Mouse Amyloid Research Articles

Impaired Spine Stability Underlies Plaque-Related Spine Loss in an Alzheimer's Disease Mouse Model

Dendritic spines, the site of most excitatory synapses in the brain, are lost in Alzheimer's disease and in related mouse models, undoubtedly contributing to cognitive dysfunction. We hypothesized that spine loss results from plaque-associated alterations of spine stability, causing an imbalance in spine formation and elimination. To investigate effects of plaques on spine stability in vivo, we observed cortical neurons using multiphoton microscopy in a mouse model of amyloid pathology before and after extensive plaque deposition. We also observed age-matched nontransgenic mice to study normal effects of aging on spine plasticity. We found that spine density and structural plasticity are maintained during normal aging. Tg2576 mice had normal spine density and plasticity before plaques appeared, but after amyloid pathology is established, severe disruptions were observed. In control animals, spine formation and elimination were equivalent over 1 hour of observation ( approximately 5% of observed spines), resulting in stable spine density. However, in aged Tg2576 mice spine elimination increased, specifically in the immediate vicinity of plaques. Spine formation was unchanged, resulting in spine loss. These data show a small population of rapidly changing spines in adult and even elderly mouse cortex; further, in the vicinity of amyloid plaques, spine stability is markedly impaired leading to loss of synaptic structural integrity.

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 A beta 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 A beta that might contribute to their differing properties in vivo. To examine the amyloidogenic potential of mouse A beta, 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 A beta could accelerate the deposition of human A beta 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 A beta but formed amyloid deposits no faster and to no greater extent than APPswe/PS1dE9 siblings. Instead, the additional mouse A beta increased the detergent solubility of accumulated amyloid and exacerbated amyloid deposition in the vasculature. These findings suggest that, although mouse A beta does not influence the rate of amyloid formation, the incorporation of A beta peptides with differing sequences alters the solubility and localization of the resulting aggregates.

Deposition of mouse amyloid β in human APP/PS1 double and single AD model transgenic mice

The deposition of amyloid beta (Abeta) peptides and neurofibrillary tangles are the two characteristic pathological features of Alzheimer's disease (AD). To investigate the relation between amyloid precursor protein (APP) production, amyloid beta deposition and the type of Abeta in deposits, i.e., human and/or mouse, we performed a histopathological analysis, using mouse and human specific antibodies, of the neocortex and hippocampus in 6, 12 and 19 months old APP/PS1 double and APP and PS1 single transgenic mice. There was a significant correlation between the human amyloid beta deposits and the intrinsic rodent amyloid beta deposits, that is, all plaques contained both human and mouse Abeta, and the diffuse amyloid beta deposits also colocalized human and mouse Abeta. Furthermore, some blood vessels (mainly leptomeningeal vessels) show labeling with human Abeta, and most of these vessels also label with mouse Abeta. Our findings demonstrate that the human amyloid deposits in APP/PS1 transgenic mice are closely associated with mouse Abeta, however, they do not precisely overlap. For instance, the core of plaques consists of primarily human Abeta, whereas the rim of the plaque contains both human and mouse amyloid beta, similarly, human and mouse Abeta are differentially localized in the blood vessel wall. Finally, as early as amyloid beta deposits can be detected, they show the presence of both human and mouse Abeta. Together, these data indicate that mouse Abeta is formed and deposited in significant amounts in the AD mouse brain and that it is deposited together with the human Abeta.

P4-005: FT-IR microspectroscopy: Analysis of Alzheimer’s disease and transgenic mouse amyloid deposition

P4-005: FT-IR microspectroscopy: Analysis of Alzheimer’s disease and transgenic mouse amyloid deposition

P4-297: Development of full-length mouse amyloid beta ELISA and determination of endogenous Abeta level in BACE1 knockout mice

