Rodent Aβ Modulates the Solubility and Distribution of Amyloid Deposits in Transgenic Mice

The amino acid sequence of amyloid precursor protein (APP) is highly conserved, and age-related Abeta aggregates have been described in a variety of vertebrate animals, with the notable exception of mice and rats. Three amino acid substitutions distinguish mouse and human Abeta that might contribute to their differing properties in vivo. To examine the amyloidogenic potential of mouse Abeta, we studied several lines of transgenic mice overexpressing wild-type mouse amyloid precursor protein (moAPP) either alone or in conjunction with mutant PS1 (PS1dE9). Neither overexpression of moAPP alone nor co-expression with PS1dE9 caused mice to develop Alzheimer-type amyloid pathology by 24 months of age. We further tested whether mouse Abeta could accelerate the deposition of human Abeta by crossing the moAPP transgenic mice to a bigenic line expressing human APPswe with PS1dE9. The triple transgenic animals (moAPP x APPswe/PS1dE9) produced 20% more Abeta but formed amyloid deposits no faster and to no greater extent than APPswe/PS1dE9 siblings. Instead, the additional mouse Abeta increased the detergent solubility of accumulated amyloid and exacerbated amyloid deposition in the vasculature. These findings suggest that, although mouse Abeta does not influence the rate of amyloid formation, the incorporation of Abeta peptides with differing sequences alters the solubility and localization of the resulting aggregates

Environmental Enrichment Mitigates Cognitive Deficits in a Mouse Model of Alzheimer's Disease

Epidemiological studies suggest that individuals with greater education or more cognitively demanding occupations have diminished risk of developing dementia. We wanted to test whether this effect could be recapitulated in rodents using environmental enrichment, a paradigm well documented to attenuate behavioral deficits induced by various pathological insults. Here, we demonstrate that learning and memory deficits observed in a transgenic mouse model of Alzheimer's disease can be ameliorated by enrichment. Female transgenic mice overexpressing amyloid precursor protein and/or presenilin-1 and nontransgenic controls were placed into enriched or standard cages at 2 months of age and tested for cognitive behavior after 6 months of differential housing. Enrichment significantly improved performance of all genotypes in the radial water maze and in the classic and repeated-reversal versions of the Morris water maze. However, enrichment did not benefit all genotypes equally. Mice overproducing amyloid-β (Aβ), particularly those with amyloid deposits, showed weaker memory for the platform location in the classic Morris water maze and learned new platform positions in the repeated-reversals task less quickly than their nontransgenic cagemates. Nonetheless, enrichment normalized the performance of Aβ-overproducing mice to the level of standard-housed nontransgenic mice. Moreover, this functional preservation occurred despite increased neuritic plaque burden in the hippocampus of double-transgenic animals and elevated steady-state Aβ levels, because both endogenous and transgene-derived Aβ are increased in enriched animals. These results demonstrate that the generation of Aβ in vivo and its impact on the function of the nervous system can be strongly modulated by environmental factors

Protein Topology of Presenilin 1

AbstractMutations in a gene encoding a multitransmembrane protein, termed presenilin 1 (PS1), are causative in the majority of early-onset cases of AD. To determine the topology of PS1, we utilized two strategies: first, we tested whether putative transmembranes are sufficient to export a protease-sensitive substrate across a lipid bilayer; and second, we examined the binding of antibodies to specific PS1 epitopes in cultured cells selectively permeabilized with the pore-forming toxin, streptolysin-O. We document that the “loop,” N-terminal, and C-terminal domains of PS1 are oriented toward the cytoplasm

Persistent Amyloidosis following Suppression of Aβ Production in a Transgenic Model of Alzheimer Disease

BACKGROUND: The proteases (secretases) that cleave amyloid-β (Aβ) peptide from the amyloid precursor protein (APP) have been the focus of considerable investigation in the development of treatments for Alzheimer disease. The prediction has been that reducing Aβ production in the brain, even after the onset of clinical symptoms and the development of associated pathology, will facilitate the repair of damaged tissue and removal of amyloid lesions. However, no long-term studies using animal models of amyloid pathology have yet been performed to test this hypothesis. METHODS AND FINDINGS: We have generated a transgenic mouse model that genetically mimics the arrest of Aβ production expected from treatment with secretase inhibitors. These mice overexpress mutant APP from a vector that can be regulated by doxycycline. Under normal conditions, high-level expression of APP quickly induces fulminant amyloid pathology. We show that doxycycline administration inhibits transgenic APP expression by greater than 95% and reduces Aβ production to levels found in nontransgenic mice. Suppression of transgenic Aβ synthesis in this model abruptly halts the progression of amyloid pathology. However, formation and disaggregation of amyloid deposits appear to be in disequilibrium as the plaques require far longer to disperse than to assemble. Mice in which APP synthesis was suppressed for as long as 6 mo after the formation of Aβ deposits retain a considerable amyloid load, with little sign of active clearance. CONCLUSION: This study demonstrates that amyloid lesions in transgenic mice are highly stable structures in vivo that are slow to disaggregate. Our findings suggest that arresting Aβ production in patients with Alzheimer disease should halt the progression of pathology, but that early treatment may be imperative, as it appears that amyloid deposits, once formed, will require additional intervention to clear

