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From the Departments of Neurology and Neuroscience* and Pathology, Weill Medical College of Cornell University, New York, New York; the Fisher Center for Alzheimer’s Research and Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York; and Gunma University School of Health Sciences, Gunma, Japan
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Abstract |
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Although Aß40 is the predominantly secreted Aß species in tissue culture, the longer Aß42 is more important in AD. Aß42 is increased in familial AD (FAD) because of mutations in the amyloid precursor protein (APP) or presenilins 1 and 2, and it accumulates as the first Aß species in Down’s syndrome and AD.1 Cell biological studies indicate that Aß generation occurs in the Golgi apparatus (Golgi), especially the trans-Golgi network, and to a lesser extent in the endoplasmic reticulum (ER). The ER appears to be more selective for Aß42 than Aß40 generation.3,4 Moreover, the ER pool of Aß42 appears to be secreted less efficiently than that from the Golgi. Aß peptide generation also occurs in the endocytic pathway after internalization from the plasma membrane.10,11
AD pathogenesis has traditionally been characterized by extracellular, aggregated, and plaque-associated Aß. However, the extent to which extracellular Aß causes cell death in AD is unclear. An increasing number of reports demonstrate behavioral, synaptic, physiological, and oxidative abnormalities months before the appearance of Aß plaques in FAD transgenic mice that develop AD-like plaque pathology.5,6,9,12
In Tg2576 mice, which express the human APP Swedish 670/671 FAD mutation,13
brain levels of Aß rise sharply as measured by enzyme-linked immunosorbent assay (ELISA) at 6 to 10 months,8
before development of plaques, which appear at 
11 to 13 months.14
Preplaque increases in soluble Aß42, but not Aß40, have been reported in Down’s syndrome brain.15
Studies of postmortem human brain show that soluble Aß correlates better than insoluble Aß or extracellular Aß plaques with cognitive dysfunction in patients with AD.16
Thus, soluble Aß42 protofibrils may play a critical pathogenic role.1
The location of the soluble Aß42 protofibrils and how they cause toxicity are of growing interest. Aß protofibrils are preferentially generated within cells rather than extracellularly.17 Neuronal NT2 cells contain abundant intracellular Aß42, which increases dramatically with time (aging) in culture.18 Transgenic mice expressing a FAD mutant presenilin 1 were reported to develop intraneuronal Aß42 accumulation and neurodegeneration.19 Intracellular Aß accumulation has been associated with pathology in inclusion body myositis20 and mucopolysaccharidosis.21 Thus, cumulative indirect evidence suggests that there may be a pathological role for neuronal Aß accumulation in AD. Using three different well-characterized pairs of polyclonal antibodies against Aß40 or Aß42, we previously reported that intraneuronal Aß42, as viewed by light microscopy, accumulates especially within AD-susceptible pyramidal neurons in postmortem tissue of patients with clinically defined early cognitive impairment.2 Intraneuronal Aß42, but not Aß40, accumulation with AD pathology now has been reported by light microscopy by several other groups.22-26 In the present study, the subcellular localization of Aß within neurons was examined using immunogold electron microscopy (EM) to localize where brain Aß increases occur before the development of plaques, in a mouse model of AD-like Aß pathology. We provide evidence implicating intraneuronal Aß42 accumulation in synaptic pathology both in AD transgenic mouse and human AD brains.
