Abstract
Alzheimer’s disease is a debilitating neurodegenerative condition that progressively causes synaptic loss and major neuronal damage. Immunotherapy utilising Aβ as an active immunogen or via passive treatment utilising antibodies raised to amyloid have shown therapeutic promise. The migratory properties of peripheral blood-borne monocytes and their ability to enter the central nervous system, suggests a beneficial role in mediating tissue damage and neuroinflammation. However, the intrinsic phagocytic properties of such cells have pre-disposed them to internalise misfolded amyloidogenic peptides that could act as seeds capable of nucleating amyloid formation in the brain. Mechanisms governing the cellular fate of amyloid therefore, may prove to be key in the development of future vaccination regimes. Herein, we have developed unequivocal and direct conformation-sensitive fluorescent molecular probes that reveal the intracytoplasmic and intranuclear persistence of amyloid in a monocytic T helper 1 (THP-1) cell line. Use of the pathogenic Aβ42 species as a model antigen in simulated vaccine formulations suggested differing mechanisms of cellular internalisation, in which fibrillar amyloid evaded lysosomal capture, even when co-deposited on particulate adjuvant materials. Taken collectively, direct fluorescent labelling of antigen-adjuvant complexes may serve as critical tools in understanding subsequent immunopotentiation in vaccines directed against amyloidosis and wider dementia.
Highlights
Alzheimer’s disease (AD) is the most prevalent form of dementia manifesting neurologically and symptomatically arising through the loss of spatial and short-term memory
Thioflavin T (ThT) fluorescence of the amyloidogenic Aβ42 peptide was first assessed in R10 cell culture medium, to ensure the formation of mature amyloid fibrils in treatment conditions compatible with subsequent T helper 1 (THP-1) cell culture
Aβ42 pre-incubated for 24 h in R10 medium containing thioflavin T (ThT) and co-cultured (1:1) with THP-1 cells for 1 h (37 °C, 5% CO2), revealed the cellular uptake of amyloid in a β-sheet conformation as identified through a green fluorescence emission (482 nm) contained in cell cytosol (Fig. 1)
Summary
Alzheimer’s disease (AD) is the most prevalent form of dementia manifesting neurologically and symptomatically arising through the loss of spatial and short-term memory. Through a cascade of deleterious effects, the amyloid plaques formed are suggested to break down releasing neurotoxic oligomeric species of Aβ, resulting in synaptic loss and neuronal damage[1,3,4] While this theory has been disputed with calls for its reassessment, current consensus supports the hypothesis that the accumulation of the Aβ peptides in vivo, contributes to the pathogenesis of both familial and sporadic forms of AD1,5. The recapitulation of the mechanisms underlying the onset of AD is most commonly achieved through the use of transgenic mouse models of the disease state[6,7] These animals typically overexpress mutant human forms of APP and the presenilin genes, triggering the progressive development of many of the pathological hallmarks of AD. Immunisation trials continue to highlight the potential of vaccines directed against Aβ in the effective clearance of AD neuropathology
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