Transgenic mice expressing mutant human amyloid precursor protein (APP) are widely used in the evaluation of amyloid beta (Abeta)–lowering strategies, such as beta and gamma secretase inhibitors. There is no clear evidence that APP expression is upregulated in sporadic Alzheimer's disease (AD) cases. Therefore, testing therapeutic agents under more physiologically relevant conditions, i.e. in mice with physiological APP expression, is important. Three amino acid residues differ between human and mouse Abeta peptides, and commonly used human Abeta ELISAs do not have optimal sensitivity to mouse Abeta. We aimed to develop a mouse Abeta ELISA, and to determine endogenous Abeta changes in non–transgenic (wild–type) mice. A monoclonal antibody against the N terminus of mouse Abeta was developed using synthetic peptide as an antigen. The antibody was characterized using standard protocols. Using an ELISA developed with this monoclonal antibody, we determined Abeta levels in BACE1 knockout mice. We identified clone 14F1, which recognized both mouse and human Abeta. The epitope is located within amino acid residues 1–4 of Abeta peptide. Clone 14F1 reacted more strongly with mouse Abeta than human Abeta. A sandwich ELISA composed of Abeta 40–specific antibody (clone 1A10) and 14F1 achieved single digit fmol/ml (equivalent to sub pg/ml) sensitivity, which is 1/10th of the endogenous Abeta level in wild–type mouse brain. We determined endogenous mouse Abeta level in BACE1 knockout mice using this ELISA. We determined that there is virtually no Abeta (background level in colorimetric assay) detectable in the brain of homozygous BACE1 knockout mice–/–. In heterozygous mice+/–, brain Abeta level was similar to that of wildtype control mice+/+. We developed a highly sensitive full–length mouse Aβ ELISA, which detected endogenous Abeta in non–transgenic (wild–type) mice. Heterozygous BACE1 gene deletion resulted in minimal Abeta reduction (in contrast to the effect of heterozygous BACE1 gene deletion in mutant APP transgenic mice). Determination of Abeta levels in non–transgenic mice is useful to elucidate the pharmacodynamic effects of secretase modulation; however, disease–modifying effects of therapeutic candidates should be characterized in plaque–forming transgenic mice.

Conditional inactivation of presenilin 1 prevents amyloid accumulation and temporarily rescues contextual and spatial working memory impairments in amyloid precursor protein transgenic mice.

Accumulation of beta-amyloid (Abeta) peptides in the cerebral cortex is considered a key event in the pathogenesis of Alzheimer's disease (AD). Presenilin 1 (PS1) plays an essential role in the gamma-secretase cleavage of the amyloid precursor protein (APP) and the generation of Abeta peptides. Reduction of Abeta generation via the inhibition of gamma-secretase activity, therefore, has been proposed as a therapeutic approach for AD. In this study, we examined whether genetic inactivation of PS1 in postnatal forebrain-restricted conditional knock-out (PS1 cKO) mice can prevent the accumulation of Abeta peptides and ameliorate cognitive deficits exhibited by an amyloid mouse model that overexpresses human mutant APP. We found that conditional inactivation of PS1 in APP transgenic mice (PS1 cKO;APP Tg) effectively prevented the accumulation of Abeta peptides and formation of amyloid plaques and inflammatory responses, although it also caused an age-related accumulation of C-terminal fragments of APP. Short-term PS1 inactivation in young PS1 cKO;APP Tg mice rescued deficits in contextual fear conditioning and serial spatial reversal learning in a water maze, which were associated with APP Tg mice. Longer-term PS1 inactivation in older PS1 cKO;APP Tg mice, however, failed to rescue the contextual memory and hippocampal synaptic deficits and had a decreasing ameliorative effect on the spatial memory impairment. These results reveal that in vivo reduction of Abeta via the inactivation of PS1 effectively prevents amyloid-associated neuropathological changes and can, but only temporarily, improve cognitive impairments in APP transgenic mice.

Induction of AApoAII and AA amyloidosis by the injections of various amyloid fibrils

Preformed amyloid fibrils accelerate conformational changes of amyloid precursor proteins and result in a rapid extension of amyloid fibrils in vitro. In this paper, we show the induction of AApoAII amyloidosis in vivo by various human and mouse amyloid fibrils isolated from human and mouse tissues or formed in vitro from synthetic peptides and recombinant proteins. These fibrils were injected intravenously into R1.P1- Apoa2 c mice. At 3 and 6 months after injection, amyloid deposition was examined. Severe amyloid depositions were detected in the tissues of mice injected with AApoAII (C) amyloid fibrils. Mild or slight amyloid depositions were also detected in the tissues of mice, which were injected with other types of fibrils, including synthetic peptides. However, no amyloid depositions were found in the control mice, which were injected with non-amyloid fibril proteins. In addition, AA amyloidosis was accelerated by the various amyloid fibrils in C57BL/6 mice, which were injected simultaneously and subcutaneously with 1.0% AgNO 3. These results demonstrated that various exogenous substances with a common structure could work as seeds for amyloid fibril formation in vivo. These findings suggested a prion-like mechanism of protein conformation in the pathogenesis of mouse AApoAII and AA amyloidosis.