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Materials and Methods |
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Monoclonal antibodies to the C-terminus of Aß40 and Aß42 were MBC40 and MBC42, respectively, generated by Dr. Haruyasu Yamaguchi, Gunma University, Gunma, Japan. MBC42 does not recognize Aß40 or Aß43.27 The specificities of MBC40 and MBC42 antibodies were determined by immunoprecipitation of human APP–transfected neuroblastoma (N2a) cells, synthetic Aß40 or Aß42 with MBC40 or MBC42 antibody, followed by Western blotting with 6E10 monoclonal antibody (Signet Laboratories, Dedham, MA) as previously described.28 Aß42 immuno-EM results were confirmed using a commercially available polyclonal anti-Aß42 antibody (Chemicon Inc., Temecula, CA). The C-terminus of full-length APP was recognized using antibody 369.29
Animals
Well-established Tg2576 mice (n = 22) with the human APP Swedish 670/671 mutation of varying ages (four, 2 months; three, 3 months; two, 4 months; one, 8 months; three, 9 months; four, 10 months; two, 11 to 12 months; two, 16 to 17 months; one, 20 months) were used in this study. APP knockout mice and wild-type littermates (n = 4, each; two 6-month-old and two 12-month-old mice, each) were generously provided by Dr. Hui Zheng, Baylor College of Medicine, Houston, TX. Sprague-Dawley rats (300 to 325 g) were obtained from Taconic Farms (Germantown, NY). All methods were approved by the Weill Medical College of Cornell University Institutional Animal Care and Use Committee and conformed to National Institute of Health guidelines.
Human Brain Tissue
Human cortical brain tissue was obtained from the Department of Pathology, Weill Medical College of Cornell University as a result of neurosurgical procedures unrelated to this study. Neurosurgical AD cortical tissue was obtained (n = 2, ages 54 and 62 years). Relatively normal cortical brain tissue was obtained from emergency aneurysm clipping and benign tumor resection (n = 2, ages 44 and 54 years).
Pre-Embedding Immunogold Electron Microscopy
Mice and rats were anesthetized with sodium pentobarbital (150 mg/kg, i.p.) and perfused via the ascending aorta with 3.75% acrolein (Polyscience, Warrington, PA) and 2% paraformaldehyde in 0.1 mol/L of phosphate buffer (pH 7.4). Human brain biopsy tissue was rapidly immersion-fixed with 1.875% acrolein and 2% paraformaldehyde in 0.1 mol/L of phosphate buffer. Brain tissue was cut (40-µm thick) on a vibrating microtome and treated with 1% sodium borohydride as previously described.30
Some sections were processed for EM using immunoperoxidase, as described previously,30 that revealed an analogous staining pattern (predominantly MVBs) with MBC42 antibody, but the subcellular localization was not as discreet as with immunogold. For immunogold labeling, free-floating sections were labeled with either MBC42, MBC40, 369, or Chemicon Aß42 antibodies by the immunogold-silver procedure of Chan and colleagues.31 For the processing, the tissue was incubated in goat anti-mouse (for MBC40 and MBC42) or goat anti-rabbit (for 369 and Chemicon anti-Aß42 antibody) IgG conjugated to 1-nm gold particles (Amersham, Arlington Heights, IL) in 0.01% gelatin and 0.08% bovine serum albumin in phosphate-buffered saline (PBS). The conjugated gold particles were enhanced by treatment with silver solution (IntenSE, Amersham).
Dual immuno-EM localization was performed as previously described30
now using polyclonal rabbit anti-APP (369) and monoclonal anti-Aß42 (MBC42). Sections were incubated with both 369 and MBC42, and then processed first for immunoperoxidase localization of APP and then with the immunogold-silver method for localization of Aß42. Aß42 antibody concentrations were 1:50, Aß40 antibody concentrations were 1:20, and APP antibody concentrations were 1:500. Sections were fixed in 2% osmium tetroxide in phosphate buffer, embedded in EMBed 812, sectioned (65- to 76-nm thick) and counterstained with uranyl acetate and Reynolds’ lead citrate.30
Final preparations were examined with a Philips CM10 electron microscope. Morphological terminology is consistent with Peters and colleagues.32
Quantification was done by counting gold particles from random images taken at the same magnification from analogous cerebral cortical regions with neuronal soma (layer 5) or processes of either 2- or 10-month-old Tg2576 mice. Student’s t-test was used for statistical analyses. Final illustrations were generated from scanned photographic prints (for Figure 1E
; Figure 3, B and C
; Figure 4A
; and Figure 5, A, B, and C
) or from a high-resolution digital imaging charge-coupled device camera system (Advanced Microscopy Techniques Corp., Danvers, MA) and processed on a Power Macintosh 8500/120 using Adobe Photoshop 6.0 (Adobe System, Mountain View, CA) and Quark X-Press 3.32 (Quark, Denver, CO).
