Longitudinal assessment of cognitive function in the APPswe/PS1dE9 mouse model of Alzheimer's-related beta-amyloidosis.

Preclinical models of Alzheimer's disease (AD)-related cognitive decline can be useful for developing therapeutics. The current study longitudinally assessed short-term memory, using a delayed matching-to-position (DMTP) task, and attention, using a 3-choice serial reaction time (3CSRT) task, from approximately 18 weeks of age through death or 72 weeks of age in APPswe/PS1dE9 mice, a widely used mouse model of AD-related amyloidosis. Both transgenic (Tg) and non-Tg mice exhibited improvements in DMTP accuracy over time. Breaks in testing reduced DMTP accuracy but accuracy values quickly recovered in both Tg and non-Tg mice. Both Tg and non-Tg mice exhibited high accuracy in the 3CSRT task with breaks in testing briefly reducing accuracy values equivalently in the 2 genotypes. The current results raise the possibility that deficits in Tg APPswe/PS1dE9 mice involve impairments in learning rather than declines in established performances. A better understanding of the factors that determine whether deficits develop will be useful for designing evaluations of potential pharmacotherapeutics and may reveal interventions for clinical application.

Quantitative in vivo assessment of amyloid-beta phagocytic capacity in an Alzheimer's disease mouse model.

Alzheimer's disease is characterized by the deposition of extracellular amyloid-beta (Aß) plaques. While microglial phagocytosis is a major mechanism through which Aß is cleared, there is no method for quantitatively assessing Aß phagocytic capacity of microglia in vivo. Here, we present a flow cytometry-based method for investigating the Aß phagocytic capacity of microglia in vivo. This method enables the direct comparison of Aß phagocytic capacity between different microglial subpopulations as well as the direct isolation of Aß phagocytic microglia for downstream applications. For complete details on the use and execution of this protocol, please refer to Lau et al. (2020).

Cerebral Microhemorrhage at MRI in Mild Cognitive Impairment and Early Alzheimer Disease: Association with Tau and Amyloid ß at PET Imaging.

Background Growing evidence indicates an association between cerebral microhemorrhages (MHs) and amyloid ß accumulation in Alzheimer disease (AD), but to the knowledge of the authors the association with tau burden is unknown. Purpose To investigate the association between cerebral MH load and tau pathologic structure measured in healthy older individuals and individuals along the AD spectrum, stratified by using the A (amyloid ß)/T (tau)/N (neurodegeneration) biomarker classification system. Materials and methods In this prospective cohort study, participants from the AD Neuroimaging Initiative were included (healthy control participants, participants with mild cognitive impairment, and participants with AD dementia; data from October 2005 to January 2019). T2*-weighted gradient-echo MRI was performed to quantify MH, fluorine 18 (18F) flortaucipir (AV-1451) PET was performed to quantify tau, and 18F-florbetaben/18F- florbetapir (AV45) PET was performed to quantify amyloid ß to study associations of MH with regional and global tau and amyloid ß load. Associations with cerebrospinal fluid (CSF) biomarkers (amyloid ß1-42, total tau, phosphorylated tau 181) were also assessed. Analysis of covariance and Spearman rank correlation test for cross-sectional analysis and Wilcoxon signed rank test for longitudinal analyses were used, controlling for multiple comparisons (Bonferroni significance threshold, P < .008). Results Evaluated were 343 participants (mean age, 75 years ± 7; 186 women), including 205 participants who were A-TN- (mean age, 73 years ± 7; 115 women), 80 participants who were A+TN- (mean age, 76 years ± 7; 38 women), and 58 participants who were A+TN+ (mean age, 77 ± 8; 34 women). MH count was associated with global (Spearman ρ = 0.27; P = .004) and frontal (ρ = 0.27; P = .005) amyloid ß load and global tau load (ρ = 0.31; P = .001). In a longitudinal analysis, MH count increased significantly over approximately 5 years in the entire cohort (T-1, 81 [range, 0-6 participants]; T0, 214 [range, 0-58 participants]; P < .001), in A+TN+ (T-1, 20 [range, 0-5 participants]; T0, 119 [range, 1-58 participants]; P < .001), A+TN- (T-1, 31 [range, 0-6 participants]; T0, 43 [range, 0-8 participants]; P = .03), and A-TN- (T-1, 30 [range, 0-4 participants]; T0, 52 [range, 0-6 participants]; P = .007). A higher MH count was associated with higher future global (ρ = 0.29; P = .008) and parietal (ρ = 0.31; P = .005) amyloid ß and parietal tau load (ρ = 0.31; P = .005). Conclusion Cerebral microhemorrhage load is associated spatially with tau accumulation, both cross-sectionally and longitudinally. © RSNA, 2020 Online supplemental material is available for this article.

