Purpose: Alzheimer’s disease (AD), the most common neurodegenerative disorder is characterized by the extracellular deposition of dense amyloid beta (Abeta) plaques. Because AD is still poorly understood, there are currently no treatment options to either delay the onset, or slow the progression of the disease. Current research suggests the progression of the disease is caused by the production and aggregation of the amyloid-beta peptide, otherwise known as the amyloid cascade hypothesis. The monomeric species of this peptide is generally regarded as non-toxic, but its presence in aggregated forms, such as oligomers, protofibrils and mature fibrils, are believed to induce neurotoxicity and dystrophy. Spontaneous Abeta fibrillation in vivo remains poorly understood, but it has been suggested that fibril nucleation in vitro occurs via an intracellular pathway initiated by monomeric Abeta endocytosis. All previous endocytosis studies assess Abeta uptake with cells plated on traditional tissue culture plastic; however, brain tissue is distinctly soft with a low-kPa stiffness. The traditional use of a plastic, ultra-stiff substrate prompts a mechanosensitive response (increased cell spreading, cell stiffness and membrane tension) that potentially distorts a cell’s endocytic behavior from that observed in vivo or in a more physiologically relevant mechanical environment. Considering the brain softens with age, this mechanosensitive endocytosis of Abeta has significant implications for understanding age-related neurodegeneration and the mechanism behind Abeta uptake and fibril production. Overall, identifying these physical factors that contribute to the pathology of AD may offer novel avenues of therapeutic intervention.
Methods: It has been previously shown that cells grown on softer substrates have a reduced membrane tension, and show a modification of both particle and peptide uptake. To model the varying environment of the ECM, polyacrylamide gels were made with varying mechanical moduli, done by adjusting the percent of polyacrylamide (5-10%) and N,N'-methylenebisacrylamide (BIS) (0.3-0.03%). The stiffness of each gel was determined by measuring the shear modulus using rheology. An ECM linker protein (collagen) was then conjugated to the gel surface using sulfo-SANPAH. Integrin expression for SH-SY5Y plated on each substrate was determined using SDS-PAGE. Cell stiffness on each of the substrates was determined using atomic force microscopy. The extent of Abeta internalization over 24 hours was quantified using flow cytometry, and qualitatively confirmed using confocal microscopy.
Results: SH-SY5Y neuroblastoma grown on these gels show a sensitivity to their mechanical environment. An increase in cell spreading, production of stress fibers, and an increase in cell stiffness were observed with an increasing substrate stiffness. Along with these mechanosensitive responses, both Abeta isoforms displayed a significant, endocytic increase for SH-SY5Y cells plated on our softest substrate. Moreover, substrate softening led to the selective uptake of Abeta(1-42) which significantly outpaced Abeta(1-40) after a 24 hour exposure period.
Conclusion: Intracellular Abeta processing remains a central question to understanding the early-stage events in AD pathogenesis. Our studies clearly identify that Abeta endocytosis is sensitive to substrate stiffness; the preferential uptake of Abeta(1-42) into softened neuroblastoma has immediate implications toward the potential aggregation of this Abeta variant and the production to neurotoxic, high-molecular weight aggregates. This work demonstrating the biophysical role of ECM stiffness in modifying Abeta internalization offers an improved in vitro model to correlate with further in vivo studies, and could potentially offer novel avenues of therapeutic intervention, once the role of the ECM is understood.
Kendra Bell– Student, University of Iowa
Thiranjeewa Lansakara Mudiyanselage– University of Iowa, Iowa
Alexei Tivanski– Associate Professor, University of Iowa, Iowa
Lewis Stevens– Assistant Professor, University of Iowa, Iowa