Neuritic and Glial Pathology Are Unchanged following Transgene Suppression

<p>Dystrophic neurites and activated astrocytes surround most compact plaques in tet-off APP mice (line 107). Dark-stained, ubiquitin-filled neurites and reactive astrocytes form a halo around cored, fibrillar deposits by 6 mo of age that worsens with time in untreated mice. Both plaque-associated pathologies are arrested, although not reversed, by transgene suppression. Hirano silver stain (top row); GFAP immunohistochemistry (middle row); ubiquitin immunohistochemistry (bottom row).</p

Suppression of Transgenic APP Arrests Progression of Amyloid Pathology

<p>(A) Aggregated Aβ was quantified in cortical tissue from dox-treated and control tTA/APP mice (line 107) using a filter trap assay. Serial dilutions of protein homogenate were passed through a cellulose acetate filter; protein aggregates larger than the pore size were trapped and immunostained for Aβ.</p> <p>(B) Quantitation of signal intensity in the linear range of each filter trap dilution series (arrow in [A]) was used to compare aggregate load across treatment groups. Aggregated Aβ increased significantly between 6 and 9 mo of age in untreated mice (significant effect of group ANOVA <i>F</i>_3,18 = 7.85, <i>p <</i> 0.002). This progression of pathology was completely prevented by transgene suppression. The amount of aggregated Aβ was identical in untreated mice at 6 mo of age to that in 9- or 12-mo-old animals treated with dox (<i>p ></i> 0.9, Tukey post-hoc test). Single transgenic tTA samples were included as negative controls and showed no signal above background. *, <i>p <</i> 0.01; **, <i>p <</i> 0.005 versus 9-mo-old untreated mice, Tukey post-hoc test; ***, <i>p <</i> 0.001 versus 9-mo-old untreated mice, Student's <i>t</i>-test.</p> <p>(C) Amyloid pathology in the hippocampus of representative mice from each treatment group: Hirano silver stain (top row), thioflavin-S (middle row), and Aβ immunohistochemistry (bottom row). Amyloid burden increases dramatically between 6 and 9 mo of age in untreated animals, but remains stable in transgene-suppressed mice over the same period (6 mo + 3 mo dox and 6 mo + 6 mo dox). Single transgenic animals (tTA only shown here) show no sign of amyloid pathology at any age tested.</p

Robust Transgene Suppression in Older Mice with Preexisting Amyloid Pathology

<p>(A) Cortical homogenates from 6- to 12-mo-old animals used for pathology studies described below (line 107) were immunoblotted with human-specific antibody 6E10 to examine transgene suppression following 3 or 6 mo of dox treatment. The blot was co-immunostained for endogenous superoxide dismutase 1 (SOD1) as a control for loading.</p> <p>(B) Quantitation of signal intensity from the Western blot shown in (A). Transgenic APP levels are significantly suppressed following 3 or 6 mo of dox treatment (96.9% and 97.6%, respectively). *, <i>p <</i> 0.001 compared to 6-mo-old untreated animals, Tukey post-hoc test applied to significant effect of group ANOVA <i>F</i>_3,12 = 107.22, <i>p <</i> 0.001. These data demonstrate that strong transgene suppression is attained both before and after the onset of amyloid pathology (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0020355#pmed-0020355-g001" target="_blank">Figure 1</a> for predeposit experiments).</p> <p>(C) Experimental design. To examine the effects of chronic Aβ suppression on amyloid pathology after the onset of deposition, we compared untreated controls harvested at 6 and 9 mo of age to animals placed on dox at 6 mo of age and harvested after 3 or 6 mo of treatment.</p> <p>(D) Dox treatment leads to rapid transgene suppression even in 6-mo-old tTA/APP mice. Immunostaining with 6E10 shows APPswe/ind levels are dramatically reduced in 6-mo-old mice treated for 1 wk with dox (upper panel). A separate blot was immunostained for APP C-terminal fragments with CT15 antibody to show that the precursors to Aβ cleavage are decreased in parallel with the full-length protein (middle panel). Costaining for superoxide dismutase 1 was used as an internal control for loading (lower panel, taken from bottom half of 6E10 blot).</p

Transgene Suppression Attenuates Hyperactivity in tTA/APP Mice

<p>(A) A 48-h measure of ambulation records extreme hyperactivity in untreated double transgenic mice compared to single transgenic and nontransgenic controls (line 107). This phenotype is completely eliminated by rearing the double transgenic mice on dox.</p> <p>(B) The same data shown in (A) are replotted to magnify data from untreated control and dox-treated groups.</p> <p>(C and D) Activity levels in the combined control groups of (A) and (B) are here separated by genotype. None of the single transgenic or nontransgenic control groups display the hyperactivity present in untreated tTA/APP animals. Again, note the <i>y</i>-axes have been enlarged for detail compared to (A).</p