Development of GMP-1 a molecular chaperone network modulator protecting mitochondrial function and its assessment in fly and mice models of Alzheimer's disease.

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. It has been shown that amyloid beta peptide (Aß) and amyloid precursor protein (APP) interact with mitochondria contributing to the mitochondrial dysfunction in AD. Prevention of abnormal protein targeting to mitochondria can protect normal mitochondrial function, increase neuronal survival and at the end, ameliorate symptoms of AD and other neurodegenerative disorders. First steps of mitochondrial protein import are coordinated by molecular chaperones Hsp70 and Hsp90 that bind to the newly synthesized mitochondria-destined proteins and deliver them to the protein import receptors on the surface of organelle. Here, we have described the development of a novel compound named GMP-1 that disrupts interactions between Hsp70/Hsp90 molecular chaperones and protein import receptor Tom70. GMP-1 treatment of SH-SY5Y cells results in decrease in mitochondria-associated APP and protects SH-SY5Y cells from toxic effect of Aß1-42 exposure. Experiments in drosophila and mice models of AD demonstrated neuroprotective effect of GMP-1 treatment, improvement in memory and behaviour tests as well as restoration of mitochondrial function.

Alzheimer's Protective Cross-Interaction between Wild-Type and A2T Variants Alters Aß42 Dimer Structure.

Whole genome sequencing has recently revealed the protective effect of a single A2T mutation in heterozygous carriers against Alzheimer's disease (AD) and age-related cognitive decline. The impact of the protective cross-interaction between the wild-type (WT) and A2T variants on the dimer structure is therefore of high interest, as the Aß dimers are the smallest known neurotoxic species. Toward this goal, extensive atomistic replica exchange molecular dynamics simulations of the solvated WT homo- and A2T hetero- Aß1-42 dimers have been performed, resulting into a total of 51 µs of sampling for each system. Weakening of a set of transient, intrachain contacts formed between the central and C-terminal hydrophobic residues is observed in the heterodimeric system. The majority of the heterodimers with reduced interaction between central and C-terminal regions lack any significant secondary structure and display a weak interchain interface. Interestingly, the A2T N-terminus, particularly residue F4, is frequently engaged in tertiary and quaternary interactions with central and C-terminal hydrophobic residues in those distinct structures, leading to hydrophobic burial. This atypical involvement of the N-terminus within A2T heterodimer revealed in our simulations implies possible interference on Aß42 aggregation and toxic oligomer formation, which is consistent with experiments. In conclusion, the present study provides detailed structural insights onto A2T Aß42 heterodimer, which might provide molecular insights onto the AD protective effect of the A2T mutation in the heterozygous state.

Analyzing and Modeling the Kinetics of Amyloid Beta Pores Associated with Alzheimer's Disease Pathology.

Amyloid beta (Aß) oligomers associated with Alzheimer's disease (AD) form Ca2+-permeable plasma membrane pores, leading to a disruption of the otherwise well-controlled intracellular calcium (Ca2+) homeostasis. The resultant up-regulation of intracellular Ca2+ concentration has detrimental implications for memory formation and cell survival. The gating kinetics and Ca2+ permeability of Aß pores are not well understood. We have used computational modeling in conjunction with the ability of optical patch-clamping for massively parallel imaging of Ca2+ flux through thousands of pores in the cell membrane of Xenopus oocytes to elucidate the kinetic properties of Aß pores. The fluorescence time-series data from individual pores were idealized and used to develop data-driven Markov chain models for the kinetics of the Aß pore at different stages of its evolution. Our study provides the first demonstration of developing Markov chain models for ion channel gating that are driven by optical-patch clamp data with the advantage of experiments being performed under close to physiological conditions. Towards the end, we demonstrate the up-regulation of gating of various Ca2+ release channels due to Aß pores and show that the extent and spatial range of such up-regulation increases as Aß pores with low open probability and Ca2+ permeability transition into those with high open probability and Ca2+ permeability.

Effects of Zn2+ binding on the structural and dynamic properties of amyloid ß peptide associated with Alzheimer's disease: Asp1 or Glu11?

