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First published online 3 August 2005
doi: 10.1242/dev.01946

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Selective expression of presenilin 1 in neural progenitor cells rescues the cerebral hemorrhages and cortical lamination defects in presenilin 1-null mutant mice

¹ Department of Psychiatry, Mount Sinai School of Medicine, New York, NY 10029, USA
² Psychiatry Service, Bronx VA Medical Center, Bronx, NY 10468, USA
³ Department of Neuroscience, Mount Sinai School of Medicine, New York, NY 10029, USA
⁴ Department of Geriatrics and Adult Development, Mount Sinai School of Medicine, New York, NY 10029, USA

View larger version (49K): [in a new window]   Fig. 1. Expression of human PSEN1 protein in transgenic mice under the control of the nestin intron 2 enhancer. Western blotting was performed on brain extracts from E12.5 nestin-Psen1 transgenic (Tg/+) embryos from line 9 and line 14, as well as non-transgenic controls (+/+). Blots were probed with NT.1 (upper panel) an antibody that recognizes the 30 kDa N-terminal fragment (NTF) of the transgenic human (Hu) PSEN1 but not the endogenous mouse (Mu) Psen1 (Wen et al., 2002b). The two wild-type (+/+) embryos are immunonegative. The lower panel was probed with a rabbit polyclonal antibody 222 that recognizes both the mouse and human NTFs (Wen et al., 2002b). Antibody 222 was raised against amino acids 2-12 of the human NTF, a region that contains one mismatch between the mouse and human proteins (Wen et al., 2002b). Thus, the apparent elevation in total Psen1 (Mu + Hu) in the transgenic (Tg/+) embryos may in part reflect a higher affinity of this antibody for the human protein.

View larger version (118K): [in a new window]   Fig. 2. Expression pattern of an NSE-Psen1 transgene. (A,B) Horizontal sections through the lateral telencephalon of an E16.5 NSE-Psen1 transgenic (A) or non-transgenic littermate control (B) embryos hybridized with a human-specific PSEN1 probe. In the transgenic embryo there is prominent hybridization in the intermediate zone (IZ), cortical plate (CP) and marginal zone (MZ), and an absence of signal in the ventricular zone (VZ). Scale bar: 50 µm.

View larger version (38K): [in a new window]   Fig. 3. A nestin-Psen1 transgene rescues the cerebral hemorrhages but not the caudal defects in the Psen1–/– embryo. (A-C) E18.5 wild-type (A), Psen1–/– (B) embryos and an embryo with the nestin-Psen1 transgene on the Psen1–/– background (C). The Psen1–/– embryo has an abnormal tail region (arrow) and a cerebral hemorrhage (arrowhead). By contrast, the embryo with the nestin-Psen1 transgene on the Psen1–/– background still exhibits the caudal defects (arrow) but no cerebral hemorrhages.

View larger version (96K): [in a new window]   Fig. 4. Normal appearance of the developing cortical plate in E12.5 embryos with or without Psen1. Cresyl Violet-stained horizontal sections from E12.5 wild-type (A,E) and Psen1–/– (B,F) embryos, as well embryos with the nestin-Psen1 transgene on the Psen1+/+ (C,G) and Psen1–/– backgrounds (D,H). (E-H) Higher power views through the lateral wall of the telencephalon. The ventricular zone (VZ) and preplate (PP) are indicated. No differences were apparent between any of the genotypes. Scale bar: 200 µm for A-D; 25 µm for E-H.

View larger version (117K): [in a new window]   Fig. 5. Rescue of cerebral hemorrhages and cortical lamination defects in Psen1–/– embryos with a nestin-Psen1 transgene. Cresyl Violet-stained coronal sections of E18.5 embryos from wild-type (A,F,K), Psen1+/– (B,G,L), Psen1–/– (C,H,M), nestin-Psen1 transgene on Psen1–/– background (D,I,N) and nestin-Psen1 transgene on Psen1+/+ background (E,J,O). (F-O) Higher power views through the lateral telencephalon. In the E18.5 embryos, the wild-type embryo shows a clear lamination pattern with a recognizable ventricular zone (VZ), intermediate zone (IZ), subplate (SP), cortical plate (CP) and marginal zone (MZ). The Psen1–/– animal has multiple areas of hemorrhage (one indicated by arrow in M) and lacks distinct cortical layers. A cortical ectopia in the Psen1–/– embryo is indicated by an arrowhead (M). By contrast, the embryo with the nestin-Psen1 transgene on the Psen1–/– background, as well as the Psen1+/– and the embryo with the nestin-Psen1 transgene on the Psen1+/+ background, are normal in appearance. Scale bars: 500 µm in A-E; 100 µm in F-J; 50 µm in K-O.

