Article Figures & Data
Figures
- Figure 1.
Aβ-induced cytotoxicity in organotypic hippocampal cultures. A, A hematoxylin stain of a hippocampal slice after 2 weeks in vitro (25× magnification). The inset shows a CA neuronal field at 1000× magnification. B, Laser scanning confocal analysis was performed on live hippocampal slices stained with calcein AM (green) or EthD-1 (red). Treatment with 25 μm Aβ leads to many EthD-1-positive nuclei in a CA neuronal field. Scale bar, 50 μm. C, Confocal visualization reveals costaining of NeuN (green) and EthD-1 (red) in a CA neuronal field of a slice treated with 50 μm Aβ. Few EthD-1-positive cells are found in slices treated with 50 μm reverse Aβ. Scale bar, 10 μm. D, Using confocal microscopy, no TUNEL-positive cells were observed in 50 μm reverse Aβ-treated slices. However, many cells within the hippocampal neuronal fields of 50 μm Aβ-treated slices are TUNEL positive. This TUNEL staining is prevented by pretreatment of 1 nm sAPPα. Scale bar, 10 μm. E, Counts of Nissl-stained neurons within the neuronal fields of hippocampal slices reveal a dramatic increase in the percentage of cells with condensed chromatin in Aβ-treated slices. The increase in cells with chromatin condensation is prevented by pretreatment with 1 nm sAPPα. Data are presented as mean ± SEM of three slices per treatment. *p < 0.05 compared with 50 μm reverse Aβ treatment; #p < 0.05 compared with 25 μm Aβ treatment.
- Figure 2.
Aβ treatment induces phosphorylation of endogenous tau in mouse hippocampal neurons. A, Cells within the hippocampal neuronal fields stain positively with the AT8 antibody (green) and ethidium homodimer (red) in 50 μm Aβ-treated slices. The tau phosphorylation and plasma membrane permeability occurs infrequently in reverse Aβ-treated slices and is prevented by pretreatment with 1 nm sAPPα. The DNA-binding dye ToPro3 (blue) reveals the neuronal field architecture. Scale bar, 20 μm. B, Several neurons in 50 μm Aβ-treated slices stain positively with an antibody recognizing tau phosphorylated at Thr231 (red). The arrow points to one such neuron with NeuN staining (green) that has become diffuse and less intense. Another phospho-tau-positive neuron demonstrates diffuse NeuN staining and nuclear pyknosis (arrowhead). No neurons stain positively for phospho-tau in 50 μm reverse Aβ-treated slices, and tau phosphorylation is prevented by pretreatment with 1 nm sAPPα. Scale bar, 20 μm. C, Many neurons in Aβ-treated slices demonstrate AT8 staining in their cell bodies and in beaded processes (arrows and inset). The inset shows a higher-magnification view of one AT8 cell with beaded processes. Similar AT8 staining is observed in the CA hippocampal neurons of human patients with AD. Scale bar, 20 μm; inset, 5 μm. D, A higher magnification of the neurons containing Aβ-induced phospho-tau shown in B. Similarly, phospho-tau(Thr231) is shown in red, NeuN is shown in green, and the nuclei are shown in blue. Yellow indicates regions of phospho-tau and NeuN costaining. Scale bar, 10 μm.
- Figure 3.
Increased levels of sAPPα and the neuroprotective proteins TTR, IGF-2, and phospho-BAD in 12-month-old APPSw mice. A, Immunohistochemistry with laser scanning confocal analysis demonstrates little to no TTR (red) in nontransgenic control hippocampus. However, APPSw mice possess dramatically increased levels of TTR in and around the NeuN (green)-positive hippocampal neurons of CA1. B, Little to no IGF-2 (red) is present in nontransgenic control hippocampus. APPSw mice have increased levels of IGF-2 in the extracellular space of the hippocampus and around the NeuN (green)-positive hippocampal neurons of CA1, including several neuronal processes (arrows). C, A small amount of phosphorylated BAD (red) is found within the CA1 neurons (green) of control mice. However, levels of phospho-BAD are dramatically increased within the neurons of the APPSw mice. D, Total BAD levels (red) are unchanged between nontransgenic and APPSw mice. Scale bar: (in D) A-D, 20 μm. E, TTR (green) costains with an Aβ plaque (arrowhead; red) and with intracellular Aβ in a neighboring neuron (arrow) in hippocampal sections from postmortem AD patients. Yellow indicates regions of costaining. F, A hippocampal neuron from an AD patient has accumulated intracellular Aβ that costains with TTR (arrow). Vertical sections through the plaque (E) and hippocampal neuron (F) are shown in the top panels. Scale bar: (in F) E, F, 10 μm.
