Intraneuronal Alzheimer abeta42 accumulates in multivesicular bodies and is associated with synaptic pathology

Abstract

A central question in Alzheimer's disease concerns the mechanism by which beta-amyloid contributes to neuropathology, and in particular whether intracellular versus extracellular beta-amyloid plays a critical role. Alzheimer transgenic mouse studies demonstrate brain dysfunction, as beta-amyloid levels rise, months before the appearance of beta-amyloid plaques. We have now used immunoelectron microscopy to determine the subcellular site of neuronal beta-amyloid in normal and Alzheimer brains, and in brains from Alzheimer transgenic mice. We report that beta-amyloid 42 localized predominantly to multivesicular bodies of neurons in normal mouse, rat, and human brain. In transgenic mice and human Alzheimer brain, intraneuronal beta-amyloid 42 increased with aging and beta-amyloid 42 accumulated in multivesicular bodies within presynaptic and especially postsynaptic compartments. This accumulation was associated with abnormal synaptic morphology, before beta-amyloid plaque pathology, suggesting that intracellular accumulation of beta-amyloid plays a crucial role in Alzheimer's disease.

Figure 1.

Ultrastructural localization of Aβ42 in neurons of normal mouse, rat, and human brain. A: Prominent Aβ42 immunogold labeling in MVBs (arrows) within a neuronal perikaryon in hippocampus (CA1) of a normal 11-month-old mouse. Note also an isolated gold particle in the ER. B: Perikaryon in CA1 region of hippocampus of a normal young rat; note Aβ42 immunogold labeling of two MVBs adjacent to a larger unlabelled endosome. Occasional gold particles also stained small clear vesicles and at times, gold particles are found in poorly identifiable locations. Inset demonstrates an example of MVB Aβ42 staining in another hippocampal perikaryon of rat brain; this MVB contains several intravesicular vesicles (arrow) that are characteristic of MVBs. C: Aβ42 immunoreactivity in a MVB of a normal human perikaryon derived from a rapidly fixed specimen of cerebral cortex from a 44-year-old with surgical resection in the setting of an acute aneurysm. D: Monoclonal anti-Aβ42 antibody MBC42 does not cross-react with APP CTFs in APP Swedish-transfected N2a cells (top) or Tg2576 mouse brain (bottom). For N2a cells, total Aβ (left) or Aβ42 (right) are shown in lysate (L) and conditioned media (M). Only the antibody directed at the mid-portion of total Aβ (4G8) but not anti-Aβ42 (MBC42) reacts against APP CTFs. The lack of MBC42 staining of CTFs is also demonstrated by this immunoprecipitation/Western blot of 12-month-old Tg2576 mouse brain, where Aβ42 is evident but not CTFs or full-length APP (flAPP). IgG bands represent nonspecific immunoglobulin bands observed as a results of the IP/Western blot. E: Dual-labeling immuno-EM showing full-length APP and APP CTFs (using C-terminal APP antibody 369) with dark immunoperoxidase reaction product primarily in Golgi, and immunogold labeling of Aβ42 (using monoclonal antibody MBC42) demonstrating labeling of an MVB (arrow) close to Golgi in this neuron from mouse cerebral cortex. Abbreviations: MVB, multivesicular body; Golgi, Golgi apparatus; ER, endoplasmic reticulum; mit, mitochondrion; Endo, endosome; N, nucleus. Scale bars: 300 nm (A–C); 500 nm (E).

Figure 2.

Absence of intraneuronal Aβ42 in APP knockout mice. A: Immunogold labeling of several MVBs (arrows) in a neuronal perikaryon from a wild-type mouse brain. B: Absence of immunogold labeling of MVBs in a neuron from an APP knockout mouse. A and B were CA1 hippocampal sections from 12-month-old littermates processed in parallel. Insets in A and B represent lower power views to emphasize the lack of Aβ42 immunoreactivity in several neurons of APP knockout mouse brain; Aβ42 gold particles within several neuronal perikarya from wild-type mouse are evident (inset in A) compared with lack of gold particles in several neurons of a knockout mouse (inset in B) processed in parallel. Arrows indicate MVBs. Abbreviations: MVBs, multivesicular bodies; N, nucleus. Scale bars, 1 μm.