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Equal amounts (25 mg) of brain tissue snap-frozen in liquid nitrogen derived from analogous cortical regions from brains of Tg2576 mice sacrificed at different ages (3, 9, and 16 months) or N2a cells stably transfected with Swedish 670/671 FAD mutation human APP (generously provided by Drs. Gopal Thinakaran and Sangram Sisodia, University of Chicago, Chicago, IL) were assayed for determination of Aß levels as previously described for cell lysates28 with slight modifications. Samples from frontal and parietal cortex were analyzed for Aß. Brain samples or N2a cells were lysed in 6% or 4% sodium dodecyl sulfate, respectively, vortexed, and heated at 95°C for several minutes, followed by sonication. Samples were spun at 15,000 x g, and supernatants adjusted to 190 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 8.3, 6 mmol/L ethylenediaminetetraacetic acid, and 2.5% Triton X-100 and incubated overnight with antibody 4G8 (Signet Laboratories), followed by addition of secondary rabbit anti-mouse antibody (Cappell) for 1 hour and then addition of protein A-Sepharose beads (Pharmacia) for 2 hours (all at 4°C). Samples were run on 10 to 20% Tris-Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels followed by electrophoretic transfer to polyvinylidene-difluoride membrane (Millipore, Bedford, MA). Polyvinylidene-difluoride membranes were boiled in PBS for 5 minutes, followed by Western blotting with 6E10 for human Aß and visualization after enhanced chemiluminescence on Kodak X-OMAT AR5 film (Eastman-Kodak, Rochester, NY).
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Results |
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In agreement with a previous report,27
standard fixation of normal rat brain for EM with paraformaldehyde/glutaraldehyde revealed primarily isolated Aß42 immunolabeling at the plasma membrane (data not shown). In contrast, fixation with a mixture of acrolein and paraformaldehyde used in the present study, which allows for more optimal antigen detection by immuno-EM,33
revealed marked intraneuronal Aß, especially Aß42. In normal mouse (Figure 1A)
and rat (Figure 1B)
brain, Aß42 was most prominent on multivesicular bodies (MVBs), especially of pyramidal neurons in the cerebral cortex and hippocampus, which are particularly prone to AD neuropathology. Intraneuronal Aß42 was less pronounced in neurons known to be relatively resistant to AD, such as in the basal ganglia. More than half of Aß42 gold particles labeling identifiable organelles within neurons in the brain of normal mouse or rat were found on MVBs. Moreover, rapidly fixed brain tissue from patients undergoing brain surgery for noninvasive neurological disease (n = 2, ages 44 and 54) also revealed Aß42 especially on MVBs within neurons (Figure 1C)
. In contrast to Aß42, neuronal Aß40 was not convincingly evident in normal mouse, rat, or human brain.
The monoclonal anti-Aß42 antibody27
did not show cross-reactivity to full-length APP or APP C-terminal fragments (CTFs) by Western analysis of either APP-transfected neuroblastoma (N2a) cells or Tg2576 mouse brain (Figure 1D)
. Peptide competition abolished Aß42 detection both by Western blot and by immunohistochemistry. Lack of cross-reactivity with APP also can be deduced from the fact that full-length APP/CTFs localized predominantly to the Golgi, although a minor amount was also detected in MVBs, of neuronal cell preparations (using antibody 369 directed at the C-terminus of APP).34
Golgi predominance of full-length APP staining by immuno-EM with antibody 369 was now observed within neurons of the brain, and immuno-EM double labeling was used to demonstrate the differential staining of full-length APP/CTFs and Aß42 (Figure 1E)
. We confirmed our MBC42 Aß42 immuno-EM labeling with the widely used polyclonal Aß42 antibody from Chemicon, which gave analogous Aß42 immunoreactivity (data not shown). In addition to the predominant site of Aß42 on the outer membrane of small MVBs (60% of identifiable labeling in young 2- to 3-month-old mouse neuronal soma), Aß42 immunogold particles were observed on very small vesicles (35% of identifiable labeling). Furthermore, immunogold labeling also was observed on ER (5% of identifiable labeling; Figure 1A
), similar to that previously observed in cultured neurons infected transiently with Semliki Forest virus containing human APP.35
Aß42 was only occasionally seen at the plasma membrane, and in contrast to full-length APP, only a minor amount of Aß42 was observed on the Golgi apparatus compared with its predominance on MVBs (Figure 1)
.