- Figure 4.
sAPPα induces the expression of TTR and IGF-2 ex vivo and likely in vivo. A, Immunoblotting with the 6E10 antibody demonstrates dramatically increased sAPPα in 12-month-old APPSw mice. Below is the densitometric analysis showing the relative intensity levels of sAPPα. Values are presented as mean ± SEM (n = 4). *p < 0.05 compared with nontransgenic mice. B, SOM clustering suggests sAPPα-driven genes. The expression levels of genes and ESTs that were significantly increased (rank, ≥9) by 1 nm sAPPα in hippocampal slice cultures were clustered to identify patterns of expression. Expression patterns were examined in vehicle-treated (n = 3) and sAPPα-treated (n = 3) hippocampal slices, 6-month-old nontransgenic control (n = 3) and APPSw (n = 3) mice, and 12-month-old nontransgenic control (n = 2) and APPSw (n = 2) mice. Plotted on the ordinate is the average expression level for each cluster of genes (circular data points). The outer lines indicate the SD for each cluster. Cluster 4 (highlighted in gray) lists genes upregulated by sAPPα treatment as well as in 6- and 12-month-old APPSw mice.
- Figure 5.
TTR and IGF-2 are necessary for sAPPα-induced protection against Aβ. The percentage of death for each treatment was quantified in neuronal fields of live hippocampal slices by counting the number of membrane-permeable, EthD-1-positive cells as well as the number of live cells that stained positively with calcein AM. Data are expressed as mean ± SEM (n = 3-5 slices per treatment). A, Aβ results in a significant increase in the percentage of death, whereas sAPPα, TTR, or IGF-2 protect against the Aβ-induced toxicity. An antibody directed against the C-terminal region of sAPPα (6E10) prevents protection by sAPPα. Finally, a fragment in the C terminus of sAPPα (SEVKMDAEFR) mimics the protective effects of sAPPα. *p < 0.05 compared with 50 μm reverse Aβ; #p < 0.01 compared with 50 μm Aβ; **p < 0.05 compared with 1 nm sAPPα plus mouse IgG plus 25 μm Aβ; ##p < 0.05 compared with 25 μm Aβ. B, Antibodies against TTR (anti-TTR) and IGF-2 (anti-IGF-2) prevent the protective effect of 1 nm sAPPα against 25 μm Aβ-induced cell death. *p < 0.01 compared with the corresponding vehicle-treated slices; #p < 0.01 compared with 1 nm sAPPα plus goat IgG plus 25 μm Aβ. C, siRNA knock-downs of TTR, IGF-2, and IGF-1R block the protective effect of 1 nm sAPPα against 25 μm Aβ-induced cell death. A scrambled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA was transfected as a control. *p < 0.05 compared with the corresponding vehicle-treated slices; #p < 0.05 compared with 1 nm sAPPα plus scrambled GAPDH plus 25 μm Aβ.
- Figure 6.
Chronic infusion of an anti-TTR antibody into the hippocampus of APPSw mice results in antibody deposition and Aβ accumulation within the infused hippocampus. Laser scanning confocal analysis was performed on hippocampal sections double immunolabeled for goat IgG or TTR (anti-goat IgG; green) and Aβ (4G8; red). Yellow indicates regions of costaining. The DNA-binding dye ToPro3 (blue) was used to stain nuclei. A, An anti-goat IgG antibody reveals that control goat IgG infused into the hippocampus is cleared after the 2 week infusion. Some Aβ staining occurs within the extracellular space and around the CA1 neurons in goat IgG-infused hippocampi and in the noninfused hippocampi of the anti-TTR-infused mice. However, mice infused with the anti-TTR antibody demonstrated a dramatic deposition of the anti-TTR antibody within the extracellular space of the infused, but not the noninfused, hippocampi. In addition, infusion of the anti-TTR antibody increased the amount of Aβ around and within the CA1 neurons near the infusion site. Scale bar, 20 μm. B, The anti-TTR antibody colabeled Aβ plaques in the infused hippocampus. Scale bar, 20 μm.
- Figure 7.
