Fig.4.

Internalization of exogenous Aβ₄₂ is enhanced by binding of IgG autoantibodies. A, B) Differentiated SH-SY5Y cells treated with a physiologically relevant dose (100 nM) of FITC-labeled Aβ₄₂ for 3 and 72 h in medium without human serum showing that Aβ₄₂ internalization can occur in the absence of serum. A) At 3 h, FITC-Aβ₄₂ was localized to small, dispersed and uniformly sized granules. B) As is typically seen in Aβ₄₂-overburdened neurons in regions with AD pathology, a significant fraction of internalized Aβ₄₂ was concentrated within aggregates at 72 h. C) Addition of AD serum to media containing 100 nM FITC-Aβ₄₂ increased the rate and extent of Aβ₄₂ internalization with very little detected on the cell surface. D) Quantification of the effects of different human sera on the rate and extent of FITC-Aβ₄₂ internalization after 72 h of treatment. Exposure of differentiated SH-SY5Y cells to 100 nM FITC-labeled Aβ₄₂ in media without serum (controls) resulted in detection of a relatively low level of intracellular Aβ₄₂. Addition of human serum to the media greatly increased the rate and extent of FITC-labeled Aβ₄₂ internalization observed at 72 h. Comparison of the effects of serum from an AD patient, an age-matched non-demented subject, and a younger non-demented control individual revealed no significant differences in the amount of Aβ₄₂ accumulation over 72 h, and intracellular Aβ₄₂ levels in these groups were all significantly higher than in cells treated with Aβ₄₂ alone. In addition, IgG purified from AD serum showed comparable Aβ₄₂ internalization rates to that of whole serum, suggesting that, among serum components, IgG is largely responsible for the observed Aβ₄₂ internalization in SH-SY5Y cells. Scale bar = 10 μm. ***p < 0.001. AD, Alzheimer’s disease serum; OC, old-aged matched, non-demented control; YC, young-aged, non-demented control; ADpurIgG, purified IgG from AD sera; white arrow, newly internalized/plasma-membrane bound Aβ₄₂; yellow arrow, aggregates of Aβ₄₂-containing granules.