APP Knockout Mice Lack Intraneuronal MVB Aß42
To further confirm the specificity of the Aß immunoreactivity, well-characterized APP knockout mice (n = 4)36
were compared with wild-type mice (n = 4) of equal age (two 6-month-old and two 12-month-old littermates) that were perfused and processed for immunogold EM in parallel. APP knockout mice did not demonstrate the Aß42 on MVBs observed in normal wild-type mice (compare Figure 2, A and B
).
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Aß was examined by immuno-EM in Tg2576 transgenic mice. Two independent previous Aß ELISA studies demonstrated exponential brain Aß increases occurring before Aß plaque deposition in Tg2576 mice.8,9
We used Aß immunoprecipitation and Western blot to further confirm brain Aß increases in our Tg2576 mice and observed analogous increases of brain Aß (Figure 3A)
as reported by ELISA, including preplaque between 3 and 9 months of age. To determine the location of Aß increases, mice were perfused at different ages ranging from 2 months to 20 months (n = 16) and processed for immunogold EM localization of Aß42. Relatively less Aß42 was found in 2- to 4-month-old mice. In contrast, significantly greater amounts of intraneuronal Aß42 were found in 10-month-old mice before the onset of plaque pathology (Figure 3, B and C)
. The Aß42 increases were most prominent on MVBs, particularly at nerve terminals (presynaptic and especially postsynaptic compartments). The immunogold labeling of Aß42 with aging in brain tissue was quantified from representative young (2-month-old) and older (10-month-old) Tg2576 mice. The MVB-associated gold particles in neuronal cell bodies (n = 20 each, from young and old) or processes of cerebral cortex were counted. Neuronal soma from 2-month-old mice had 1.48 ± 0.3 gold particles per MVB whereas neurons from older mice had 2.02 ± 0.4 gold particles per MVB (Figure 3D)
. This increase of MVB-associated gold particles in neuronal soma with aging did not reach statistical significance. Aß42 increases were most pronounced within neuronal processes (profiles), especially small dendrites (<1.5 µm in diameter) and axon terminals. Thirty images of dendritic profiles were taken each from 2-month-old and from 10-month-old mice at random at the same magnification. The number of gold particles per MVB in processes was 1.50 ± 0.29 and 3.42 ± 0.45 for 2- and 10-month-old mice, respectively (Figure 3D
; P < 0.033). MVB-associated gold particles were only rarely found in dendrites of 2-month-old mice. In contrast, numerous MVBs labeled with several gold particles were found in 10-month-old mouse brain. No MVBs with three or more gold particles were observed in 2-month-old dendritic profiles, whereas nine MVBs with three or more gold particles were observed in 10-month-old processes (Figure 3E)
. Lack of a specific marker for MVBs precludes biochemical quantification for potential changes in number of MVBs with aging in Tg2576 mice. Immuno-EM is not an optimal method for quantification and counting of MVBs by EM is further confounded by our observation that MVBs in distal processes of 10-month-old Tg2576 mice tended to be larger and contained a greater number of intravesicular vesicles than in processes of 2-month-old mice (compare Figure 3, B and C
). To reduce potential bias from gold particles, we quantified total MVBs in dendrites based on morphology by EM without immunogold labeling. The number of MVBs almost doubled in dendrites of young 2-month-old compared to older 10-month-old preplaque Tg2576 mice (1.96-fold increase; P < 0.03).