Chronic infusion of an anti-TTR antibody into the hippocampus of APPSw mice results in tau phosphorylation, apoptosis, and neuronal loss within the infused hippocampus. A, After a 2 week infusion of goat IgG, no phosphorylated tau [AT8, green; or phospho-tau(Thr231), red] is observed within the hippocampal neuronal fields. The CA1 region is shown, and the DNA-binding dye ToPro3 (blue) was used to stain nuclei. In contrast, a 2 week infusion of an antibody to TTR resulted in AT8 staining within occasional CA1 neurons. Many of these neurons possess pyknotic nuclei (ToPro3, blue; arrows). The inset shows a higher magnification within the CA1 neuronal field of a phosphorylated tau-stained neuron with a condensed nucleus (arrowhead) and beaded processes indicative of degeneration. Many neurons within the CA1 field also stain intensely for tau phosphorylated at Thr231 (phospho-tau, red). No AT8 staining and little phospho-tau(Thr231) is observed in the noninfused hippocampus. Scale bar, 20 μm; inset, 5 μm. B, CA1 neurons within the anti-TTR-infused hippocampus stain intensely for phospho-tau(Thr231) (red) and the neuronal marker NeuN (green). Scale bar, 10 μm. C, Unbiased stereology was used to determine the total number of cells with pyknotic nuclei within the infused CA1 pyramidal neuronal fields of goat IgG- (n = 4) and anti-TTR- (n = 4) infused mice. *p < 0.01, two-tailed Wilcoxon signed rank test. D, The total number of CA1 neurons was significantly reduced in anti-TTR-(n = 4) compared with goat IgG-(n = 4) infused mice. *p < 0.05, two-tailed Wilcoxon signed rank test.
Tables
- Table 1.
Differentially expressed genes in sAPP α-treated organotypic hippocampal slice cultures
Classification Gene name FC Rank Amyloid sequestration Transthyretin 8.94 ± 4.49 12 Apoptosis Apoptosis inhibitor 6 2.09 ± 1.12 10 Calcium binding Calmodulin-like 4 1.75 ± 0.42 12 Cell cycle Cyclin D2 4.22 ± 0.88 11 Cyclin-dependent kinase 4 1.26 ± 0.05 10 Detoxification Glutathione S-transferase, α4 3.52 ± 1.11 14 Glutathione S-transferase, μ5 1.38 ± 0.40 9 Peroxiredoxin 2 1.19 ± 0.31 10 Extracellular matrix and tissue remodeling Elastin 1.66 ± 1.22 9 Chondroitin sulfate proteoglycan 2 1.62 ± 0.21 11 Glycolysis Phosphofructokinase, platelet 1.32 ± 0.06 12 Galactokinase 1 1.28 ± 0.30 10 Growth Insulin-like growth factor 2 2.58 ± 0.42 14 Insulin-like growth factor binding protein 2 1.53 ± 0.08 14 Immune related Lymphocyte antigen 6 complex, locus A 1.90 ± 0.97 12 Intercellular adhesion molecule 2 1.71 ± 0.73 9 Ion channel FXYD domain-containing ion transport regulator 1 1.20 ± 0.30 10 Peroxisome (fatty acid transport) ATP-binding cassette, subfamily D, member 3 1.35 ± 0.12 9 Prostaglandin synthesis Prostaglandin D2 synthase (brain) 1.97 ± 0.53 12 Protein catabolism Proteasome (prosome, macropain) 28 subunit, α 2.67 ± 0.73 10 Praja 1, RING-H2 motif containing 1.20 ± 0.03 9 Receptor Cytokine receptor-like factor 1 1.54 ± 0.45 9 Receptor (calcitonin) activity modifying protein 1 1.27 ± 0.06 9 Retionic acid binding Cellular retinoic acid binding protein II 2.37 ± 0.29 16 Retinol binding protein 1, cellular 1.69 ± 0.15 16 Miscellaneous Eyes absent 4 homolog (Drosophila) 19.26 ± 8.88 11 H19 fetal liver mRNA 1.95 ± 0.23 17 Ectonucleotide pyrophosphatase/phosphodiesterase 2 1.88 ± 0.82 11 Phosphatidic acid phosphatase 2a 1.83 ± 0.78 10 Heterogeneous nuclear ribonucleoproteins methyltransferase-like 2 (Saccharomyces cerevisiae) 1.34 ± 0.35 10 Topoisomerase (DNA) II α 1.34 ± 0.96 10 Neuronal protein 15.6 1.33 ± 0.05 10 Ribosomal protein L8 1.32 ± 0.08 11 Ena-vasodilator stimulated phosphoprotein 1.28 ± 0.35 10 Thymus cell antigen 1, θ 1.27 ± 0.32 11 ADP-ribosylation-like factor 6 interacting protein 5 1.25 ± 0.04 14 STIP1 homology and U-box containing protein 1 1.19 ± 0.05 9 ESTs AW060684 4.54 ± 1.78 9 AW121164 2.54 ± 0.86 9 AW121336 2.50 ± 0.34 9 AW060956 1.35 ± 0.11 9 AW120814 1.32 ± 0.11 9 AW124069 1.26 ± 0.34 9 AW048976 1.24 ± 0.08 10 AI226264 1.21 ± 0.03 10 -
Rank is based on the p value for each comparison (3 × 3). Rank values ranging from 9 to 18 indicate significantly increased gene expression. FC is expressed as mean ± SEM.
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