Efflux of human and mouse amyloid β proteins 1–40 and 1–42 from brain: impairment in a mouse model of alzheimer's disease

Brain to blood transport is believed to be a major determinant of the amount of amyloid β protein (AβP) found in brain. Impaired efflux has been suggested as a mechanism by which AβP can accumulate in the CNS and so lead to Alzheimer's disease (AD). To date, however, no study of the efflux of the form of AβP most relevant to AD, AβP1–42, has been conducted, even though a single amino acid substitution in AβP can greatly alter efflux. Here, we examined the efflux of AβP mouse1–42, mouse1–40, human1–42, and human1–40 in young CD-1, young senesence accelerated mouse (SAM) P8, and aged SAMP8 mice. The SAMP8 mouse with aging spontaneously overproduces AβP and develops cognitive impairments reversed by AβP-directed antibody or phosphorothioate antisense oligonucleotide. CD-1 mice transported all forms of AβP, although mouse1–42 and human1–40 were transported faster than the other forms. There was a decrease in the saturable transport of mouse1–42 in SAMP8 mice regardless of age. Efflux of mouse1–40 and human1–42 was only by a non-saturable mechanism in young SAMP8 mice and their efflux was totally absent in aged SAMP8 mice. These differences in the efflux of the various forms of AβP among the three groups of mice supports the hypothesis that impaired efflux is an important factor in the accumulation of AβP in the CNS.

Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein.

Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.

Amyloid and tau mice: phenotype and phenotype modulation

Amyloid and tau mice: phenotype and phenotype modulation

APLP2, a member of the Alzheimer precursor protein family, is required for correct genomic segregation in dividing mouse cells

The mouse amyloid precursor-like protein 2 (APLP2) belongs to the Alzheimer peptide precursor family. A possible role in pre-implantation development had been suggested previously, and was investigated further by creating a large deletion in the genomic locus. While heterozygous mice developed normally, homozygous embryos were arrested before reaching the blastocyst stage. One-cell embryos which contained protein of maternal origin underwent a limited number of cleavages. The progressive disappearance of the protein at stages 4 and beyond correlated with the appearance of extensive cytopathological effects. Nuclear DNA contents of the arrested embryos departed widely from the normal 2-4C value, thus suggesting a role for the protein in replication and/or segregation of the embryonic genome. Embryonic mortality was not due to the untimely initiation of programmed cell death, and it occurred before the stage at which apoptotic cells normally appear. The same abnormal distribution of DNA contents was seen in primary cultures of Aplp2 +/- embryonic fibroblasts following transfection of an expression vector for Aplp2 antisense RNA with green fluorescent protein (GFP) expressed from a co-transfected construct. Daughter cells derived from a GFP-positive cell showed abnormal DNA contents both >4C and <2C, thus indicating a role for the protein in the mitotic segregation of the genome and establishment of the proper nuclear structure.

Adenoviral expression of murine serum amyloid A proteins to study amyloid fibrillogenesis.

Serum amyloid A (SAA) proteins are one of the most inducible acute-phase reactants and are precursors of secondary amyloidosis. In the mouse, SAA1 and SAA2 are induced in approximately equal quantities in response to amyloid induction models. These two isotypes differ in only 9 of 103 amino acid residues; however, only SAA2 is selectively deposited into amyloid fibrils. SAA expression in the CE/J mouse species is an exception in that gene duplication did not occur and the CE/J variant is a hybrid molecule sharing features of SAA1 and SAA2. However, even though it is more closely related to SAA2 it is not deposited as amyloid fibrils. We have developed an adenoviral vector system to overexpress SAA proteins in cell culture to determine the ability of these proteins to form amyloid fibrils, and to study the structural features in relation to amyloid formation. Both the SAA2 and CE/J SAA proteins were synthesized in large quantities and purified to homogeneity. Electron microscopic analysis of the SAA proteins revealed that the SAA2 protein was capable of forming amyloid fibrils, whereas the CE/J SAA was incapable. Radiolabelled SAAs were associated with normal or acute-phase high-density lipoproteins (HDLs); we examined them for their clearance from the circulation. In normal mice, SAA2 had a half-life of 70 min and CE/J SAA had a half-life of 120 min; however, in amyloid mice 50% of the SAA2 cleared in 55 min, compared with 135 min for the CE/J protein. When the SAA proteins were associated with acute-phase HDLs, SAA2 clearance was decreased to 60 min in normal mice compared with 30 min in amyloidogenic mice. Both normal and acute-phase HDLs were capable of depositing SAA2 into preformed amyloid fibrils, whereas the CE/J protein did not become associated with amyloid fibrils. This established approach opens the doors for large-scale SAA production and for the examination of specific amino acids involved in the fibrillogenic capability of the SAA2 molecule in vitro and in vivo.