Aß42 Accumulation within Nerve Terminals Is Associated with Neuropathology
Before the development of AD plaque pathology, we observed accumulation of Aß42 presynaptically within axon terminals and especially postsynaptically within distal dendrites. At 10 months of age and before Aß plaques, dendrites were found that displayed multiple gold particles and morphological alterations, such as atypically dark Aß42 immunogold-associated MVBs, unusual clusters of ER in the vicinity of the Aß42 immunogold-associated MVBs, and abnormally collapsed, dark membranes consistent with degenerating processes in the vicinity of especially numerous gold particles (Figure 4A)
. At later ages, when plaques were forming, Aß42-accumulating processes were more numerous and showed strikingly abnormal morphological changes in the absence of an associated Aß plaque (Figure 4
; B, C, and D). Increased intraneuronal Aß42 immunogold labeling often was affiliated with swollen processes, sometimes within areas lacking normal appearing organelles or including dark structures resembling degeneration (Figure 4
; B, C, and D). Aß42 accumulation could also be seen associated with disrupted appearing MVBs (Figure 4B)
. The presence of abnormal appearing APP-containing neurites within plaques has been known37
and isolated Aß fibrils have been noted in such dystrophic neurites,38
but this is the first study showing that Aß42 accumulates in synaptic compartments and is associated with morphological changes. Interestingly, in a recent study isolated neurons in Down’s syndrome brain with marked intraneuronal Aß42 accumulation were reported to display concomitant terminal dUTP nick-end labeling staining indicative of apoptosis.26
In contrast to Aß42, Aß40 increases were less evident in Aß42-accumulating neurites, but Aß40 was seen to accumulate in Aß extracellular plaques and vessel walls (data not shown).
In human AD cortical brain tissue (n = 2, ages 54 and 62), as in the case of the transgenic mice, we observed Aß42 accumulation especially within neuronal processes and associated abnormal morphological changes (Figure 5)
. Compared with non-AD cortical biopsy tissue, AD cortex had marked increases of Aß42 immunogold particles both in soma and processes, even including areas not directly associated with Aß plaques. MVBs with 5 to 20 gold particles, not seen in normal human brain, could be seen in human AD brain (Figure 5B)
. In AD brain, Aß42 immunogold accumulation was seen in disrupted swollen dendritic profiles displaying degenerative changes (Figure 5C)
. In AD and older plaque bearing Tg2576 mouse brain, astrocytes and microglia in the vicinity of Aß plaques contained Aß42; this Aß42 was found especially within bundles of intermediate filaments in fibrous astrocytes (data not shown).
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Discussion |
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Aß peptides were initially believed to be an abnormal and toxic by-product of APP metabolism, until it was found in the early 1990s that all cells seem to normally secrete Aß.1
Aß was subsequently thought to be generated and rapidly secreted at the plasma membrane, until cell biological studies demonstrated that Aß peptides can be found intracellularly3
and even increase in amount with neuronal maturation or time in culture.3,18
Our observation of Aß42 associated with MVBs in normal brain raises the possibility that this small hydrophobic peptide may have a normal biological function. Even though APP has been studied intensively since its identification as the precursor to Aß in the late 1980s, the normal function of APP remains unknown. APP knockout mice were not found to have an obvious abnormal phenotype,36
but subsequent studies have indicated that APP knockout neurons have decreased neurite extension in tissue culture40
and deficits in synaptic plasticity.41
The normal presence and increases with aging of Aß42, the first Aß peptide found in early diffuse plaques in Down’s syndrome and AD, within neurons in Tg2576 mice and in AD brain, also in areas not adjacent to plaques, suggests that this intracellular Aß may be the site of Aß aggregation and neurotoxicity, subsequently leading to "extracellular" appearing Aß plaques. We and others reported images of Aß plaques seemingly originating from within neuronal soma in AD.2,22
We did not observe such images in Tg2576 mice. We hypothesize that the formation of plaques and lack of neuron cell death seen in APP FAD mutant transgenic mice may reflect Aß42 accumulation within distant neuronal processes with synaptosis and plaque formation, with lesser Aß accumulation within soma, thereby avoiding cell death, possibly secondary to the absence of human 
.42,43
Our study does not provide information on the NH2-terminus of MVB-associated Aßx-42, with AßAsp1 and AßGlu11 being major NH2-terminal Aß peptides generated by neurons.46,47 Because the earliest plaques with AD pathology have been reported to be NH2 terminally truncated Aßx-42,46 we, and others, have hypothesized that NH2-terminally truncated Aß42, such as AßGlu11-42, may be especially important in AD pathogenesis. This study again suggests, as did our and other previous studies,22-26 that Aßx-42, and not Aßx-40, is the major intraneuronal Aß peptide in brain. Tissue culture studies and brain ELISA studies of AD and Tg2576 mouse brain demonstrate considerable levels of both Aß40 and Aß42 peptides. Brain Aß40 may either not be visualized as well by immunohistochemical methods or may mainly reflect that associated with blood vessels and Aß plaques. In the present study, some Aß40 immunogold labeling, although less apparent than Aß42, was observed on neuronal MVBs in brain with aging.