View larger version (177K): [in a new window]   Fig. 6. Failure of an NSE-Psen1 transgene to rescue the brain pathology in Psen1–/– mice. Shown are Cresyl Violet-stained horizontal sections of E18.5 embryos that are wild type (A,D,G), Psen1–/– (B,E,H) or have the NSE-Psen1 transgene on Psen1–/– background (C,F,I). (D-I) Higher power views of the lateral cerebral wall. The embryo with the NSE-Psen1 transgene on the Psen1–/– background exhibits multiple areas of hemorrhage (one indicated by arrow), as well as disrupted cortical lamination in a pattern that is indistinguishable from that of the Psen1–/– embryo. Cortical layers (MZ, CP, SP, IZ) are indicated as in Fig. 5. Scale bar: 500 µm in A-C; 100 µm in D-F; 50 µm in G-I.

View larger version (33K): [in a new window]   Fig. 7. Rescue of cell migration defects in Psen1–/– embryos with a nestin-Psen1 transgene. Pregnant females were given one dose of BrdU at E12.5 and the embryos were collected at E18.5 and immunostained with anti-BrdU antibodies. Shown are BrdU-immunostained sections through the lateral cerebral wall of E18.5 embryos from wild-type (A), Psen1+/– (B), Psen1–/– (C), nestin-Psen1 transgene on Psen1–/– background (D) and nestin-Psen1 transgene on Psen1+/+ background (E). BrdU labeling is dispersed in the Psen1–/– embryo and normalized by the nestin-Psen1 transgene. Cortical layers (MZ, CP, SP, IZ, VZ) are indicated as in Fig. 5. Scale bar: 100 µm.

View larger version (139K): [in a new window]   Fig. 8. Lack of detectable transgene expression in cephalic mesenchyme by in situ hybridization with a human-specific Psen1 cRNA. Shown are horizontal sections through the telencephalon of an E12.5 nestin-Psen1 transgenic embryo from line 9 (A,B,D) or a non-transgenic littermate control (C,E) embryo hybridized with a human specific Psen1 probe. (A) A section through the anterior telencephalon. The lateral ventricle (LV) and interhemispheric fissure (IH) are indicated. There is prominent hybridization in the brain parenchyma. Hybridization in the ventricular zone from a transgenic (Tg) and non-transgenic (+/+) embryo is shown in B and C. The transgenic embryo is strongly hybridized. (D) Hybridization at the juncture between the brain parenchyma and the overlying cephalic mesenchyme in the interhemispheric fissure from a transgenic embryo. (E) A comparable region from a non-transgenic embryo (+/+) is shown to indicate the level of background staining. An arrow indicates the border between the brain parenchyma (showing hybridization product in D but not in E) and the cephalic mesenchyme. Scale bar: 20 µm for A; 50 µm for B-E.

View larger version (90K): [in a new window]   Fig. 9. Lack of the nestin intron 2 enhancer activity in vascular progenitor cells or endothelial cells. NesCrenls line 27 was crossed with a Cre/loxP reporter line cActXstopXEGFP44. The hippocampal CA1 region of an adult double transgenic mouse is shown in A-C. Spontaneous EGFP fluorescence (A) was imaged and combined with immunohistochemistry for aquaporin 4 (B). EGFP is prominently expressed in the pyramidal cells but not in the aquaporin 4 outlined vessels (merged image is shown in C). (D-F) Combined immunohistochemical staining is shown for EGFP (D), and von Willebrand factor (E) on an adult brain from a NesCrenls line 27 mouse crossed to the Z/EG reporter line (merged image is shown in F). Immunostaining in D-F was performed on sections cut from tissue perfusion fixed with paraformaldehyde. A large penetrating vessel is visible at the cortical surface. There is no EGFP labeling of the von Willebrand factor-stained vessel. Scale bar: 50 µm for A-C; 10 µm for D-F.

View larger version (108K): [in a new window]   Fig. 10. Rescue of brain vascular abnormalities in embryos with the nestin-Psen1 transgene on the Psen1–/– background. (A-C) Staining with isolectin B4 was performed on paraffin-embedded E12.5 embryos from wild-type (A) and Psen1–/– (B) embryos, and embryos with the nestin-Psen1 transgene on the Psen1–/– background (C). Sections through the lateral telencephalon are shown. Vessels in the Psen1–/– embryo are enlarged. (D-L) Isolectin staining (E,H,K) was combined with nestin immunostaining (D,G,J) on paraffin-embedded E18.5 embryos from wild-type (D-F) and Psen1–/– (G-I) embryos, and embryos with the nestin-Psen1 transgene on the Psen1–/– background (J-L). Merged images are shown in F,I,L. Regions along the cortical surface of the lateral telencephalon are illustrated. The lectin-stained vessels in the Psen1–/– brain are distorted and lobular appearance, and this pattern is normalized by the nestin-Psen1 transgene. The pattern of nestin staining is altered in the Psen1–/– embryo. Scale bar: 100 µm for A-C; 50 µm for D-L.