Massive CA1/2 neuronal loss with intraneuronal and N-terminal truncated Abeta42 accumulation in a novel Alzheimer transgenic model

Abstract

Alzheimer's disease (AD) is characterized by a substantial degeneration of pyramidal neurons and the appearance of neuritic plaques and neurofibrillary tangles. Here we present a novel transgenic mouse model, APP(SL)PS1KI that closely mimics the development of AD-related neuropathological features including a significant hippocampal neuronal loss. This transgenic mouse model carries M233T/L235P knocked-in mutations in presenilin-1 and overexpresses mutated human beta-amyloid (Abeta) precursor protein. Abeta(x-42) is the major form of Abeta species present in this model with progressive development of a complex pattern of N-truncated variants and dimers, similar to those observed in AD brain. At 10 months of age, an extensive neuronal loss (>50%) is present in the CA1/2 hippocampal pyramidal cell layer that correlates with strong accumulation of intraneuronal Abeta and thioflavine-S-positive intracellular material but not with extracellular Abeta deposits. A strong reactive astrogliosis develops together with the neuronal loss. This loss is already detectable at 6 months of age and is PS1KI gene dosage-dependent. Thus, APP(SL)PS1KI mice further confirm the critical role of intraneuronal Abeta(42) in neuronal loss and provide an excellent tool to investigate therapeutic strategies designed to prevent AD neurodegeneration.

Figure 1-4263

Gene-targeting strategy for PS1KI mouse generation and effect of Ps1 mutations on PS1 expression and APP metabolism in the mouse brain. A: Schematic representation of the structure and restriction map of the wild-type mouse Ps1 gene (WT Ps1) around the exon (black box) 7 (top line) and the targeting vector used (immediately below line, vector) with the neo-cassette (gray box). Base pair changes were performed by directed mutagenesis to create M233T and L235P mutations (**) in the exon 7. The modified locus containing the point mutations in Ps1 gene is illustrated in the bottom line (Ps1^{M233T, L235T).} The position of a 230-bp DNA fragment used as a probe for ES cells and mice screening is indicated. Sizes of DNA fragments generated by enzymatic digestion are also indicated. Restriction enzymes: E, EcoRI; X, XbaI; B, BamHII; H, HindIII. B: Southern blot hybridization to discriminate among the WT (single 9.2-kb band), homozygous (Ho, single 7.4-kb band), and heterozygous (He, both bands) mice. C: Western blot of 20-kd C-terminal PS1 protein showing normal levels in mutant mice. D: Analysis of APP holoprotein and APP-CTFs in APP^SLPS1KI mice by Western blot on 6-month-old mouse brain. Detection was performed with APP-CTF C17 antiserum. The three bands detected for the full-length APP correspond to the murine APP₆₉₅ and the immature and mature human APP₇₅₁ isoforms that are not modified. In middle panel, arrows indicate the α-, β-, β′- and αP-, β′P-, βP-APP-CTFs that are increased in the PS1KI. E: Quantification of the Western blot in D was done after signal normalization with tubulin and the value means ± SEM (n = 4 mice for each group) obtained for the APP^SL (in white) and APP^SLPS1KI mice (in black) are represented as histograms. Statistical significance was analyzed with the Student’s t-test.

Figure 2-4263

Accelerated Aβ accumulation in APP^SLPS1KI mouse brain. Whole brain (minus cerebellum) Aβ₄₂ levels (A) or Aβ₄₂/total Aβ ratio (B) were quantified by electrochemiluminescence (average ± SEM) in APP^SL and APP^SLPS1KI homozygous (Ho) or heterozygous (He) for Ps1^M233T/L235P. Asterisks indicate the significance of the difference whether none, one, or two copies of the mutated PS1 allele are present (*, P < 0.05; ***, P < 0.005; Mann-Whitney test, n = 5 to 7). Note: 6- and 10-month-old hemizygous APP^SLPS1KI animals were not investigated.

Figure 3-4263

Proteomic analysis of Aβ₄₂ species in the APP^SL and APP^SLPS1KI transgenic mice. Two-dimensional Western blots of Aβ₄₂ species were performed from acid formic-solubilized brain homogenates from APP^SL (top) and APP^SLPS1KI (remaining panels) transgenic mice at the indicated ages. Two APP^SL, one APP^SLPS1KI at 2.5 and 4 months of age, and two at 6 and 10 months of age were analyzed. According to the characterization of Aβ₄₂ species performed in the human brain of patients with AD and confirmed by mass spectrometry analysis (see supplemental Figure S1), the identity of human Aβ₄₂ species (hAβ) is summarized at the bottom. Isoelectric points were determined using internal standards. Spot at pI 4.3 putatively corresponds to the endogenous mouse Aβ₄₂ (mAb). Arrowheads point (down leftwards) to the weak staining of spots at pI 5.8 and 6.3. Monomeric species of Aβ₄₂ are visualized at 4 kd, whereas dimeric species are resolved at 8 kd.

