Quaternary Structure Defines a Large Class of Amyloid-β Oligomers Neutralized by Sequestration

SummaryThe accumulation of amyloid-β (Aβ) as amyloid fibrils and toxic oligomers is an important step in the development of Alzheimer’s disease (AD). However, there are numerous potentially toxic oligomers and little is known about their neurological effects when generated in the living brain. Here we show that Aβ oligomers can be assigned to one of at least two classes (type 1 and type 2) based on their temporal, spatial, and structural relationships to amyloid fibrils. The type 2 oligomers are related to amyloid fibrils and represent the majority of oligomers generated in vivo, but they remain confined to the vicinity of amyloid plaques and do not impair cognition at levels relevant to AD. Type 1 oligomers are unrelated to amyloid fibrils and may have greater potential to cause global neural dysfunction in AD because they are dispersed. These results refine our understanding of the pathogenicity of Aβ oligomers in vivo

Three-Dimensional Cell Entrapment as a Function of the Weight Percent of Peptide-Amphiphile Hydrogels

The design of scaffolds which mimic the stiffness, nanofiber structure, and biochemistry of the native extracellular matrix (ECM) has been a major objective for the tissue engineering field. Furthermore, mimicking the innate three-dimensional (3D) environment of the ECM has been shown to significantly altered cellular response compared to that of traditional two-dimensional (2D) culture. We report the development of a self-assembling, fibronectin-mimetic, peptide-amphiphile nanofiber scaffold for 3D cell culture. To form such a scaffold, 5 mol % of a bioactive PR_g fibronectin-mimetic peptide-amphiphile was mixed with 95 mol % of a diluent peptide-amphiphile (E2) whose purpose was to neutralize electrostatic interactions, increase the gelation kinetics, and promote cell survival. Atomic force microscopy verified the fibrilar structure of the gels, and the mechanical properties were characterized for various weight percent (wt %) formulations of the 5 mol % PR_g–95 mol % E2 peptide-amphiphile mixture. The 0.5 wt % formulations had an elastic modulus of 429.0 ± 21.3 Pa whereas the 1.0 wt % peptide-amphiphile hydrogels had an elastic modulus of 808.6 ± 38.1 Pa. The presence of entrapped cells in the gels decreased the elastic modulus, and the decrease was a function of cell loading. Although both formulations supported cell proliferation, the 0.5 wt % gels supported significantly greater NIH3T3/GFP fibroblast cell proliferation throughout the gels than the 1.0 wt % gels. However, compared to the 0.5 wt % formulations, the 1.0 wt % hydrogels promoted greater increases in mRNA expression and the production of fibronectin and type IV collagen ECM proteins. This study suggests that this fibronectin-mimetic scaffold holds great promise in the advancement of 3D culture applications and cell therapies

Characterization of a Novel Mouse Model of Alzheimer’s Disease—Amyloid Pathology and Unique β-Amyloid Oligomer Profile

<div><p>Amyloid plaques composed of β-amyloid (Aβ) protein are a pathological hallmark of Alzheimer’s disease. We here report the generation and characterization of a novel transgenic mouse model of Aβ toxicity. The rTg9191 mice harbor a transgene encoding the 695 amino-acid isoform of human amyloid precursor protein (APP) with the <i>Swedish</i> and <i>London</i> mutations (APP_NLI) linked to familial Alzheimer’s disease, under the control of a tetracycline-response element, as well as a transgene encoding the tetracycline transactivator, under the control of the promoter for calcium-calmodulin kinase IIα. In these mice, APP_NLI is expressed at a level four-fold that of endogenous mouse APP and its expression is restricted to forebrain regions. Transgene expression was suppressed by 87% after two months of doxycycline administration. Histologically, we showed that (1) Aβ plaques emerged in cerebral cortex and hippocampus as early as 8 and 10.5-12.5 months of age, respectively; (2) plaque deposition progressed in an age-dependent manner, occupying up to 19% of cortex at ~25 months of age; and (3) neuropathology—such as abnormal neuronal architecture, tau hyperphosphorylation and misfolding, and neuroinflammation—was observed in the vicinity of neuritic plaques. Biochemically, we determined total Aβ production at varied ages of mice, and we showed that mice produced primarily fibrillar Aβ assemblies recognized by conformation-selective OC antibodies, but few non-fibrillar oligomers (e.g., Aβ*56) detectable by A11 antibodies. Finally, we showed that expression of the tetracycline transactivator resulted in reduced brain weight and smaller dentate-gyrus size. Collectively, these data indicate that rTg9191 mice may serve as a model for studying the neurological effects of the fibrillar Aβ assemblies <i>in situ</i>.</p></div

The APP_NLI responder transgene construct.

<p>The 695 amino acid-long amyloid precursor protein cDNA harboring the <i>Swedish</i> mutation (APP_NL695) was inserted into the <i>Xho</i>I site of MoPrP.Xho fragment, which was further excised at two <i>Xba</i>I sites. The resulting fragment of prnp.APP_NL was cloned into the unique <i>Xba</i>I site in the inducible expression vector pTRE. The <i>London</i> mutation (V717I) was further introduced into the pTRE.prnp.APP_NL plasmid using site-directed mutagenesis.</p

TTA expression results in reduced forebrain weight and dentate-gyrus size.

