ABSTRACT: In this study, we have evaluated amyloidosis-relevant Aβ proteoform flux using immunoenrichment and quantitative Top-Down Mass Spectrometry in a well-studied 5xFAD mouse model of age-dependent Aβ amyloidosis.
Project description:Amyloid-beta (Aβ) plays a key role in the neuropathogenesis of Alzheimer’s disease, but little is known about the proteoforms (i.e., all protein variants of a single gene including post-translational modifications and sequence variants) present in human AD brain. We used high-resolution mass spectrometry to analyze intact, whole Aβ from soluble and insoluble aggregates in brains of 6 cases with severe dementia and pathologically confirmed Alzheimer’s disease. We found an extraordinary diversity of Aβ peptides totaling 91 unique proteoforms including various N- and C-terminal truncations, 17 different types of post-translational modifications (PTMs), and remarkably 5 amino acid variants in the peptide sequence never before described. Ratios of Aβ proteoforms did not distinguish between soluble and insoluble aggregates, but four individual Aβ proteoforms, three in the insoluble and one in the soluble fraction, were markedly increased when compared across aggregates. Such heterogeneity of the Aβ peptide both deepens our understanding of AD, but perhaps also warrants pause and consideration in our investigation into pathological mechanisms of the disease and therapeutic development.
Project description:Amyloid-beta (Aβ) plays a key role in the pathogenesis of Alzheimer’s disease (AD), but little is known about the proteoforms present in human AD brain. We used high-resolution mass spectrometry to analyze intact, undigested Aβ from purified soluble aggregates and insoluble material in brains of 6 cases with severe dementia and pathologically confirmed AD. The soluble aggregates are especially relevant because they are believed to be the most toxic form of Aβ. We found a diversity of Aβ peptides, with 26 unique proteoforms including various N- and C-terminal truncations. N- and C-terminal truncations comprised 73% and 30%, respectively, of the number of Aβ proteoforms detected. The Aβ proteoforms segregated between the soluble and more insoluble aggregates with N-terminal truncations predominating in the insoluble material and C- terminal truncations segregating into the soluble aggregates. In contrast, canonical Aβ comprised the minority of the number of identified proteoforms (15.3%) and did not distinguish between the soluble and more insoluble aggregates. The relative abundance of many truncated Aβ proteoforms did not correlate with post-mortem interval, suggesting they are not artefacts. This heterogeneity of Aβ proteoforms deepens our understanding of AD and offers many new avenues for investigation into pathological mechanisms of the disease, with implications for therapeutic development.
Project description:Brain-derived amyloid-β (Aβ) dimers are associated with Alzheimer´s disease (AD). However, their covalent nature remains controversial. This feature is relevant, as a covalent cross-link would make brain-derived dimers (native dimers) more synaptotoxic than Aβ monomers and would make them suitable candidates for biomarker development. To resolve this controversy, we here present a three-step approach. First, we validated a type of synthetic cross-linked Aβ (CL Aβ) dimers, obtained by means of the photo-induced cross-linking of unmodified proteins (PICUP) reaction, as well-defined mimics of putative native CL Aβ dimers. Second, we used these PICUP CL Aβ dimers as standards to improve the isolation of native Aβ dimers and to develop state-of-the-art mass spectrometry (MS) strategies to allow their characterization. Third, we applied these MS methods to the analysis of native Aβ dimer samples allowing the detection of the CL [Aβ(6-16)]2 peptide comprising a dityrosine cross-link. This result demonstrates the presence of CL Aβ dimers in the brains of patients with AD and opens up avenues for establishing new therapeutic targets and developing novel biomarkers for this disease.
Project description:Microglia, the innate immune cells of the central nervous system, perform critical inflammatory and non-inflammatory functions to maintain homeostasis and normal neural function. However in Alzheimer’s disease (AD), these beneficial functions become progressively impaired, contributing to synapse and neuron loss and cognitive impairment. The inflammatory cyclooxygenase-PGE2 pathway, including the PGE2 receptor EP2, is implicated in AD development, both in human epidemiology and in transgenic models of AD. To test the transcriptional responses of EP2-deficient microglia to Aβ in vivo, we used mice in which the EP2 receptor is conditionally deleted in microglia using the CD11b-Cre transgene and floxed alleles of the EP2 gene. By injecting these mice with Aβ ICV and isolating microglia from the brains, we have been able to establish the transcriptional response of microglia to Aβ in vivo and test how EP2 deletion in microglia affects this response. 8 month-old C57BL/6 mice, of the genotype CD11b-Cre; EP2+/+ or CD11b-Cre; EP2lox/lox, were injected I.C.V. with either Aβ or vehicle. 48 hours after injection, the mice were sacrificed and transcardially perfused with cold heparinized 0.9% NaCl. Brains were then removed from the mice and pooled, two brains of the same genotype per sample, to ensure adequate cell and RNA yield. The brains were then enzymatically dissociated for microglia isolation using the Neural Tissue Dissociation Kit (P), MACS Separation Columns (LS), and magnetic CD11b Microbeads from Miltenyi Biotec according to the manufacturer's protocol. Immediately after isolating the microglia, RNA was extracted from the cells for microarray analysis.
