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1.
Proc Natl Acad Sci U S A ; 120(41): e2300258120, 2023 10 10.
Article in English | MEDLINE | ID: mdl-37801475

ABSTRACT

Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.


Subject(s)
Alzheimer Disease , Single-Domain Antibodies , Supranuclear Palsy, Progressive , Humans , Animals , Mice , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , tau Proteins/metabolism , Single-Domain Antibodies/pharmacology , Single-Domain Antibodies/metabolism , Neurofibrillary Tangles/metabolism , Supranuclear Palsy, Progressive/metabolism , Antibodies/metabolism , Brain/metabolism
3.
Alzheimers Dement ; 19(6): 2239-2252, 2023 06.
Article in English | MEDLINE | ID: mdl-36448627

ABSTRACT

INTRODUCTION: The inositol polyphosphate-5-phosphatase D (INPP5D) gene encodes a dual-specificity phosphatase that can dephosphorylate both phospholipids and phosphoproteins. Single nucleotide polymorphisms in INPP5D impact risk for developing late onset sporadic Alzheimer's disease (LOAD). METHODS: To assess the consequences of inducible Inpp5d knockdown in microglia of APPKM670/671NL /PSEN1Δexon9 (PSAPP) mice, we injected 3-month-old Inpp5dfl/fl /Cx3cr1CreER/+ and PSAPP/Inpp5dfl/fl /Cx3cr1CreER/+ mice with either tamoxifen (TAM) or corn oil (CO) to induce recombination. RESULTS: At age 6 months, we found that the percent area of 6E10+ deposits and plaque-associated microglia in Inpp5d knockdown mice were increased compared to controls. Spatial transcriptomics identified a plaque-specific expression profile that was extensively altered by Inpp5d knockdown. DISCUSSION: These results demonstrate that conditional Inpp5d downregulation in the PSAPP mouse increases plaque burden and recruitment of microglia to plaques. Spatial transcriptomics highlighted an extended gene expression signature associated with plaques and identified CST7 (cystatin F) as a novel marker of plaques. HIGHLIGHTS: Inpp5d knockdown increases plaque burden and plaque-associated microglia number. Spatial transcriptomics identifies an expanded plaque-specific gene expression profile. Plaque-induced gene expression is altered by Inpp5d knockdown in microglia. Our plaque-associated gene signature overlaps with human Alzheimer's disease gene networks.


Subject(s)
Alzheimer Disease , Mice , Humans , Animals , Infant , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Microglia/metabolism , Mice, Transgenic , Plaque, Amyloid/metabolism , Disease Models, Animal , Amyloid beta-Peptides/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/genetics , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/metabolism
4.
J Biol Chem ; 296: 100168, 2021.
Article in English | MEDLINE | ID: mdl-33298522

ABSTRACT

Antibodies against Aß amyloid are indispensable research tools and potential therapeutics for Alzheimer's disease. They display several unusual properties, such as specificity for aggregated forms of the peptide, the ability to distinguish polymorphic aggregate structures, and the ability to recognize generic aggregation-related epitopes formed by unrelated amyloid sequences. Understanding the mechanisms underlying these unusual properties and the structures of their corresponding epitopes is crucial for the understanding why antibodies display different therapeutic activities and for the development of more effective therapeutic agents. Here we employed a novel "epitomic" approach to map the fine structure of the epitopes of 28 monoclonal antibodies against amyloid-beta using immunoselection of random sequences from a phage display library, deep sequencing, and pattern analysis to define the critical sequence elements recognized by the antibodies. Although most of the antibodies map to major linear epitopes in the amino terminal 1 to 14 residues of Aß, the antibodies display differences in the target sequence residues that are critical for binding and in their individual preferences for nontarget residues, indicating that the antibodies bind to alternative conformations of the sequence by different mechanisms. Epitomic analysis also identifies discontinuous, nonoverlapping sequence Aß segments that may constitute the conformational epitopes that underlie the aggregation specificity of antibodies. Aggregation-specific antibodies recognize sequences that display a significantly higher predicted propensity for forming amyloid than antibodies that recognize the monomer, indicating that the ability of random sequences to aggregate into amyloid is a critical element of their binding mechanism.


Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Antibodies, Monoclonal/metabolism , Epitope Mapping/methods , Epitopes/metabolism , Peptide Fragments/metabolism , Plaque, Amyloid/metabolism , Alzheimer Disease/diagnosis , Alzheimer Disease/pathology , Amino Acid Sequence , Amyloid beta-Peptides/chemistry , Antibodies, Monoclonal/chemistry , Binding Sites , Brain/metabolism , Brain/pathology , Epitopes/chemistry , Humans , Microtomy , Neurons/metabolism , Neurons/pathology , Peptide Fragments/chemistry , Peptide Library , Plaque, Amyloid/pathology , Protein Aggregates , Protein Array Analysis , Protein Binding
5.
J Biol Chem ; 295(31): 10662-10676, 2020 07 31.
Article in English | MEDLINE | ID: mdl-32493775

ABSTRACT

Soluble oligomers of aggregated tau accompany the accumulation of insoluble amyloid fibrils, a histological hallmark of Alzheimer disease (AD) and two dozen related neurodegenerative diseases. Both oligomers and fibrils seed the spread of Tau pathology, and by virtue of their low molecular weight and relative solubility, oligomers may be particularly pernicious seeds. Here, we report the formation of in vitro tau oligomers formed by an ionic liquid (IL15). Using IL15-induced recombinant tau oligomers and a dot blot assay, we discovered a mAb (M204) that binds oligomeric tau, but not tau monomers or fibrils. M204 and an engineered single-chain variable fragment (scFv) inhibited seeding by IL15-induced tau oligomers and pathological extracts from donors with AD and chronic traumatic encephalopathy. This finding suggests that M204-scFv targets pathological structures that are formed by tau in neurodegenerative diseases. We found that M204-scFv itself partitions into oligomeric forms that inhibit seeding differently, and crystal structures of the M204-scFv monomer, dimer, and trimer revealed conformational differences that explain differences among these forms in binding and inhibition. The efficiency of M204-scFv antibodies to inhibit the seeding by brain tissue extracts from different donors with tauopathies varied among individuals, indicating the possible existence of distinct amyloid polymorphs. We propose that by binding to oligomers, which are hypothesized to be the earliest seeding-competent species, M204-scFv may have potential as an early-stage diagnostic for AD and tauopathies, and also could guide the development of promising therapeutic antibodies.


Subject(s)
Alzheimer Disease , Protein Multimerization , Single-Chain Antibodies/chemistry , tau Proteins/chemistry , Crystallography, X-Ray , Humans
6.
Am J Pathol ; 189(8): 1621-1636, 2019 08.
Article in English | MEDLINE | ID: mdl-31108099

ABSTRACT

Apolipoprotein E (apoE) colocalizes with amyloid-ß (Aß) in Alzheimer disease (AD) plaques and in synapses, and evidence suggests that direct interactions between apoE and Aß are important for apoE's effects in AD. The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Aß complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Aß complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Aß was confirmed by coimmunoprecipitation experiments. The apoE receptors low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early- and late-stage AD. Dual-labeling flow cytometry analysis of LRP1- and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)-positive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Aß concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Aß was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Aß complex and associated lipids into synaptic terminals, with subsequent Aß clearance in control synapses and accumulation in AD synapses.


Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Apolipoprotein E4/metabolism , Apolipoproteins E/metabolism , Cerebral Cortex/metabolism , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , Receptors, LDL/metabolism , Synapses/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/pathology , Cerebral Cortex/pathology , Disks Large Homolog 4 Protein/metabolism , Female , Humans , Male , Middle Aged , Synapses/pathology , Synaptosomes/metabolism , Synaptosomes/pathology
7.
Circ Res ; 122(10): e75-e83, 2018 05 11.
Article in English | MEDLINE | ID: mdl-29483093

ABSTRACT

RATIONALE: Disrupted proteostasis is one major pathological trait that heart failure (HF) shares with other organ proteinopathies, such as Alzheimer and Parkinson diseases. Yet, differently from the latter, whether and how cardiac preamyloid oligomers (PAOs) develop in acquired forms of HF is unclear. OBJECTIVE: We previously reported a rise in monophosphorylated, aggregate-prone desmin in canine and human HF. We now tested whether monophosphorylated desmin acts as the seed nucleating PAOs formation and determined whether positron emission tomography is able to detect myocardial PAOs in nongenetic HF. METHODS AND RESULTS: Here, we first show that toxic cardiac PAOs accumulate in the myocardium of mice subjected to transverse aortic constriction and that PAOs comigrate with the cytoskeletal protein desmin in this well-established model of acquired HF. We confirm this evidence in cardiac extracts from human ischemic and nonischemic HF. We also demonstrate that Ser31 phosphorylated desmin aggregates extensively in cultured cardiomyocytes. Lastly, we were able to detect the in vivo accumulation of cardiac PAOs using positron emission tomography for the first time in acquired HF. CONCLUSIONS: Ser31 phosphorylated desmin is a likely candidate seed for the nucleation process leading to cardiac PAOs deposition. Desmin post-translational processing and misfolding constitute a new, attractive avenue for the diagnosis and treatment of the cardiac accumulation of toxic PAOs that can now be measured by positron emission tomography in acquired HF.


