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Amyloid-? protofibrils: size, morphology and synaptotoxicity of an engineered mimic.

ABSTRACT: Structural and biochemical studies of the aggregation of the amyloid-? peptide (A?) are important to understand the mechanisms of Alzheimer's disease, but research is complicated by aggregate inhomogeneity and instability. We previously engineered a hairpin form of A? called A?cc, which forms stable protofibrils that do not convert into amyloid fibrils. Here we provide a detailed characterization of A?42cc protofibrils. Like wild type A? they appear as smooth rod-like particles with a diameter of 3.1 (±0.2) nm and typical lengths in the range 60 to 220 nm when observed by atomic force microscopy. Non-perturbing analytical ultracentrifugation and nanoparticle tracking analyses are consistent with such rod-like protofibrils. A?42cc protofibrils bind the ANS dye indicating that they, like other toxic protein aggregates, expose hydrophobic surface. Assays with the OC/A11 pair of oligomer specific antibodies put A?42cc protofibrils into the same class of species as fibrillar oligomers of wild type A?. A?42cc protofibrils may be used to extract binding proteins in biological fluids and apolipoprotein E is readily detected as a binder in human serum. Finally, A?42cc protofibrils act to attenuate spontaneous synaptic activity in mouse hippocampal neurons. The experiments indicate considerable structural and chemical similarities between protofibrils formed by A?42cc and aggregates of wild type A?42. We suggest that A?42cc protofibrils may be used in research and applications that require stable preparations of protofibrillar A?.

SUBMITTER: Dubnovitsky A

PROVIDER: S-EPMC3699592 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Amyloid-β protofibrils: size, morphology and synaptotoxicity of an engineered mimic.

Dubnovitsky Anatoly A Sandberg Anders A Rahman M Mahafuzur MM Benilova Iryna I Lendel Christofer C Härd Torleif T

PloS one 20130702 7

Structural and biochemical studies of the aggregation of the amyloid-β peptide (Aβ) are important to understand the mechanisms of Alzheimer's disease, but research is complicated by aggregate inhomogeneity and instability. We previously engineered a hairpin form of Aβ called Aβcc, which forms stable protofibrils that do not convert into amyloid fibrils. Here we provide a detailed characterization of Aβ42cc protofibrils. Like wild type Aβ they appear as smooth rod-like particles with a diameter o ...[more]

PMID: 23843949

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Project description:Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimer's disease (AD). Studies employing patient-specific human iPSCs as models of familial and sporadic forms of AD described elevated levels of AD-related amyloid-? (A?). However, none of the present AD iPSC studies could recapitulate the synaptotoxic actions of A?, which are crucial early events in a cascade that eventually leads to vast brain degeneration. Here we established highly reproducible, human iPSC-derived cortical cultures as a cellular model to study the synaptotoxic effects of A?. We developed a highly efficient immunopurification procedure yielding immature neurons that express markers of deep layer cortical pyramidal neurons and GABAergic interneurons. Upon long-term cultivation, purified cells differentiated into mature neurons exhibiting the generation of action potentials and excitatory glutamatergic and inhibitory GABAergic synapses. Most interestingly, these iPSC-derived human neurons were strongly susceptible to the synaptotoxic actions of A?. Application of A? for 8 days led to a reduction in the overall FM4-64 and vGlut1 staining of vesicles in neurites, indicating a loss of vesicle clusters. A selective analysis of presynaptic vesicle clusters on dendrites did not reveal a significant change, thus suggesting that A? impaired axonal vesicle clusters. In addition, electrophysiological patch-clamp recordings of AMPA receptor-mediated miniature EPSCs revealed an A?-induced reduction in amplitudes, indicating an impairment of postsynaptic AMPA receptors. A loss of postsynaptic AMPA receptor clusters was confirmed by immunocytochemical stainings for GluA1. Incubation with A? for 8 days did not result in a significant loss of neurites or cell death. In summary, we describe a highly reproducible cellular AD model based on human iPSC-derived cortical neurons that enables the mechanistic analysis of A?-induced synaptic pathomechanisms and the development of novel therapeutic approaches.

Dataset Information

Amyloid-? protofibrils: size, morphology and synaptotoxicity of an engineered mimic.

Publications

Amyloid-β protofibrils: size, morphology and synaptotoxicity of an engineered mimic.

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