Abstract
Alzheimer's disease (AD) is characterized by significant neurodegeneration in the cortex and hippocampus; intraneuronal tangles of hyperphosphorylated tau protein; and accumulation of β-amyloid (Aβ) proteins 40 and 42 in the brain parenchyma as well as in the cerebral vasculature. The current understanding that AD is initiated by the neuronal accumulation of Aβ proteins due to their inefficient clearance at the blood-brain-barrier (BBB), places the neurovascular unit at the epicenter of AD pathophysiology. The objective of this study is to investigate cellular mechanisms mediating the internalization of Aβ proteins in the principle constituents of the neurovascular unit, neurons and BBB endothelial cells. Laser confocal micrographs of wild type (WT) mouse brain slices treated with fluorescein labeled Aβ40 (F-Aβ40) demonstrated selective accumulation of the protein in a subpopulation of cortical and hippocampal neurons via nonsaturable, energy independent, and nonendocytotic pathways. This groundbreaking finding, which challenges the conventional belief that Aβ proteins are internalized by neurons via receptor mediated endocytosis, was verified in differentiated PC12 cells and rat primary hippocampal (RPH) neurons through laser confocal microscopy and flow cytometry studies. Microscopy studies have demonstrated that a significant proportion of F-Aβ40 or F-Aβ42 internalized by differentiated PC12 cells or RPH neurons is located outside of the endosomal or lysosomal compartments, which may accumulate without degradation. In contrast, BBME cells exhibit energy dependent uptake of F-Aβ40, and accumulate the protein in acidic cell organelle, indicative of endocytotic uptake. Such a phenomenal difference in the internalization of Aβ40 between neurons and BBB endothelial cells may provide essential clues to understanding how various cells can differentially regulate Aβ proteins and help explain the vulnerability of cortical and hippocampal neurons to Aβ toxicity.
Highlights
Alzheimer’s disease (AD), the most frequent form of senile dementia associated with progressive neurodegeneration, is characterized by extracellular amyloid plaques, intra-neuronal tangles, and cerebrovascular amyloid deposits
The extracellular amyloid plaques are predominantly formed in the hippocampus, cerebral cortex and other brain regions important for cognitive function; whereas, the cerebrovascular amyloid deposits are formed in the media and adventitia of small and mid-sized arteries and arterioles present in the cerebral cortex and leptomeninges, as well as cerebral capillaries, resulting in a condition known as cerebral amyloid angiopathy (CAA) [1]
A comparison of fluorescence intensities in bovine brain microvascular endothelial (BBME) cells treated simultaneously with fluorescein labeled Ab40 (F-Ab40) and AF633-Trf at 4uC or 37uC revealed that the cellular uptake of both fluorophores decreased significantly at 4uC compared to that at 37uC (Figure 11I–II). These results demonstrated that the uptake of F-Ab40 in BBME cells is temperature dependent, which is contrary to the observation made in neuronal cells
Summary
Alzheimer’s disease (AD), the most frequent form of senile dementia associated with progressive neurodegeneration, is characterized by extracellular amyloid plaques, intra-neuronal tangles, and cerebrovascular amyloid deposits. The extracellular amyloid plaques are predominantly formed in the hippocampus, cerebral cortex and other brain regions important for cognitive function; whereas, the cerebrovascular amyloid deposits are formed in the media and adventitia of small and mid-sized arteries and arterioles present in the cerebral cortex and leptomeninges, as well as cerebral capillaries, resulting in a condition known as cerebral amyloid angiopathy (CAA) [1]. Both AD and CAA are causatively linked. The debate appears to be settling in favor of Ab proteins as the root cause of AD pathology, one important question still lingers: whether extracellular Ab deposition or intracellular Ab accumulation initiates the AD process
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