Subject(s)
Amyloid/metabolism , Desmin/metabolism , Heart Failure/metabolism , Myocytes, Cardiac/metabolism , Protein Processing, Post-Translational , Amyloid/analysis , Amyloid/drug effects , Animals , Catechin/analogs & derivatives , Catechin/pharmacology , Cells, Cultured , Desmin/genetics , Female , Genetic Vectors , Heart Failure/etiology , Humans , Male , Mass Spectrometry/methods , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Myocardial Ischemia/complications , Phosphorylation , Polymorphism, Single Nucleotide , Positron-Emission Tomography/methods , Pressure , Protein Aggregates/drug effects , Protein Folding , Rats , Recombinant Proteins/metabolism , alpha-Crystallins/deficiency , beta-Crystallins/deficiency
8.
J Biol Chem ; 293(8): 2888-2902, 2018 02 23.
Article in English | MEDLINE | ID: mdl-29282295

ABSTRACT

Amyloid-ß (Aß) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aß and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aß and hIAPP, termed Aß(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity. We observed a high degree of structural similarity between their backbone atoms (0.96-Å root mean square deviation). Moreover, fibrils of these segments induced amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment showed cross-efficacy for full-length Aß and hIAPP and reduced cytotoxicity of both proteins, although by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aß(24-34) WT and hIAPP(19-29) S20G offers a molecular model for cross-seeding between Aß and hIAPP.


Subject(s)
Amyloid beta-Peptides/metabolism , Islet Amyloid Polypeptide/metabolism , Models, Molecular , Neurofibrillary Tangles/metabolism , Peptide Fragments/metabolism , Amino Acid Substitution , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/genetics , Animals , Cell Line, Tumor , Computational Biology , Crystallography, X-Ray , Drug Design , HEK293 Cells , Humans , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/pharmacology , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Insulin-Secreting Cells/ultrastructure , Islet Amyloid Polypeptide/antagonists & inhibitors , Islet Amyloid Polypeptide/chemistry , Islet Amyloid Polypeptide/genetics , Mice , Microscopy, Electron, Transmission , Mutation , Neurofibrillary Tangles/chemistry , Neurofibrillary Tangles/drug effects , Neurofibrillary Tangles/ultrastructure , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Neurons/ultrastructure , Nootropic Agents/chemistry , Nootropic Agents/pharmacology , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Aggregation, Pathological/metabolism , Protein Aggregation, Pathological/pathology , Protein Aggregation, Pathological/prevention & control , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure
9.
Proc Natl Acad Sci U S A ; 113(34): E4976-84, 2016 08 23.
Article in English | MEDLINE | ID: mdl-27469165

ABSTRACT

Amyloid-ß (Aß) is present in humans as a 39- to 42-amino acid residue metabolic product of the amyloid precursor protein. Although the two predominant forms, Aß(1-40) and Aß(1-42), differ in only two residues, they display different biophysical, biological, and clinical behavior. Aß(1-42) is the more neurotoxic species, aggregates much faster, and dominates in senile plaque of Alzheimer's disease (AD) patients. Although small Aß oligomers are believed to be the neurotoxic species, Aß amyloid fibrils are, because of their presence in plaques, a pathological hallmark of AD and appear to play an important role in disease progression through cell-to-cell transmissibility. Here, we solved the 3D structure of a disease-relevant Aß(1-42) fibril polymorph, combining data from solid-state NMR spectroscopy and mass-per-length measurements from EM. The 3D structure is composed of two molecules per fibril layer, with residues 15-42 forming a double-horseshoe-like cross-ß-sheet entity with maximally buried hydrophobic side chains. Residues 1-14 are partially ordered and in a ß-strand conformation, but do not display unambiguous distance restraints to the remainder of the core structure.