Conserved elements in the 5′ regulatory region of the amyloid precursor protein gene in primates

Oligonucleotides corresponding to conserved sites between the human and mouse amyloid precursor protein (APP) genes have been used to polymerase chain reaction (PCR) amplify and sequence the promoter region of the APP gene from chimpanzee, gorilla, orang-utan, papio and African green monkey. Several novel conserved potentially-regulatory sequences of the APP gene have been detected after alignment of the APP promoter sequences: an apolipoprotein E-B1 (APOE-B1) element at position −450, also present in the APOE gene, two activator protein-2 (AP-2) sites at positions −374 and −301 and an intermediate early-1 gene (IE1) site at position −280. These elements are conserved in all mammalian APP promoter sequences studied. Additionally a previously detected heat shock element (HSE) at position −317, and an activator protein-1 (AP-1) site at position −350 are also conserved. Knowledge of the essential regulatory elements at the APP gene constitute the basis for understanding its transcriptional control and subsequent model studies.

CDNA cloning and chromosome mapping of the human Fe65 gene: interaction of the conserved cytoplasmic domains of the human beta-amyloid precursor protein and its homologues with the mouse Fe65 protein.

Using the yeast two hybrid system, a mouse embryo cDNA library was screened for proteins that interact with the C-terminus of the human beta-amyloid precursor protein (beta PP). A fusion protein was identified that interacts specifically with the cytoplasmic domain of beta PP and does not interact with the beta-amyloid region. The protein encoded by this partial mouse cDNA is identical to the C-terminus of the rat Fe65 protein. This mouse protein also interacts with the homologous C-terminal domains of the mouse amyloid precursor-like proteins, APLP1 and APLP2. These conserved cytoplasmic regions contain a common amino acid motif, Asn-Pro-Thr-Tyr, which has previously been shown to influence both the secretion and internalization of beta PP. Fe65 has been implicated in regulatory and cell signaling mechanisms because it contains two different motifs involved in protein binding, a WW domain (a variant of Src homology 3 domains) and a phosphotyrosine interaction domain (PID). Interestingly, the PID domain binds to the same motif present in the conserved cytoplasmic domains of the beta PP and beta PP-like proteins. RNA analyses reveal that Fe65 is predominantly expressed in brain and in the regions most affected by Alzheimer's disease (AD)-associated neuropathology. The human Fe65 mRNA was cloned from a fetal brain cDNA library. The message encodes a protein of 735 amino acids that is 95% identical to the rat Fe65 protein. The human Fe65 gene was mapped on human metaphase chromosomes to band 11p15 using fluorescence in situ hybridization.

Generation of mice with a 200-kb amyloid precursor protein gene deletion by Cre recombinase-mediated site-specific recombination in embryonic stem cells.

Gene disruptions and deletions of up to 20kb have been generated by homologous recombination with appropriate targeting vectors in murine embryonic stem (ES) cells. Because we could not obtain a deletion of about 200 kb in the mouse amyloid precursor protein gene by the classical technique, we employed strategies involving the insertion of loxP sites upstream and downstream of the region to be deleted by homologous recombination and elicited excision of the loxP-flanked region by introduction of a Cre expression vector into the ES cells. In the first approach, the loxP sequences were inserted in two successive steps and after each step, ES cell clones were isolated and characterized. Deletion of the loxP-flanked sequence was accomplished by introducing the cre gene in a third step. In the second approach, ES cells containing the upstream loxP cassette were electroporated simultaneously with the downstream loxP targeting vector and the Cre expression plasmid. ES cells were obtained that gave rise to chimeric mice capable of germ-line transmission of the deleted amyloid precursor protein allele.

