Lysosome‐autophagosome Defects Mediate Proteinopathy in Early Stages of Alzheimer’s Disease

AbstractBackgroundAlzheimer’s disease (AD) is a neurodegenerative disorder characterized by abnormal protein aggregation and synaptic deficits. To regulate normal cellular recycling of cellular debris, lysosomes require an acidic environment which is maintained by an active vacuolar H+‐ATPase (V‐ATPase proton pump). The acidic pH in lysosomes is essential for autophagy, a catabolic pathway to degrade cellular proteins. In AD, V‐ATPase disruption can lead to abnormal β‐amyloid and tau accumulation. A candidate mechanism concurrent with V‐ATPase defects is Ca2+ dyshomeostasis, which alters the ion exchange and thus pH within these organelles. We hypothesize that altered Ca2+ signaling disrupts V‐ATPase ion exchange and instigates AD pathology.MethodImmunohistochemistry in fixed brain slices was used to determine expression of V‐ATPase subunits (V1B2, V0a1), lysosomes (Lamp1), hyperphosphorylated tau (S262) and mature autophagosomes (LC3B) in 4‐month old 3xTg‐AD mice and non‐transgenic (NTg) controls with or without a 30‐day Ryanodex treatment (NAM of RyR; 10mg/kg). Lysosomal pH upon RyR stimulation with caffeine (20mM) was measured in RyR‐overexpressing HEK cells and iN using Lysosensor. Induced human neurons (iN) transformed from AD patient fibroblasts were used to measure, autophagosomes, β‐amyloid, and hyperphosphorylated tau levels after500nM bafilomycin V‐ATPase inhibitor treatment.ResultV‐ATPase and lysosome markers are diminished in brains of 3xTg‐AD mice relative to NTg, whereas LC3B expression is increased. These phenotypes were restored to NTg levels after Ryanodex treatment in 3xTg‐AD mice. Additionally, increased aberrant RyR‐Ca2+ signaling alkalizes lysosomes and increases expression of hyperphosphorylated tau and autophagosomes in iN and RyR‐overexpressing HEK cells, with no significant difference with of β‐amyloid levels.ConclusionPrior to overt histopathology or cognitive deficits, decreased V‐ATPase expression on acidic compartments and increased autophagosomes in 3xTg‐AD mice reflects impaired lysosomal activity, which is mediated through upstream RyR‐Ca2+ dyshomeostatis. Increased RyR‐mediated Ca2+ release alkalizes lysosomes pH resulting in abnormal intracellular protein aggregates, demonstrating that aberrant intracellular Ca2+ signaling in early AD disrupts autophagy and protein clearance.