Subject(s)
Amyloid beta-Peptides/ultrastructure , Peptide Fragments/ultrastructure , Amyloid beta-Peptides/genetics , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Microscopy, Electron , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/genetics , Protein Conformation, beta-Strand , Recombinant Proteins/genetics , Recombinant Proteins/ultrastructure
10.
J Biol Chem ; 292(8): 3172-3185, 2017 02 24.
Article in English | MEDLINE | ID: mdl-28049728

ABSTRACT

Most cases of Alzheimer's disease (AD) are sporadic, but a small percentage of AD cases, called familial AD (FAD), are associated with mutations in presenilin 1, presenilin 2, or the amyloid precursor protein. Amyloid precursor protein mutations falling within the amyloid-ß (Aß) sequence lead to a wide range of disease phenotypes. There is increasing evidence that distinct amyloid structures distinguished by amyloid conformation-dependent monoclonal antibodies have similarly distinct roles in pathology. It is possible that this phenotypic diversity of FAD associated with mutations within the Aß sequence is due to differences in the conformations adopted by mutant Aß peptides, but the effects of FAD mutations on aggregation kinetics and conformational and morphological changes of the Aß peptide are poorly defined. To gain more insight into this possibility, we therefore investigated the effects of 11 FAD mutations on the aggregation kinetics of Aß, as well as its ability to form distinct conformations recognized by a panel of amyloid conformation-specific monoclonal antibodies. We found that most FAD mutations increased the rate of aggregation of Aß. The FAD mutations also led to the adoption of alternative amyloid conformations distinguished by monoclonal antibodies and resulted in the formation of distinct aggregate morphologies as determined by transmission electron microscopy. In addition, several of the mutant peptides displayed a large reduction in thioflavin T fluorescence, despite forming abundant fibrils indicating that thioflavin T is a probe of conformational polymorphisms rather than a reliable indicator of fibrillization. Taken together, these results indicate that FAD mutations falling within the Aß sequence lead to dramatic changes in aggregation kinetics and influence the ability of Aß to form immunologically and morphologically distinct amyloid structures.


Subject(s)
Alzheimer Disease/genetics , Amyloid beta-Peptides/genetics , Amyloid beta-Protein Precursor/genetics , Mutation , Protein Aggregates , Alzheimer Disease/pathology , Amyloid beta-Peptides/analysis , Amyloid beta-Peptides/ultrastructure , Amyloid beta-Protein Precursor/analysis , Amyloid beta-Protein Precursor/ultrastructure , Humans , Protein Conformation
11.
Nature ; 485(7400): 651-5, 2012 May 02.
Article in English | MEDLINE | ID: mdl-22660329

ABSTRACT

Extracellular plaques of amyloid-ß and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer's disease. Plaques comprise amyloid-ß fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer's disease. Despite the importance of plaques to Alzheimer's disease, oligomers are considered to be the principal toxic forms of amyloid-ß. Interestingly, many adverse responses to amyloid-ß, such as cytotoxicity, microtubule loss, impaired memory and learning, and neuritic degeneration, are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-ß are strongly associated with Alzheimer's disease, are more toxic than amyloid-ß, residues 1-42 (Aß(1-42)) and Aß(1-40), and have been proposed as initiators of Alzheimer's disease pathogenesis. Here we report a mechanism by which pE-Aß may trigger Alzheimer's disease. Aß(3(pE)-42) co-oligomerizes with excess Aß(1-42) to form metastable low-n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aß(1-42) alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aß(3(pE)-42) plus 95% Aß(1-42) (5% pE-Aß) seed new cytotoxic LNOs through multiple serial dilutions into Aß(1-42) monomers in the absence of additional Aß(3(pE)-42). LNOs isolated from human Alzheimer's disease brain contained Aß(3(pE)-42), and enhanced Aß(3(pE)-42) formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aß(3(pE)-42) confers tau-dependent neuronal death and causes template-induced misfolding of Aß(1-42) into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aß(3(pE)-42) acts similarly at a primary step in Alzheimer's disease pathogenesis.