Extensive experimental and computational studies have suggested that multiple Zn(2+) binding modes in amyloid ß (Aß) peptides could exist simultaneously. However, consistent results have not been obtained for the effects of Zn(2+) binding on Aß structure, dynamics, and kinetics in particular. Some key questions such as why it is so difficult to distinguish the polymorphic states of metal ions binding to Aß and what the underlying rationale is, necessitate elucidation. In this work, two 3N1O Zn(2+) binding modes were constructed with three histidines (His(6), His(13), and His(14)), and Asp(1)/Glu(11) of Aß40 coordinated to Zn(2+). Results from molecular dynamics simulations reveal that the conformational ensembles of different Zn(2+)-Aß40 complexes are nonoverlapping. The formation of turn structure and, especially, the salt bridge between Glu(22)/Asp(23) and Lys(28) is dependent on specific Zn(2+) binding mode. Agreement with available NMR observations of secondary and tertiary structures could be better achieved if the two simulation results are considered together. The free energy landscape constructed by combining both conformations of Aß40 indicates that transitions between distinct Aß40 conformations thar are ready for Zn(2+) binding could be possible in aqueous solution. Markov state model analyses reveal the complex network of conformational space of Aß40 modeulated by Zn(2+) binding, suggesting various misfolding pathways. The binding free energies evaluated using a combination of quantum mechanics calculations and the MM/3D-RISM method suggest that Glu(11) is the preferred oxygen ligand of Zn(2+). However, such preference is dependent on the relative populations of different conformations with specific Zn(2+) binding modes, and therefore could be shifted when experimental or simulation conditions are altered.

1H-MRS assessment of the therapeutic effect of bilateral intraventricular BDNF infusion into APP/PS1 double transgenic mice.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by extracellular accumulation of amyloid deposits. Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor whose levels have been shown to be decreased in AD brains. BDNF supplementation can offer improvement in the course of AD. However, the means of assessment are still relatively limited. In the present study, 1H-MRS was used to evaluate the therapeutic effects of bilateral intraventricular BDNF infusion into Alzheimer's disease APP/PS1 double transgenic mice. For comparison to the 1H-MRS observations, Fluoro-Jade B staining and immunofluorescence for beta amyloid peptides (Aß), glial fibrillary acidic protein, and tropomyosin-related kinase B (TrkB) were also performed. Our results showed that N-acetylaspartate (NAA) levels increased and myoinositol levels decreased in the BDNF group compared with the PBS group. However, the BDNF group NAA level was still lower than the control group at 6 weeks after infusion. These changes correlated with increased immunoreactivity for TrkB, decreased compact Aß peptide containing plaques, and decreased Fluoro-Jade B-positive cells in the BDNF-infused mice compared to vehicle controls. These findings demonstrate that 1H-MRS may be a promising means of evaluating the therapeutic effects of BDNF on AD.

Investigating how peptide length and a pathogenic mutation modify the structural ensemble of amyloid beta monomer.

The aggregation of amyloid beta (Aß) peptides plays an important role in the development of Alzheimer's disease. Despite extensive effort, it has been difficult to characterize the secondary and tertiary structure of the Aß monomer, the starting point for aggregation, due to its hydrophobicity and high aggregation propensity. Here, we employ extensive molecular dynamics simulations with atomistic protein and water models to determine structural ensembles for Aß(42), Aß(40), and Aß(42)-E22K (the Italian mutant) monomers in solution. Sampling of a total of >700 microseconds in all-atom detail with explicit solvent enables us to observe the effects of peptide length and a pathogenic mutation on the disordered Aß monomer structural ensemble. Aß(42) and Aß(40) have crudely similar characteristics but reducing the peptide length from 42 to 40 residues reduces ß-hairpin formation near the C-terminus. The pathogenic Italian E22K mutation induces helix formation in the region of residues 20-24. This structural alteration may increase helix-helix interactions between monomers, resulting in altered mechanism and kinetics of Aß oligomerization.

Application of immunosignatures to the assessment of Alzheimer's disease.

OBJECTIVE: Accurate assessment of Alzheimer's disease (AD), both presymptomatically and at different disease stages, will become increasingly important with the expanding elderly population. There are a number of indications that the immune system is engaged in AD. Here we explore the ability of an antibody-profiling technology to characterize AD and screen for peptides that may be used for a simple diagnostic test. METHODS: We developed an array-based system to profile the antibody repertoire of transgenic mice with cerebral amyloidosis (TG) and elderly individuals with or without AD. The array consists of 10,000 random sequence peptides (20-mers) capable of detecting antibody binding patterns, allowing the identification of peptides that mimic epitopes targeted by a donor's serum. RESULTS: TG mice exhibited a distinct immunoprofile compared to nontransgenic littermates. Further, we show that dementia patients, including autopsy-confirmed AD subjects, have distinguishable profiles compared to age-matched nondemented people. Using antibodies to different forms of Aß peptide and blocking protocols, we demonstrate that most of this signature is not due to the subject's antibodies raised against Aß. INTERPRETATION: We propose that "immunosignaturing" technology may have potential as a diagnostic tool in AD.

