Nanospray-charge detection mass spectrometry was performed on α-synuclein fibrils amplified from human brains and demonstrated its synergistic combination with real-time quaking-induced conversion to characterize amyloid deposits in neuropathology.
Selective neuronal vulnerability of hippocampal Cornu Ammonis (CA)-1 neurons is a pathological hallmark of Alzheimer's disease (AD) with an unknown underlying mechanism. We interrogated the expression of tuberous sclerosis complex-1 (TSC1; hamartin) and mTOR-related proteins in hippocampal CA1 and CA3 subfields.A human post-mortem cohort of mild (n = 7) and severe (n = 10) AD and non-neurological controls (n = 9) was used for quantitative and semi-quantitative analyses. We also developed an in vitro TSC1 knockdown model in rat hippocampal neurons, and transcriptomic analyses of TSC1 knockdown neuronal cultures were performed.We found a selective increase of TSC1 cytoplasmic inclusions in human AD CA1 neurons with hyperactivation of one of TSC1's downstream targets, the mammalian target of rapamycin complex-1 (mTORC1), suggesting that TSC1 is no longer active in AD. TSC1 knockdown experiments showed accelerated cell death independent of amyloid-beta toxicity. Transcriptomic analyses of TSC1 knockdown neuronal cultures revealed signatures that were significantly enriched for AD-related pathways.Our combined data point to TSC1 dysregulation as a key driver of selective neuronal vulnerability in the AD hippocampus. Future work aimed at identifying targets amenable to therapeutic manipulation is urgently needed to halt selective neurodegeneration, and by extension, debilitating cognitive impairment characteristic of AD.
Abstract Alzheimer’s disease (AD) is the most common cause of dementia worldwide. AD brains display deposits of insoluble amyloid plaques consisting mainly of aggregated amyloid-β (Aβ) peptides, and Aβ oligomers are likely a toxic species in AD pathology. AD patients display altered metal homeostasis, and AD plaques show elevated concentrations of metals such as Cu, Fe, and Zn. Yet, the metal chemistry in AD pathology remains unclear. Ni(II) ions are known to interact with Aβ peptides, but the nature and effects of such interactions are unknown. Here, we use numerous biophysical methods—mainly spectroscopy and imaging techniques—to characterize Aβ/Ni(II) interactions in vitro, for different Aβ variants: Aβ(1–40), Aβ(1–40)(H6A, H13A, H14A), Aβ(4–40), and Aβ(1–42). We show for the first time that Ni(II) ions display specific binding to the N-terminal segment of full-length Aβ monomers. Equimolar amounts of Ni(II) ions retard Aβ aggregation and direct it towards non-structured aggregates. The His6, His13, and His14 residues are implicated as binding ligands, and the Ni(II)·Aβ binding affinity is in the low µM range. The redox-active Ni(II) ions induce formation of dityrosine cross-links via redox chemistry, thereby creating covalent Aβ dimers. In aqueous buffer Ni(II) ions promote formation of beta sheet structure in Aβ monomers, while in a membrane-mimicking environment (SDS micelles) coil–coil helix interactions appear to be induced. For SDS-stabilized Aβ oligomers, Ni(II) ions direct the oligomers towards larger sizes and more diverse (heterogeneous) populations. All of these structural rearrangements may be relevant for the Aβ aggregation processes that are involved in AD brain pathology.
Les amyloidoses sont des maladies caracterisees par l’agregation structuree de proteines, sous forme de fibres amyloides. Le diagnostic precoce de ces maladies represente un enjeu important, pour la prise en charge de nombreuses pathologies associees. Dans ce travail, nous avons montre le ciblage et la detection de plusieurs fibres amyloides, de l’in vitro a l’in vivo. Pour cela, nous avons utilise des nanoparticules multimodales pour l’imagerie medicale (TEP, IRM), greffees avec diverses molecules ciblant les fibres. Trois types de fibres amyloides ont ete testees, formees a partir du peptide amyloide β (maladie d’Alzheimer), d’amyline (diabete de type 2), et de la transthyretine (polyneuropathie familiale). Comme montre par des techniques de spectroscopie et de resonance plasmonique de surface (fluorescence et Biacore), des nanoparticules generiques (du au greffage du Pittsburgh compound B ou d’un nanocorps) ciblent avec une bonne affinite les trois types de fibres in vitro, tandis que des nanoparticules specifiques (du au greffage de peptides) ciblent avec une affinite moindre les fibres d’amyloide β ou de transthyretine. Le ciblage et la detection des depots amyloides par les nanoparticules ont ete confirmes par microscopie a fluorescence, sur des tissus de souris presentant chacune des trois maladies. Le suivi par imageries in vivo (par IRM) et post-mortem (par microscopie optique), apres injection de nanoparticules generiques chez la souris Alzheimer, supposent un ciblage des depots amyloides intracerebraux. Par ailleurs, nous avons detecte les fibres amyloides sans aucun marquage. Des etudes spectroscopiques in vitro ont permis de montrer des proprietes luminescentes intrinseques des fibres amyloides, dans l’UV-visible et le proche infrarouge. Ces caracteristiques ont ete observees sur des coupes de tissus de cerveau de souris Alzheimer par microscopie a fluorescence, et les etudes in vivo en cours semblent prometteuses (par imagerie photo-acoustique, et en temps resolu). Que cela soit par l’utilisation de nanoparticules fonctionnalisees multimodales, ou de proprietes intrinseques des fibres amyloides suggerant une detection completement non-invasive, ces deux strategies innovantes semblent adaptees pour le diagnostic precoce des amyloidoses chez l’Homme.