Subject(s)
Amyloid beta-Peptides/chemistry , Amyloid/toxicity , Glutamic Acid/metabolism , Mutant Proteins/chemistry , Mutant Proteins/toxicity , Peptide Fragments/chemistry , Prions/metabolism , tau Proteins/metabolism , Alzheimer Disease/metabolism , Amyloid/chemistry , Amyloid/drug effects , Amyloid/metabolism , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/toxicity , Animals , Disease Models, Animal , Glutamic Acid/chemistry , Humans , Mice , Mice, Transgenic , Mutant Proteins/genetics , Mutant Proteins/metabolism , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Fragments/toxicity , Prions/chemistry , Prions/toxicity , tau Proteins/deficiency , tau Proteins/genetics
12.
Am J Pathol ; 186(1): 185-98, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26718979

ABSTRACT

Amyloid-ß (Aß) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aß and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aß and p-tau in large populations of individual synaptic terminals. Soluble Aß oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aß was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aß oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aß-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aß drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed.


Subject(s)
Alzheimer Disease/pathology , Amyloid beta-Peptides/analysis , Synapses/pathology , tau Proteins/analysis , Aged , Aged, 80 and over , Animals , Brain/pathology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Humans , Male , Microscopy, Confocal , Phosphorylation , Rats , Rats, Transgenic
13.
Acta Neuropathol ; 134(5): 769-788, 2017 11.
Article in English | MEDLINE | ID: mdl-28612290

ABSTRACT

Conventional genetic approaches and computational strategies have converged on immune-inflammatory pathways as key events in the pathogenesis of late onset sporadic Alzheimer's disease (LOAD). Mutations and/or differential expression of microglial specific receptors such as TREM2, CD33, and CR3 have been associated with strong increased risk for developing Alzheimer's disease (AD). DAP12 (DNAX-activating protein 12)/TYROBP, a molecule localized to microglia, is a direct partner/adapter for TREM2, CD33, and CR3. We and others have previously shown that TYROBP expression is increased in AD patients and in mouse models. Moreover, missense mutations in the coding region of TYROBP have recently been identified in some AD patients. These lines of evidence, along with computational analysis of LOAD brain gene expression, point to DAP12/TYROBP as a potential hub or driver protein in the pathogenesis of AD. Using a comprehensive panel of biochemical, physiological, behavioral, and transcriptomic assays, we evaluated in a mouse model the role of TYROBP in early stage AD. We crossed an Alzheimer's model mutant APP KM670/671NL /PSEN1 Δexon9 (APP/PSEN1) mouse model with Tyrobp -/- mice to generate AD model mice deficient or null for TYROBP (APP/PSEN1; Tyrobp +/- or APP/PSEN1; Tyrobp -/-). While we observed relatively minor effects of TYROBP deficiency on steady-state levels of amyloid-ß peptides, there was an effect of Tyrobp deficiency on the morphology of amyloid deposits resembling that reported by others for Trem2 -/- mice. We identified modulatory effects of TYROBP deficiency on the level of phosphorylation of TAU that was accompanied by a reduction in the severity of neuritic dystrophy. TYROBP deficiency also altered the expression of several AD related genes, including Cd33. Electrophysiological abnormalities and learning behavior deficits associated with APP/PSEN1 transgenes were greatly attenuated on a Tyrobp-null background. Some modulatory effects of TYROBP on Alzheimer's-related genes were only apparent on a background of mice with cerebral amyloidosis due to overexpression of mutant APP/PSEN1. These results suggest that reduction of TYROBP gene expression and/or protein levels could represent an immune-inflammatory therapeutic opportunity for modulating early stage LOAD, potentially leading to slowing or arresting the progression to full-blown clinical and pathological LOAD.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Alzheimer Disease/genetics , Brain/pathology , Adaptor Proteins, Signal Transducing/metabolism , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Animals , Brain/metabolism , Disease Models, Animal , Maze Learning/physiology , Mice , Mice, Knockout , Microglia/metabolism , Microglia/pathology , Mutation , Phosphorylation , tau Proteins/metabolism
14.
Biochem Biophys Res Commun ; 477(4): 700-705, 2016 09 02.
Article in English | MEDLINE | ID: mdl-27363332

ABSTRACT

In Alzheimer's disease, soluble Aß oligomers are believed to play important roles in the disease pathogenesis, and their levels correlate with cognitive impairment. We have previously shown that Aß oligomers can be categorized into multiple structural classes based on their reactivity with conformation-dependent antibodies. In this study, we analyzed the structures of Aß40 oligomers belonging to two of these classes: fibrillar and prefibrillar oligomers. We found that fibrillar oligomers were similar in structure to fibrils but were less stable towards denaturation while prefibrillar oligomers were found to be partially disordered. These results are consistent with previously proposed structures for both oligomer classes while providing additional structural information.