Figure 4-4263

Accelerated Aβ peptide deposition in APP^SLPS1KI mouse brain. Representative photomicrographs of Aβ immunostaining on sagittal brain sections taken from 6 (A)- and 10 (B)-month-old APP^SL (left) and APP^SLPS1KI mice (right) (4G8 antibody on 25-μm-thick cryostat sections). Aβ immunostaining in the entire section and in the cortical (Cx), subicular, hippocampal (Hip), and thalamic (Tha) subareas illustrate the acceleration of extracellular Aβ deposition and its widespread distribution in brain parenchyma of young (ie, 6 months of age) APP^SLPS1KI mice (higher magnification in bottom panels). Note the difference in the size and number of Aβ deposits in double- versus single-transgenic mice at 10 months of age with overall similar amyloid load (B). Scale bars: 500 μm, top panel (A); 20 μm, middle panel (A); 20 μm, bottom panel (B).

Figure 5-4263

APP^SLPS1KI transgenic mice develop massive neuronal loss in the hippocampus. A: Representative photomicrographs of cresyl violet-stained sagittal brain sections of 10-month-old PS1KI, APP^SL, and two APP^SLPS1KI mice at low magnification. Note the deeply reduced thickness of the CA1/2 pyramidal cell layer indicated between arrows in the APP^SLPS1KI brain (bottom). B: Higher magnification views of the cresyl violet-stained CA1/2 subfield of a representative APP^SL (top) and APP^SLPS1KI (bottom) mouse are shown. C: APP immunostaining of the hippocampal formation in 2 (top)- and 10 (bottom)-month-old APP^SLPS1KI mice. APP staining reveals a very strong APP expression in CA1/2 subfield with a faint labeling in CA3 where no neuronal loss was detected. Note again the reduced thickness of CA1/2 subfield with the APP neuronal immunostaining. Scale bars: 150 μm (A); 50 μm (B); 100 μm (C).

Figure 6-4263

Stereological examination of total numbers of neurons within CA1/2 hippocampal cell layer. APP^SL, PS1KI, and APP^SLPS1KI transgenic were analyzed by high precision design-based stereological assessment (see Material and Methods). M2, 2-month-old animals (PS1KI, four males; APP^SL, two males and one female; APP^SLPS1KI, two males and two females). M10, 10-month-old animals (PS1KI, four males; APP^SL, three males and one female; APP^SLPS1KI, two males and two females). Differences between the groups were tested with analysis of variance followed by posthoc Bonferoni’s multiple comparison test for pair-wise comparisons. Statistical significance was established at P < 0.05. **, P < 0.01; ***, P < 0.001.

Figure 7-4263

Relationship between neuronal loss and intraneuronal Aβ peptide accumulation in the CA1/2 subfield. Representative photomicrographs of Aβ immunostaining (4G8 antibody) on sagittal brain sections of 10-month-old APP^SL (left) and APP^SLPS1KI (right) mice. Two different mice per group are illustrated. A: Within the CA1/2 hippocampal subarea Aβ deposits are mainly present on either side of rather than within the pyramidal cell layer in both APP^SL and APP^SLPS1KI. Both the intensity and frequency of granular Aβ immunostaining within the remaining CA1/2 pyramidal cells were increased in APP^SLPS1KI mice compared to APP^SL mice. Arrows indicate the localization of the CA1/2 neuronal cell layer. B: Astrocytic staining against GFAP (blue) and Aβ staining (antiserum 692, brown) in CA1 of a 6-month-old APP^SL (left) and APP^SLPS1KI (right) 4-μm brain sections. Note the astrogliosis in the pyramidal cell layer in APP^SLPS1KI mice. Scale bars: 100 μm (A); 50 μm (B).

Figure 8-4263

Intraneuronal Aβ peptide accumulation in CA1/2. Representative photomicrographs of Aβ immunoreactivity in 2 (A)-, 6 (B)-, and 10 (C)-month-old APP^SLPS1KI mice (antibody 692). Whereas young mice show a punctate staining pattern, larger and compact granules were evident in aged mice. B: Note the reduced thickness of the CA1/2 pyramidal layer already at 6 months. D and E: Thioflavine-S staining reveals aggregated intracellular material in 2 (D)- and 10 (E)-month-old APP^SLPS1KI mice. F: High-power photomicrograph demonstrating abundant intraneuronal Aβ in the CA1 subfield of a 2-month-old APP^SLPS1KI mouse. G: Double labeling of Aβ (blue) and APP (brown) in a 2-month-old APP^SLPS1KI mouse, showing abundant intraneuronal Aβ in cortical APP-expressing neurons. H and I: High-power photomicrographs of the subiculum of a 2-month-old APP^SLPS1KI mouse showing abundant intraneuronal Aβ₄₀ (H) as well as Aβ₄₂ (I). The thickness of the sections was 4 μm in all pictures. Counter staining was performed with hematoxylin (A-C, F, H, I) and 4′,6-diamidine-2′-phenylindole dihydrochloride (D, E). Scale bars: 50 μm (A–E); 2 μm (F–I). Original magnifications: ×400 (D, E); ×1000 (insets in D, E).