in control human cerebral VSMCs cultured for 48 h under normoxic or hypoxic (1% O₂₎ conditions. (d) MYOCD, SRF and LRP levels in control cerebral VSMCs within 48 h of normoxia or hypoxia. (e) Nuclear translocation of SREBP2 (green) and downregulation of LRP (red) in hypoxic human VSMCs within 48 h of hypoxia. Nuclear DAPI staining in white. Scale bars, 20 µm. (f) MYOCD, SRF, SREBP2 and LRP levels in isolated pial vessels from 5–6 month old APPsw^± mice subjected to hypoxia (10–8% oxygen) or normoxia (21% oxygen) for 2 weeks. (g) Real-time qPCR for MYOCD and LRP mRNAs in pial vessels isolated from APPsw^± mice treated as in f. (h) Aβ40 and Aβ42 levels in isolated pial vessels from APPsw^± mice subjected to hypoxia or normoxia, as in f. In, Data represent mean ± s.e.m. of 3 independent cultures per group (a–d) or mean ± s.e.m. of 5 animals per group (f–h).

Figure 8

SRF and MYOCD control blood flow and cerebral amyloid angiopathy (CAA). (a) Effects on cerebral blood flow (CBF). SRF and MYOCD overexpression in cerebral VSMC in AD stabilizes a VSMC hypercontractile phenotype through directed expression of the SRF-dependent genes which regulate Ca²⁺ homeostasis, for example, myosin light-chain kinase (MYLK), calsequestrin 1 (CASQ1), sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (ATP2A2), and of SRF-MYOCD-regulated smooth muscle (SM) contractile genes, for example, SM myosin heavy chain (MHC), SM α-actin, SM22α and SM calponin, resulting in arterial hypercontractility and reductions in cerebral blood flow (CBF). (b) Pathological effects on CAA. SRF and MYOCD promote an Aβ non-clearing VSMC phenotype through directed expression of SREBP2, which acts as a transcriptional suppressor of LRP, a major clearance receptor for Aβ. This pathway leads to the development of CAA and limits local clearance of Aβ from brain tissue resulting in focal Aβ brain accumulations.