Relatively little is known about the biological function of MVBs, which are defined by their ultrastructural appearance on EM. No clear distinction exists between MVBs and endosomes, except that MVBs tend to be regarded as smaller than, and potentially even be derived from, endosomes. In normal brain, Aß42 appeared to localize especially to small MVBs, but with aging Aß42 could also be observed on larger MVB/endosomes. MVBs are generally viewed as part of the endosomal/lysosomal system and both early and, more typically, late endosomes have been described as MVBs.47,48
Aß is known to be generated in the endosomal-lysosomal system10,11
and early lysosomal abnormalities in AD have been described.49,50
APP metabolism within Niemann-Pick cells demonstrated a pool of accumulating intracellular Aß42 in endosomes that is differentially regulated from the constitutive Aß secretory pathway.51,52
Possibly this pool of Aß42 represents the MVB-associated pool of Aß42. The ER has been described as an important site of Aß42 formation and we observed some Aß42 in ER of neurons in brain. Indeed, MVBs are often seen in the vicinity of the ER and may represent extensions of smooth ER.32
Similarly, the Golgi is a major site of Aß generation in the secretory pathway and MVBs also are Golgi associated (Figure 1E)
.32
We also observed Aß42 in very small vesicles that may represent recycling vesicles between MVBs and major intracellular organelles and/or the plasma membrane. With Aß42 accumulation on MVBs with aging and AD pathology, structures representing disrupted MVBs were observed (Figure 4B)
. Because pre-embedding immuno-EM generally underestimates actual quantity of antigen, such MVB disruption could lead to release of enough Aß42 to induce neurotoxicity. Specifically Aß42 treatment of cells was reported to induce leakage of lysosomal contents into the cytoplasm.53
Recently, it was demonstrated that Aß1-42, but not Aß1-40 or Aß42-1, is exquisitely neurotoxic when introduced into the cytosol of cultured primary neurons.54
Thus, leakage of Aß42 from MVBs into the cytosol may be a critical neurotoxic event in AD.
The mechanism whereby intracellular Aß accumulates, aggregates, and is neurotoxic to neurons remains to be elucidated. Our studies suggest but do not prove that Aß42 is the toxic entity. It was recently reported that BACE, presenilin 1, and APP are all transported along axons in membrane vesicles that require APP/kinesin-1 interactions and that vesicle preparations from peripheral nerves can generate Aß.55
Based on these findings and the fact that these vesicles also carry additional vital cargo proteins, such as the neurotrophin receptor, TrkA, Kamal and colleagues55
hypothesized that loss of trophic support to distal synapses from aberrant Aß accumulation or altered APP function could hold up this cargo and lead to neurodegeneration in AD.55,56
A neuron-specific C-terminal type kinesin superfamily protein (KIFC2) was found to be associated with MVBs by immuno-EM and to be important for dendritic transport of MVBs.57
Our study supports the scenario that accumulating Aß42 located on the outer membrane of MVBs could directly associate and disrupt kinesin (and possibly even -)-mediated transport of MVBs.