Project description:The aggregation of amyloid beta (Aβ) peptide is associated with Alzheimer’s disease (AD) pathogenesis. Cell membrane composition, especially monosialotetrahexosylganglioside (GM1), is known to promote the formation of Aβ fibrils, yet little is known about the roles of GM1 in the early steps of Aβ oligomer formation. Here, by using GM1-contained liposomes as a mimic of neuronal cell membrane, we demonstrate that GM1 is a critical trigger of Aβ oligomerization and aggregation. We find that GM1 not only promotes the formation of Aβ fibrils, but also facilitates the maintenance of Aβ oligomers on liposome membranes. We structurally characterize the Aβ oligomers formed on the membrane and find that GM1 captures Aβ by binding to its arginine-5 residue. To interrogate the mechanism of Aβ oligomer toxicity, we design a new liposome-based Ca2+-encapsulation assay and provide new evidence for the Aβ ion channel hypothesis. Finally, we conduct cell viability assay to determine the toxicity of Aβ oligomers formed on membranes. Overall, by uncovering the roles of GM1 in mediating early Aβ oligomer formation and maintenance, our work provides a novel direction for pharmaceutical research for AD.
Project description:Alzheimer’s disease (AD) is the most common form of neurodegeneration presented mainly as late-onset AD (LOAD), and in younger individuals early-onset AD (EOAD). The pathological hallmarks of AD are the hyperphosphorylated tau neurofibrillary tangles and the amyloid beta (Aβ) plaques. We examined changes in the proteome of post-mortem temporal cortex from individuals with EOAD and LOAD, and revealed that EOAD represents a more aggressive form of the disease. Notably, we observed up-regulation of AD biomarkers p-tau181, p-tau217, and p-tau23, and tau MTBR243, in AD tissues. Two abundant tau peptides phosphorylated at p-231/p-235 and p-231/p-235/p-237 were present largely in AD tissues, which may be developed as biomarkers. Aβ was detected in about half of the cognitively unimpaired elderly controls with subtle proteome changes that resembled in part the changes of biological processes in AD. This could indicate an early stage of neurodegeneration preceding clinical AD symptoms or represent an AD-resilient state.
Project description:Alzheimer’s disease (AD) is the most common form of neurodegeneration presented mainly as late-onset AD (LOAD), and in younger individuals early-onset AD (EOAD). The pathological hallmarks of AD are the hyperphosphorylated tau neurofibrillary tangles and the amyloid beta (Aβ) plaques. We examined changes in the proteome of post-mortem temporal cortex from individuals with EOAD and LOAD, and revealed that EOAD represents a more aggressive form of the disease. Notably, we observed up-regulation of AD biomarkers p-tau181, p-tau217, and p-tau23, and tau MTBR243, in AD tissues. Two abundant tau peptides phosphorylated at p-231/p-235 and p-231/p-235/p-237 were present largely in AD tissues, which may be developed as biomarkers. Aβ was detected in about half of the cognitively unimpaired elderly controls with subtle proteome changes that resembled in part the changes of biological processes in AD. This could indicate an early stage of neurodegeneration preceding clinical AD symptoms or represent an AD-resilient state.
Project description:Alzheimer’s disease (AD) is the most common form of neurodegeneration presented mainly as late-onset AD (LOAD), and in younger individuals early-onset AD (EOAD). The pathological hallmarks of AD are the hyperphosphorylated tau neurofibrillary tangles and the amyloid beta (Aβ) plaques. We examined changes in the proteome of post-mortem temporal cortex from individuals with EOAD and LOAD, and revealed that EOAD represents a more aggressive form of the disease. Notably, we observed up-regulation of AD biomarkers p-tau181, p-tau217, and p-tau23, and tau MTBR243, in AD tissues. Two abundant tau peptides phosphorylated at p-231/p-235 and p-231/p-235/p-237 were present largely in AD tissues, which may be developed as biomarkers. Aβ was detected in about half of the cognitively unimpaired elderly controls with subtle proteome changes that resembled in part the changes of biological processes in AD. This could indicate an early stage of neurodegeneration preceding clinical AD symptoms or represent an AD-resilient state.
Project description:Alzheimer’s disease (AD) is the most common form of neurodegeneration presented mainly as late-onset AD (LOAD), and in younger individuals early-onset AD (EOAD). The pathological hallmarks of AD are the hyperphosphorylated tau neurofibrillary tangles and the amyloid beta (Aβ) plaques. We examined changes in the proteome of post-mortem temporal cortex from individuals with EOAD and LOAD, and revealed that EOAD represents a more aggressive form of the disease. Notably, we observed up-regulation of AD biomarkers p-tau181, p-tau217, and p-tau23, and tau MTBR243, in AD tissues. Two abundant tau peptides phosphorylated at p-231/p-235 and p-231/p-235/p-237 were present largely in AD tissues, which may be developed as biomarkers. Aβ was detected in about half of the cognitively unimpaired elderly controls with subtle proteome changes that resembled in part the changes of biological processes in AD. This could indicate an early stage of neurodegeneration preceding clinical AD symptoms or represent an AD-resilient state.
Project description:Alzheimer’s disease (AD) is the most common form of neurodegeneration presented mainly as late-onset AD (LOAD), and in younger individuals early-onset AD (EOAD). The pathological hallmarks of AD are the hyperphosphorylated tau neurofibrillary tangles and the amyloid beta (Aβ) plaques. We examined changes in the proteome of post-mortem temporal cortex from individuals with EOAD and LOAD, and revealed that EOAD represents a more aggressive form of the disease. Notably, we observed up-regulation of AD biomarkers p-tau181, p-tau217, and p-tau23, and tau MTBR243, in AD tissues. Two abundant tau peptides phosphorylated at p-231/p-235 and p-231/p-235/p-237 were present largely in AD tissues, which may be developed as biomarkers. Aβ was detected in about half of the cognitively unimpaired elderly controls with subtle proteome changes that resembled in part the changes of biological processes in AD. This could indicate an early stage of neurodegeneration preceding clinical AD symptoms or represent an AD-resilient state.