<p>(A) Weights of rTg9191 mice (APP/TTA), littermates harboring only the activator gene (TTA), only the responder gene (APP), and neither gene (non-Tg). There were no genotype-related differences in body weight in young and mid-aged mice. Aged rTg9191 mice, however, had lower body weights compared to their littermates. (B) rTg9191 mice and TTA littermates have lower forebrain weights than their APP and non-Tg littermates at all ages studied. The numbers of mice examined are shown for each genotype. # p < 0.05, * p < 0.01, ** p < 0.001, *** p < 0.0001, two-way ANOVA followed by Fisher’s <i>post hoc</i> analysis. The percentage of female mice in the genotype of APP/TTA, TTA, APP, and non-Tg is 50%, 38%, 67%, and 55%, respectively for the 2–6 month-old; 51%, 35%, 48%, and 50%, respectively for the 13–17 month-old; 46%, 61%, 47%, and 51%, respectively for the 24–27 month-old. Chi square/Fisher exact tests showed no significant difference in gender distribution between genotypes. (C-F) Representative photomicrographs showing hematoxylin and eosin staining of the hippocampal regions of 16.5-month-old rTg9191 mice and their age-matched littermates. Sections at ~1.20 mm lateral from the midline were used. The sizes of dentate gyri of rTg9191 (F) and TTA (E) mice are decreased compared to those of APP (D) and non-Tg (C) littermates. Scale bar: 200 μm, applies to C-F. Representative photomicrographs show hippocampus hematoxylin and eosin staining of female mice, and similar results were observed in male mice.</p

Regional expression pattern of the APP transgene in rTg9191 mice.

<p>The regional pattern of APP_NLI expression in four distinct anatomical structures (cerebral cortex (Ctx.), hippocampus (Hpp.), olfactory bulb (Olf.), and cerebellum (Cbm.)) of brain was analyzed using mouse monoclonal antibody LN27, which specifically recognizes human APP, and 6E10. The APP transgene was expressed in cerebral cortex and hippocampus with a minor portion in olfactory bulb; however, no expression was observed in cerebellum. No immunoreactivity using these human-specific antibodies was seen in non-transgenic littermates (neg). Alpha-tubulin served as the loading control. Representative blots show the APP_NLI expression pattern of female mice, and similar results were found in male mice.</p

Plaque-associated tau pathology in rTg9191 mice.

<p>Brain sections from rTg9191 mice of 24 months of age (B, F, J, N, R, V, Z), their age-matched non-transgenic littermates (A, E, I, M, Q, U, Y), 23-month-old Tg2576 mice (C, G, K, O, S, W, AA) and 15-month-old rTg4510 mice (D, H, L, P, T, X, AB) were stained with a variety of antibodies directed against pathological conformation- and phosphorylation-dependent epitopes of tau: AT8 (A-D), CP13 (E-H), PG5 (I-L), PHF-1 (M-P), Alz50 (Q-T), MC1 (U-X) and TG-3 (Y-AB). Representative photomicrographs showed that hyperphosphorylated and/or misfolded tau proteins accumulated (brown puncta) around dense-core plaques visualized using Congo red (pink). Neuronal staining (brown) in rTg4510 mice served as positive control. Scale bars: 20 μm. Images in the same row have the same magnification. Representative photomicrographs show tau pathology of female mice, and similar results were found in male mice.</p

Age-related production of Aβ proteins in water-soluble, detergent-soluble, and detergent-insoluble fractions of brain extracts from rTg9191 mice.

<p>The levels of total Aβ38, Aβ40, and Aβ42 proteins in water-soluble (A), detergent-soluble (B) and detergent-insoluble (C) fractions of brain extracts of rTg9191 mice at 4, 12, 21, and 24 months of age were measured using ELISA.</p

rTg9191 mice produce Aβ dimers in an age-dependent manner, but lack Aβ*56.

<p>(A-B) Production of Aβ dimers. (A) A representative immunoblot (upper panel) shows levels of Aβ dimers in young, mid-age, and old mice; α-tubulin served as the loading control (lower panel). (B) Quantification. rTg9191 mice exhibit an age-dependent progression in levels of Aβ dimers. # p < 0.05, *** p < 0.0001 (compared to 26M), one-way ANOVA, followed by Fisher’s <i>post hoc</i> analysis. (C-D) rTg9191 mice lack Aβ*56. (C) Representative immunoblot shows levels of Aβ*56 in transgenic mouse lines rTg9191, TgArc6, and hAPP-J20. Although a faint band at ~56kDa was seen occasionally in extracts from rTg9191 mice, the intensity of this band was comparable to that seen in some samples from non-transgenic mice. As monoclonal antibody 6E10 recognizes human, but not mouse Aβ, this faint band represents non-specific background noise, and not a true Aβ signal. (D) Quantification. The intensity of the ~56 kDa band in rTg9191 mice (4, 12, 21, 24 and 26M of age) is comparable to that of non-transgenic littermates (neg, 26M) and TgArc6 (4M), but is significantly lower than that of hAPP-J20 (4M). *** p < 0.0001, each group compared to hAPP-J20, one-way ANOVA, followed by Fisher’s <i>post hoc</i> analysis. Neg, non-transgenic littermates of rTg9191 mice; pos, mice that express APP transgenes. Samples from both genders were loaded onto the representative blots, aligning in the order of left to right for (A): F, M, F, F, M, M, F, M, F, M, F, F (M, male; F, female), and for (C): F, F, M, M, F, M, F, M, F, M, F, F, M, F, M, F.</p