Figure 3.

Neuronal Aβ42 immunoreactivity increases with aging in the brains of Tg2576 mice containing the APP Swedish 670/671 FAD mutation. A: Immunoprecipitation and Western blot of 4-kd human Aβ in equivalent brain tissue (parietal cortex) of Tg2576 mice at 3, 9, and 16 months. Note the total Aβ increases in preplaque mouse brains between 3 and 9 months. B and C: Aβ42 immunogold MVB labeling (arrows) of representative dendrites from young (2 months; B) and older preplaque (10 months; C) Tg2576 mice. Abbreviations: Den, dendrite; MVB, multivesicular body. D: Bar graph indicating the number of gold particles per MVB in neuronal soma (left) or dendrites (right) in 2-month-old (open bars) and 10-month-old (filled bars) Tg2576 mice (asterisk denotes statistical significance). E: Frequency distribution of total number of MVBs containing various numbers of gold particles in distal dendritic processes of young (2 to 3 months, open circles) and old (10 months, filled circles) Tg2576 mice. Scale bars, 500 nm (A–C).

Figure 4.

Aβ42 accumulates within neuronal processes in Tg2576 mice before plaque formation. A: A dendritic process with significant MVB-associated Aβ42 shows several apparent morphological abnormalities in the cortex of a preplaque 10-month-old mouse (arrowheads indicate outer membrane of this dendrite). Increased Aβ42 immunogold labeling is seen within this dendrite, which, based on its relative darkness (asterisk), appears to be degenerating. Unusual images of Aβ42 MVBs associated with lysosomes (such as the MVB under the single arrow at the far right) and abnormally dark Aβ42 immunogold MVBs (see two MVBs in inset and MVB under the single arrow) are observed. Inset is higher magnification of two darker appearing Aβ42 immunogold-associated MVBs above the paired arrows. B: In a 17-month-old mouse (B to D), a distended dendritic profile traversing the image with Aβ42 immunogold, and absence of normal cytoskeletal organelles. A second dendrite with Aβ42 is seen at the left with an abnormal dense body (asterisk) close to multiple MVB-associated gold particles; inset is a higher magnification of this MVB, in which characteristic small intravesicular vesicles can be seen (arrow). C: Axon terminal (At1) with substantial Aβ42 immunogold and some electron-dense components suggestive of degeneration. Another axon terminal (At2) with Aβ42 accumulation close to a synapse (arrow) with a dendritic spine. At left is a dendrite with abnormally stacked membranes (curved arrow) and Aβ42 accumulation. Inset shows increase in Aβ42 immunogold within dendritic and axonal compartments close to another synaptic density (arrow). D: At left, is a poorly defined process (asterisk) with Aβ42 accumulation also containing clusters of abnormal, darker membranes. Inset shows degenerating dark profiles (asterisk) seemingly within an axon terminal that contains clusters of Aβ42 gold labeling. Abbreviations: Den, dendrite; At, axon terminal. Scale bars, 300 nm.

Figure 5.

Aβ42 accumulates within neuronal soma and processes in human AD brain. A: Human AD cortical brain specimen showing substantial amounts of Aβ42 labeling within MVBs of an abnormal appearing neuronal soma (arrow); curved arrow indicates lipofuscin devoid of specific immunogold labeling. Nucleus has abnormal appearing accumulation of heterochromatin near the outer membrane. Insets represent higher power views of Aβ42 immunogold labeling of MVBs in neuronal soma. B: Multiple Aβ42 gold particles are associated with two MVBs in a dendrite. C: Multiple aggregated Aβ42 immunogold particles are found within a disrupted, swollen dendrite containing electron-dense material (asterisk), indicative of degeneration. Abbreviations: Den, dendrite; mit, mitochondrion; N, nucleus. Scale bars: 1 μm (A); 500 nm (B and C).