Intracellular aggregation of proteins is increasingly being linked to neurodegenerative diseases,58
including AD. FAD mutations increase not only secreted, but also intracellular Aß42.59,60
Recently, a FAD mutation was discovered that leads not to increased but decreased levels of Aß secretion, and to increased generation of protofibrils, leading the authors to suggest that intracellular Aß increases may be especially important for AD.61
The present study supports the concept that intracellular Aß is important in the genesis of AD. Previous experiments indicated that radiolabeled Aß42 can be internalized by neurons,62
suggesting that one route of intraneuronal Aß may be via neuronal uptake and subsequent intracellular accumulation. A recent study demonstrated that epidermal growth factor and transforming growth factor-
internalize after binding to the epidermal growth factor receptor initially via clathrin-coated vesicles, but at 30 minutes of chase are found on the outer membrane of MVBs,63
analogous to the location of Aß42 on the outside membrane of MVBs. Thus, MVB-associated Aß42 may be derived from endocytosis at the plasma membrane. Internally generated Aß might be expected to be prominent at synapses, because APP, and therefore Aß, is transported down axons64
and dendrites.65
Indeed, the Aß domain of APP appears critical for this sorting of APP for transport.65
Progressive accumulation and aggregation of intracellular Aß42 in dendrites/distal processes and presynaptic and postsynaptic compartments could lead to dystrophic neurite swellings, further APP conversion to Aß42 within these processes, release of this Aß42 from normal compartments, disruption of distal processes and synapses, and then eventually to dissolution of processes/synapses with the emergence of "extracellular" Aß plaques. The mechanism(s) by which Aß42 increases on MVBs, aggregates, and becomes neurotoxic within neurons and their processes, remain(s) to be determined. A better understanding of the biogenesis and biological role(s) of MVBs and a more detailed understanding of the subcellular events involved in Aß-induced AD neuropathology may be of value in the development of more effective therapies for AD.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (grants NS02037, AG09464, and HL18974), the Alzheimer’s Association, the American Academy of Neurology Education and Research Foundation, and Paul Beeson Physician Faculty Scholar Award (to G. K. G.).
Accepted for publication July 22, 2002.
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V. Vingtdeux, M. Hamdane, A. Loyens, P. Gele, H. Drobeck, S. Begard, M.-C. Galas, A. Delacourte, J.-C. Beauvillain, L. Buee, et al. Alkalizing Drugs Induce Accumulation of Amyloid Precursor Protein By-products in Luminal Vesicles of Multivesicular Bodies J. Biol. Chem., June 22, 2007; 282(25): 18197 - 18205. [Abstract] [Full Text] [PDF]
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C. V. Zerbinatti, S. E. Wahrle, H. Kim, J. A. Cam, K. Bales, S. M. Paul, D. M. Holtzman, and G. Bu Apolipoprotein E and Low Density Lipoprotein Receptor-related Protein Facilitate Intraneuronal Abeta42 Accumulation in Amyloid Model Mice J. Biol. Chem., November 24, 2006; 281(47): 36180 - 36186. [Abstract] [Full Text] [PDF]
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H. Oakley, S. L. Cole, S. Logan, E. Maus, P. Shao, J. Craft, A. Guillozet-Bongaarts, M. Ohno, J. Disterhoft, L. Van Eldik, et al. Intraneuronal beta-Amyloid Aggregates, Neurodegeneration, and Neuron Loss in Transgenic Mice with Five Familial Alzheimer's Disease Mutations: Potential Factors in Amyloid Plaque Formation J. Neurosci., October 4, 2006; 26(40): 10129 - 10140. [Abstract] [Full Text] [PDF]
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 E. Cohen, J. Bieschke, R. M. Perciavalle, J. W. Kelly, and A. Dillin Opposing Activities Protect Against Age-Onset Proteotoxicity Science, September 15, 2006; 313(5793): 1604 - 1610. [Abstract] [Full Text] [PDF]