Multiple sclerosis (MS) and Alzheimer's disease (AD) are neurodegenerative diseases demonstrating age-related accumulation of disability. Inflammation lasting decades is a paradigmatic feature of MS pathology that variably relates to neurodegeneration, while the accumulation of Amyloid-beta (A-beta) plaques and neurofibrillary tangles (NFT) are cornerstones of AD pathology. However, few studies investigated the accumulation of amyloids in MS. We investigated A-beta deposition and NFT density in temporal or frontal cortices derived from a large post-mortem cohort of MS (n=78) and age and sex-matched control (n=65) cases. We found reduced A-beta burden in MS cases compared with controls in particular in cases below 65 years of age. NFT was similarly reduced in MS compared to controls, in particular in cases above 65 years of age. Higher A-beta expression predicted greater NFT density in MS. These findings suggest that MS-related factors may influence A-beta and NFT deposition and/or clearance. This work highlights new therapeutic perspectives relevant for both MS and AD.
Abstract The anterior optic pathway is one of the preferential sites of involvement in CNS inflammatory demyelinating diseases, such as multiple sclerosis and neuromyelitis optica, with optic neuritis being a common presenting symptom. What is more, optic nerve involvement in these diseases is often subclinical, with optical coherence tomography demonstrating progressive neuroretinal thinning in the absence of optic neuritis. The pathological substrate for these findings is poorly understood and requires investigation. We had access to post-mortem tissue samples of optic nerves, chiasms and tracts from 29 multiple sclerosis (mean age 59.5, range 25–84 years; 73 samples), six neuromyelitis optica spectrum disorders (mean age 56, range 18–84 years; 22 samples), six acute disseminated encephalomyelitis (mean age 25, range 10–39 years; 12 samples) cases and five non-neurological controls (mean age 55.2, range 44–64 years; 16 samples). Formalin-fixed paraffin-embedded samples were immunolabelled for myelin, inflammation (microglial/macrophage, T- and B-cells, complement), acute axonal injury and astrocytes. We assessed the extent and distribution of these markers along the anterior optic pathway for each case in all compartments (i.e. parenchymal, perivascular and meningeal), where relevant. Demyelinated plaques were classified as active based on established criteria. In multiple sclerosis, demyelination was present in 82.8% of cases, of which 75% showed activity. Microglia/macrophage and lymphocyte inflammation were frequently found both in the parenchymal and meningeal compartments in non-demyelinated regions. Acute axonal injury affected 41.4% of cases and correlated with extent of inflammatory activity in each compartment, even in cases that died at advanced age with over 20 years of disease duration. An antero-posterior gradient of anterior optic pathway involvement was observed with optic nerves being most severely affected by inflammation and acute axonal injury compared with the optic tract, where a higher proportion of remyelinated plaques were seen. In neuromyelitis optica spectrum disorder, cases with a history of optic neuritis had extensive demyelination and lost aquaporin-4 reactivity. In contrast, those without prior optic neuritis did not have demyelination but rather diffuse microglial/macrophage, T- and B-lymphocyte inflammation in both parenchymal and meningeal compartments, and acute axonal injury was present in 75% of cases. Acute demyelinating encephalomyelitis featured intense inflammation, and perivenular demyelination in 33% of cases. Our findings suggest that chronic inflammation is frequent and leads to neurodegeneration in multiple sclerosis and neuromyelitis optica, regardless of disease stage. The chronic inflammation and subsequent neurodegeneration occurring along the optic pathway broadens the plaque-centred view of these diseases and partly explains the progressive neuroretinal changes observed in optic coherence tomography studies.
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