Monte Carlo study of the formation and conformational properties of dimers of Aß42 variants.

Small soluble oligomers, and dimers in particular, of the amyloid ß-peptide (Aß) are believed to play an important pathological role in Alzheimer's disease. Here, we investigate the spontaneous dimerization of Aß42, with 42 residues, by implicit solvent all-atom Monte Carlo simulations, for the wild-type peptide and the mutants F20E, E22G and E22G/I31E. The observed dimers of these variants share many overall conformational characteristics but differ in several aspects at a detailed level. In all four cases, the most common type of secondary structure is intramolecular antiparallel ß-sheets. Parallel, in-register ß-sheet structure, as in models for Aß fibrils, is rare. The primary force driving the formation of dimers is hydrophobic attraction. The conformational differences that we do see involve turns centered in the 20-30 region. The probability of finding turns centered in the 25-30 region, where there is a loop in Aß fibrils, is found to increase upon dimerization and to correlate with experimentally measured rates of fibril formation for the different Aß42 variants. Our findings hint at reorganization of this part of the molecule as a potentially critical step in Aß aggregation.

Comparing the folding free-energy landscapes of Abeta42 variants with different aggregation properties.

The properties of the amyloid-beta peptide that lead to aggregation associated with Alzheimer's disease are not fully understood. This study aims at identifying conformational differences among four variants of full-length Abeta42 that are known to display very different aggregation properties. By extensive all-atom Monte Carlo simulations, we find that a variety of beta-sheet structures with distinct turns are readily accessible for full-length Abeta42. In the simulations, wild type (WT) Abeta42 preferentially populates two major classes of conformations, either extended with high beta-sheet content or more compact with lower beta-sheet content. The three mutations studied alter the balance between these classes. Strong mutational effects are observed in a region centered at residues 23-26, where WT Abeta42 tends to form a turn. The aggregation-accelerating E22G mutation associated with early onset of Alzheimer's disease makes this turn region conformationally more diverse, whereas the aggregation-decelerating F20E mutation has the reverse effect, and the E22G/I31E mutation reduces the turn population. Comparing results for the four Abeta42 variants, we identify specific conformational properties of residues 23-26 that might play a key role in aggregation.

Longitudinal, quantitative assessment of amyloid, neuroinflammation, and anti-amyloid treatment in a living mouse model of Alzheimer's disease enabled by positron emission tomography.

We provide the first evidence for the capability of a high-resolution positron emission tomographic (PET) imaging system in quantitatively mapping amyloid accumulation in living amyloid precursor protein transgenic (Tg) mice. After the intravenous administration of N-[11C]methyl-2-(4'-methylaminophenyl)-6-hydroxybenzothiazole (or [11C]PIB for "Pittsburgh Compound-B") with high-specific radioactivity, the Tg mice exhibited high-level retention of radioactivity in amyloid-rich regions. PET investigation for Tg mice over an extended range of ages, including longitudinal assessments, demonstrated age-dependent increase in radioligand binding consistent with progressive amyloid accumulation. Reduction in amyloid levels in the hippocampus of Tg mice was also successfully monitored by multiple PET scans along the time course of anti-amyloid treatment using an antibody against amyloid beta peptide (Abeta). Moreover, PET scans with [18F]fluoroethyl-DAA1106, a radiotracer for activated glia, were conducted for these individuals parallel to amyloid imaging, revealing treatment-induced neuroinflammatory responses, the magnitude of which intimately correlated with the levels of pre-existing amyloid estimated by [11C]PIB. It is also noteworthy that the localization and abundance of [11C]PIB autoradiographic signals were closely associated with those of N-terminally truncated and modified Abeta, AbetaN3-pyroglutamate, in Alzheimer's disease (AD) and Tg mouse brains, implying that the detectability of amyloid by [11C]PIB positron emission tomography is dependent on the accumulation of specific Abeta subtypes. Our results support the usefulness of the small animal-dedicated PET system in conjunction with high-specific radioactivity probes and appropriate Tg models not only for clarifying the mechanistic properties of amyloidogenesis in mouse models but also for preclinical tests of emerging diagnostic and therapeutic approaches to AD.