Subject(s)
Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/ultrastructure , Amyloid/chemistry , Amyloid/ultrastructure , Peptide Fragments/chemistry , Peptide Fragments/ultrastructure , Dimerization , Protein Conformation
15.
Proc Natl Acad Sci U S A ; 110(8): 3077-82, 2013 Feb 19.
Article in English | MEDLINE | ID: mdl-23365139

ABSTRACT

Aggregation of misfolded proteins is characteristic of a number of neurodegenerative diseases, including Huntington disease (HD). The CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring) chaperonin complex can inhibit aggregation and cellular toxicity induced by expanded repeat Huntingtin (mHtt) fragments. The substrate-binding apical domain of CCT/TRiC subunit CCT1, ApiCCT1, is sufficient to inhibit aggregation of expanded repeat mHtt fragments in vitro, providing therapeutic promise for HD. However, a key hurdle in considering ApiCCT1 as a potential treatment is in delivery. Because ApiCCT1 has a region of similarity to the HIV Tat protein cell-transduction domain, we tested whether recombinant ApiCCT1 (ApiCCT1(r)) protein could enter cells following exogenous delivery and modulate an established panel of mHtt-mediated cell-based phenotypes. Cell fractionation studies demonstrate that exogenous ApiCCT1(r) can penetrate cell membranes and can localize to the nucleus, consistent with a strategy that can target both cytosolic and nuclear pathogenic events in HD. ApiCCT1(r) application does indeed modulate HD cellular phenotypes by decreasing formation of visible inclusions, fibrillar oligomers, and insoluble mHtt derived from expression of a truncated mHtt exon 1 fragment. ApiCCT1(r) also delays the onset of inclusion body formation as visualized via live imaging. ApiCCT1(r) reduces mHtt-mediated toxicity in immortalized striatal cells derived from full-length knock-in HD mice, suggesting that therapeutic benefit may extend beyond effects on aggregation. These studies provide the basis for a potentially robust and unique therapeutic strategy to target mHtt-mediated protein pathogenesis.


Subject(s)
Chaperonins/administration & dosage , Mutation , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Amino Acid Sequence , Animals , Blotting, Western , Cell Survival , Chaperonins/chemistry , Electrophoresis, Polyacrylamide Gel , Huntingtin Protein , Mice , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , PC12 Cells , Phenotype , Rats
16.
J Mol Cell Cardiol ; 79: 295-302, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25463275

ABSTRACT

Rapid activation causes remodeling of atrial myocytes resembling that which occurs in experimental and human atrial fibrillation (AF). Using this cellular model, we previously observed transcriptional upregulation of proteins implicated in protein misfolding and amyloidosis. For organ-specific amyloidoses such as Alzheimer's disease, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species. In the setting of oxidative stress, highly-reactive lipid-derived mediators known as γ-ketoaldehydes (γ-KAs) have been identified that rapidly adduct proteins and cause PAO formation for amyloid ß1-42 implicated in Alzheimer's. We hypothesized that rapid activation of atrial cells triggers oxidative stress with lipid peroxidation and formation of γ-KAs, which then rapidly crosslink proteins to generate PAOs. To investigate this hypothesis, rapidly-paced and control, spontaneously-beating atrial HL-1 cells were probed with a conformation-specific antibody recognizing PAOs. Rapid stimulation of atrial cells caused the generation of cytosolic PAOs along with a myocyte stress response (e.g., transcriptional upregulation of Nppa and Hspa1a), both of which were absent in control, unpaced cells. Rapid activation also caused the formation of superoxide and γ-KA adducts in atriomyocytes, while direct exposure of cells to γ-KAs resulted in PAO production. Increased cytosolic atrial natriuretic peptide (ANP), and the generation of ANP oligomers with exposure to γ-KAs and rapid atrial HL-1 cell stimulation, strongly suggest a role for ANP in PAO formation. Salicylamine (SA) is a small molecule scavenger of γ-KAs that can protect proteins from modification by these reactive compounds. PAO formation and transcriptional remodeling were inhibited when cells were stimulated in the presence of SA, but not with the antioxidant curcumin, which is incapable of scavenging γ-KAs. These results demonstrate that γ-KAs promote protein misfolding and PAO formation as a component of the atrial cell stress response to rapid activation, and they provide a potential mechanistic link between oxidative stress and atrial cell injury.