Expression in brain of amyloid precursor protein mutated in the alpha-secretase site causes disturbed behavior, neuronal degeneration and premature death in transgenic mice.

A double mutation in the alpha-secretase site in the betaA4 region of mouse amyloid precursor protein (APP) reduced its secretion from COS cells, polarized MDCK cells and rat primary neurons. Expression of this mutant in the brain of mice, using the neuron-specific elements of the mouse Thy-1 gene promoter, resulted in transgenic mice that became progressively hyperactive, displayed seizures and died prematurely. In three different transgenic lines the severity of the phenotype was related directly to the expression levels of the transgene, estimated by both mRNA and protein levels. In addition, homozygous mice derived from each transgenic strain showed more severe symptoms which also occurred earlier in life than in heterozygotes. The observed symptoms were, however, not essentially different in the different lines. Increased aggressiveness, disturbed responses to kainic acid and N-methyl-D-aspartate, neophobia and deficiency in exploratory behavior were demonstrated in these mice. In the brain, the observed neuropathological changes included necrosis, apoptosis and astrogliosis in the hippocampus, cortex and other areas. The data demonstrate that incomplete or incorrect alpha-secretase processing of APP results in severe neurotoxicity and that this effect is expressed in a dominant manner.

Characterization of the promoter of the mouse amyloid precursor protein homologue, APLP2

Characterization of the promoter of the mouse amyloid precursor protein homologue, APLP2

Spontaneous amyloidosis in senile NSY mice.

Senile Nagoya, Shibata, Yasuda (NSY) mice developed amyloidosis and died from renal failure as a result of amyloidosis. NSY mice were first reported as experimental congenital diabetic mice by Shibata et al. in 1980. This study questioned whether NSY mice died from diabetic nephropathy. The authors of the present study investigated the life span and cause of death in these mice. The life span of NSY mice was found to be 618.7 +/- 72.5 days. NSY mice that lived for more than 400 days showed rising blood urea nitrogen and large amounts of amyloid deposits in the glomerulus of the kidneys. NSY mice died of renal amyloidosis. Immunological methods revealed that AApoAII was evident in the amyloid deposits of NSY mice. Apart from the kidneys, amyloid deposition was also found in the tongue, esophagus, stomach, small intestine, large intestine, rectum, lung, heart and adrenal glands. Amyloid deposits were found to a slight degree in the liver and the spleen. The most dominant amyloid deposition in NSY mice was seen in the glomerulus of the kidneys. From the point of view of amyloid depositional distribution, NSY mice were unique compared with other spontaneous amyloid mice.

Rapid separation of proteins and peptides by reversed‐phase microcolumn liquid chromatography

AbstractProteins and peptides were separated in the reversed‐phase mode on microcolumns packed with nonporous octadecyl‐group bonded silica gel with an average particle diameter of 4.5 or 20 μm. Separation columns were prepared from glass‐lined stainless steel tubing of 39 or 56‐mm × 0.5‐mm i.d. An artificial mixture of several proteins could be separated within 1 min by split‐flow gradient elution. The system was applied to the separation of a triptic digest of mouse amyloid A. On‐column sample enrichment allowed direct injection of 2‐5 μL of sample solution, leading to improved detectability. The limits of detection were ≤ 1 μg mL−1.

Technical communication Immunogold light and electron microscopic detection of amyloi plaques in transmissible spongiform encephalopathies

The antigenicity of the 'prion' protein amyloid fibrils was shown to be preserved after glutaraldehyde/OsO4 fixation in uranyl acetate-stained brain tissue blocks from patients with Gerstmann-Sträussler syndrome (GSS) and from mice infected with Creutzfeldt-Jakob disease (CJD). Amyloid plaques were demonstrated by light microscopy in immunogold silver-intensified semithin sections. Under the electron microscope, the amyloid fibrils were labelled in immunogold-reacted ultrathin sections using an antiserum prepared against GSS amyloid plaque cores and mouse amyloid fibrils respectively. The influence of various oxidizing agents (hydrogen peroxide, sodium metaperiodate) on the tissue preservation and the immunohistochemical detection was tested.