A synthetic peptide blocking the apolipoprotein E/beta-amyloid binding mitigates beta-amyloid toxicity and fibril formation in vitro and reduces beta-amyloid plaques in transgenic mice.

Alzheimer's disease (AD) is associated with accumulation of beta-amyloid (Abeta). A major genetic risk factor for sporadic AD is inheritance of the apolipoprotein (apo) E4 allele. ApoE can act as a pathological chaperone of Abeta, promoting its conformational transformation from soluble Abeta into toxic aggregates. We determined if blocking the apoE/Abeta interaction reduces Abeta load in transgenic (Tg) AD mice. The binding site of apoE on Abeta corresponds to residues 12 to 28. To block binding, we synthesized a peptide containing these residues, but substituted valine at position 18 to proline (Abeta12-28P). This changed the peptide's properties, making it non-fibrillogenic and non-toxic. Abeta12-28P competitively blocks binding of full-length Abeta to apoE (IC50 = 36.7 nmol). Furthermore, Abeta12-28P reduces Abeta fibrillogenesis in the presence of apoE, and Abeta/apoE toxicity in cell culture. Abeta12-28P is blood-brain barrier-permeable and in AD Tg mice inhibits Abeta deposition. Tg mice treated with Abeta12-28P for 1 month had a 63.3% reduction in Abeta load in the cortex (P = 0.0043) and a 59.5% (P = 0.0087) reduction in the hippocampus comparing to age-matched control Tg mice. Antibodies against Abeta were not detected in sera of treated mice; therefore the observed therapeutic effect of Abeta12-28P cannot be attributed to an antibody clearance response. Our experiments demonstrate that compounds blocking the interaction between Abeta and its pathological chaperones may be beneficial for treatment of beta-amyloid deposition in AD.

Assessment of the bioactivity of antibodies against beta-amyloid peptide in vitro and in vivo.

The accumulation of the beta-amyloid peptide (Abeta) is a central event in the pathogenesis of Alzheimer's disease (AD). Abeta removal from the brain by immune therapy shows promising potential for the treatment of patients with AD, although the mechanisms of the antibody action are incompletely understood. In this study we compared the biological activities of antibodies raised against various Abeta fragments for Abeta reduction in vitro and in vivo. Antibodies against Abeta enhanced the uptake of Abeta42 aggregates up to 6-fold by primary microglial cells in vitro. The kinetics of Abeta42 uptake varied considerably among antibodies. Based on the activity to mediate Abeta42 uptake by microglial cells, we identified a bioactive antibody that significantly reduced Abeta42 levels in the brains of transgenic mice with neuronal expression of an AD-related mutated amyloid precursor protein. This effect depended on the epitopes recognized by the antibody. Our data suggest that the ability to facilitate Abeta42 uptake by primary microglia cells in vitro can be used to predict the biological activity of the antibody by passive immunization in vivo. This protocol may prove useful for the rapid validation of the activity of antibodies designed to be used in immune therapy of AD.

Computationally derived structural models of the beta-amyloid found in Alzheimer's disease plaques and the interaction with possible aggregation inhibitors.

We report the modeling of and possible interactions within the solid beta-amyloid (ABeta) 1-43 fibril, the most fibrillogenic peptide known. All models proposed are consistent with the known experimental structural data, in terms of both secondary structure and packing motifs. The model containing antiparallel beta-sheets, and a beta-turn at G(25)S(26)N(27)K(28) has the lowest calculated packing energy. As such, it can be considered a reasonable model for solid beta-amyloid in Alzheimer's disease plaques. Interestingly, with the turn located at this position, the 1-43 structure is stabilized by a number of complementary intermolecular interactions between the beta-sheets. These well-defined interactions exist for the side-chain residues of 41, 42, and 43 with adjacent ABeta molecules. These interactions would not be conserved in the 1-40 peptide, and indeed, this enhanced interaction is proposed to give rise to the increased fibrillogenic nature of the ABeta 1-43 species over the 1-40 form. The models are used to explain the increased fibrillogenic nature of the Dutch family mutation of ABeta. These models are also employed to examine possible docking interactions of previously reported antiaggregation inhibitors, such as 4'-deoxy-4'-iododoxorubicin (IDOX) onto the theoretical growing surface. A docked structure of IDOX with the model of the solid fibril is described and a proposal for the mechanism of its antiaggregation properties is presented.