Subject(s)
Aldehydes/pharmacology , Amyloid/metabolism , Heart Atria/metabolism , Heart Atria/pathology , Protein Folding/drug effects , Protein Multimerization , Amines/pharmacology , Animals , Atrial Natriuretic Factor/metabolism , Cardiac Pacing, Artificial , Cell Line , Curcumin/pharmacology , Cytosol/drug effects , Cytosol/metabolism , Heart Atria/drug effects , Humans , Mice , Models, Biological , Oxidative Stress/drug effects , Superoxides/metabolism
17.
J Biol Chem ; 289(46): 32131-32143, 2014 Nov 14.
Article in English | MEDLINE | ID: mdl-25281743

ABSTRACT

Amyloidogenic proteins generally form intermolecularly hydrogen-bonded ß-sheet aggregates, including parallel, in-register ß-sheets (recognized by antiserum OC) or antiparallel ß-sheets, ß-solenoids, ß-barrels, and ß-cylindrins (recognized by antiserum A11). Although these groups share many common properties, some amyloid sequences have been reported to form polymorphic structural variants or strains. We investigated the humoral immune response to Aß42 fibrils and produced 23 OC-type monoclonal antibodies recognizing distinct epitopes differentially associated with polymorphic structural variants. These mOC antibodies define at least 18 different immunological profiles represented in aggregates of amyloid-ß (Aß). All of the antibodies strongly prefer amyloid aggregates over monomer, indicating that they recognize conformational epitopes. Most of the antibodies react with N-terminal linear segments of Aß, although many recognize a discontinuous epitope consisting of an N-terminal domain and a central domain. Several of the antibodies that recognize linear Aß segments also react with fibrils formed from unrelated amyloid sequences, indicating that reactivity with linear segments of Aß does not mean the antibody is sequence-specific. The antibodies display strikingly different patterns of immunoreactivity in Alzheimer disease and transgenic mouse brain and identify spatially and temporally unique amyloid deposits. Our results indicate that the immune response to Aß42 fibrils is diverse and reflects the structural polymorphisms in fibrillar amyloid structures. These polymorphisms may contribute to differences in toxicity and consequent effects on pathological processes. Thus, a single therapeutic monoclonal antibody may not be able to target all of the pathological aggregates necessary to make an impact on the overall disease process.


Subject(s)
Amyloid beta-Peptides/chemistry , Antibodies, Monoclonal/chemistry , Peptide Fragments/chemistry , Alzheimer Disease/immunology , Alzheimer Disease/metabolism , Amino Acid Sequence , Amyloid/chemistry , Animals , Brain/immunology , Brain/metabolism , Epitope Mapping , Epitopes/chemistry , Hot Temperature , Humans , Hydrogen Bonding , Mice , Mice, Transgenic , Molecular Sequence Data , Plaque, Amyloid/chemistry , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid , alpha-Synuclein/chemistry
18.
Neurobiol Dis ; 82: 552-560, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26369878

ABSTRACT

Increased dietary consumption of docosahexaenoic acid (DHA) is associated with decreased risk for Alzheimer's disease (AD). These effects have been postulated to arise from DHA's pleiotropic effects on AD pathophysiology, including its effects on ß-amyloid (Aß) production, aggregation, and toxicity. While in vitro studies suggest that DHA may inhibit and reverse the formation of toxic Aß oligomers, it remains uncertain whether these mechanisms operate in vivo at the physiological concentrations of DHA attainable through dietary supplementation. We sought to clarify the effects of dietary DHA supplementation on Aß indices in a transgenic APP/PS1 rat model of AD. Animals maintained on a DHA-supplemented diet exhibited reductions in hippocampal Aß plaque density and modest improvements on behavioral testing relative to those maintained on a DHA-depleted diet. However, DHA supplementation also increased overall soluble Aß oligomer levels in the hippocampus. Further quantification of specific conformational populations of Aß oligomers indicated that DHA supplementation increased fibrillar (i.e. putatively less toxic) Aß oligomers and decreased prefibrillar (i.e. putatively more toxic) Aß oligomers. These results provide in vivo evidence suggesting that DHA can modulate Aß aggregation by stabilizing soluble fibrillar Aß oligomers and thus reduce the formation of both Aß plaques and prefibrillar Aß oligomers. However, since fibrillar Aß oligomers still retain inherent neurotoxicity, DHA may need to be combined with other interventions that can additionally reduce fibrillar Aß oligomer levels for more effective prevention of AD in clinical settings.


Subject(s)
Alzheimer Disease/diet therapy , Amyloid beta-Peptides/metabolism , Dietary Supplements , Docosahexaenoic Acids , Hippocampus/metabolism , Peptide Fragments/metabolism , Plaque, Amyloid/diet therapy , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Alzheimer Disease/psychology , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Disease Models, Animal , Female , Hippocampus/pathology , Humans , Male , Maze Learning , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Plaque, Amyloid/psychology , Presenilin-1/genetics , Presenilin-1/metabolism , Protein Multimerization , Rats, Sprague-Dawley , Rats, Transgenic , Treatment Outcome
19.
Proc Natl Acad Sci U S A ; 109(51): 20913-8, 2012 Dec 18.
Article in English | MEDLINE | ID: mdl-23213214

ABSTRACT

Although aberrant protein aggregation has been conclusively linked to dozens of devastating amyloid diseases, scientists remain puzzled about the molecular features that render amyloid fibrils or small oligomers toxic. Here, we report a previously unobserved type of amyloid fibril that tests as cytotoxic: one in which the strands of the contributing ß-sheets are out of register. In all amyloid fibrils previously characterized at the molecular level, only in-register ß-sheets have been observed, in which each strand makes its full complement of hydrogen bonds with the strands above and below it in the fibril. In out-of-register sheets, strands are sheared relative to one another, leaving dangling hydrogen bonds. Based on this finding, we designed out-of-register ß-sheet amyloid mimics, which form both cylindrin-like oligomers and fibrils, and these mimics are cytotoxic. Structural and energetic considerations suggest that out-of-register fibrils can readily convert to toxic cylindrins. We propose that out-of-register ß-sheets and their related cylindrins are part of a toxic amyloid pathway, which is distinct from the more energetically favored in-register amyloid pathway.


Subject(s)
Amyloid/chemistry , Congo Red/pharmacology , Crystallography, X-Ray/methods , Fluorescent Dyes/pharmacology , Humans , Hydrogen Bonding , Microscopy, Electron, Transmission/methods , Models, Molecular , Molecular Conformation , Peptides/chemistry , Protein Conformation , Protein Structure, Secondary , Proteins/chemistry , Thermodynamics , X-Ray Diffraction , beta 2-Microglobulin/chemistry
20.
J Neurosci ; 33(15): 6245-56, 2013 Apr 10.
Article in English | MEDLINE | ID: mdl-23575824

ABSTRACT

Alzheimer's disease (AD) is hallmarked by amyloid plaques, neurofibrillary tangles, and widespread cortical neuronal loss (Selkoe, 2001). The "amyloid cascade hypothesis" posits that cerebral amyloid sets neurotoxic events into motion that precipitate Alzheimer dementia (Hardy and Allsop, 1991). Yet, faithful recapitulation of all AD features in widely used transgenic (Tg) mice engineered to overproduce Aß peptides has been elusive. We have developed a Tg rat model (line TgF344-AD) expressing mutant human amyloid precursor protein (APPsw) and presenilin 1 (PS1ΔE9) genes, each independent causes of early-onset familial AD. TgF344-AD rats manifest age-dependent cerebral amyloidosis that precedes tauopathy, gliosis, apoptotic loss of neurons in the cerebral cortex and hippocampus, and cognitive disturbance. These results demonstrate progressive neurodegeneration of the Alzheimer type in these animals. The TgF344-AD rat fills a critical need for a next-generation animal model to enable basic and translational AD research.


Subject(s)
Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Cerebral Cortex/pathology , Cognition Disorders/pathology , Hippocampus/pathology , Nerve Degeneration/pathology , Plaque, Amyloid/pathology , Tauopathies/pathology , Age Factors , Alzheimer Disease/complications , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Behavior, Animal , Cerebral Amyloid Angiopathy , Cerebral Cortex/metabolism , Cognition Disorders/complications , Cognition Disorders/genetics , Cognition Disorders/metabolism , Disease Models, Animal , Female , Gliosis/genetics , Gliosis/pathology , Hippocampus/metabolism , Humans , Male , Nerve Degeneration/genetics , Nerve Degeneration/metabolism , Plaque, Amyloid/genetics , Presenilin-1/genetics , Rats , Rats, Inbred F344 , Rats, Transgenic , Tauopathies/metabolism , tau Proteins/metabolism
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