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L. Rajendran, M. Honsho, T. R. Zahn, P. Keller, K. D. Geiger, P. Verkade, and K. Simons Alzheimer's disease beta-amyloid peptides are released in association with exosomes PNAS, July 25, 2006; 103(30): 11172 - 11177. [Abstract] [Full Text] [PDF]
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 B. A. Tate and P. M. Mathews Targeting the Role of the Endosome in the Pathophysiology of Alzheimer's Disease: A Strategy for Treatment Sci. Aging Knowl. Environ., June 28, 2006; 2006(10): re2 - re2. [Abstract] [Full Text] [PDF]
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C. G. Almeida, R. H. Takahashi, and G. K. Gouras Beta-amyloid accumulation impairs multivesicular body sorting by inhibiting the ubiquitin-proteasome system. J. Neurosci., April 19, 2006; 26(16): 4277 - 4288. [Abstract] [Full Text] [PDF]
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J.-P. Guo, T. Arai, J. Miklossy, and P. L. McGeer From the Cover: Abeta and tau form soluble complexes that may promote self aggregation of both into the insoluble forms observed in Alzheimer's disease PNAS, February 7, 2006; 103(6): 1953 - 1958. [Abstract] [Full Text] [PDF]
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K. Offe, S. E. Dodson, J. T. Shoemaker, J. J. Fritz, M. Gearing, A. I. Levey, and J. J. Lah The Lipoprotein Receptor LR11 Regulates Amyloid beta Production and Amyloid Precursor Protein Traffic in Endosomal Compartments J. Neurosci., February 1, 2006; 26(5): 1596 - 1603. [Abstract] [Full Text] [PDF]
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 K. S. Vetrivel and G. Thinakaran Amyloidogenic processing of {beta}-amyloid precursor protein in intracellular compartments Neurology, January 24, 2006; 66(1_suppl_1): S69 - S73. [Abstract] [Full Text] [PDF]
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J. Magrane, K. M. Rosen, R. C. Smith, K. Walsh, G. K. Gouras, and H. W. Querfurth Intraneuronal {beta}-Amyloid Expression Downregulates the Akt Survival Pathway and Blunts the Stress Response J. Neurosci., November 23, 2005; 25(47): 10960 - 10969. [Abstract] [Full Text] [PDF]
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 W. H. Yu, A. M. Cuervo, A. Kumar, C. M. Peterhoff, S. D. Schmidt, J.-H. Lee, P. S. Mohan, M. Mercken, M. R. Farmery, L. O. Tjernberg, et al. Macroautophagy--a novel {beta}-amyloid peptide-generating pathway activated in Alzheimer's disease J. Cell Biol., October 10, 2005; 171(1): 87 - 98. [Abstract] [Full Text] [PDF]
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C. A. Saura, G. Chen, S. Malkani, S.-Y. Choi, R. H. Takahashi, D. Zhang, G. K. Gouras, A. Kirkwood, R. G. M. Morris, and J. Shen Conditional Inactivation of Presenilin 1 Prevents Amyloid Accumulation and Temporarily Rescues Contextual and Spatial Working Memory Impairments in Amyloid Precursor Protein Transgenic Mice J. Neurosci., July 20, 2005; 25(29): 6755 - 6764. [Abstract] [Full Text] [PDF]
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V. T. Marchesi An alternative interpretation of the amyloid A{beta} hypothesis with regard to the pathogenesis of Alzheimer's disease PNAS, June 28, 2005; 102(26): 9093 - 9098. [Abstract] [Full Text] [PDF]
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J. Ashley, M. Packard, B. Ataman, and V. Budnik Fasciclin II Signals New Synapse Formation through Amyloid Precursor Protein and the Scaffolding Protein dX11/Mint J. Neurosci., June 22, 2005; 25(25): 5943 - 5955. [Abstract] [Full Text] [PDF]
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M. Arbel, I. Yacoby, and B. Solomon Inhibition of amyloid precursor protein processing by {beta}-secretase through site-directed antibodies PNAS, May 24, 2005; 102(21): 7718 - 7723. [Abstract] [Full Text] [PDF]
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S. L. Cole, A. Grudzien, I. O. Manhart, B. L. Kelly, H. Oakley, and R. Vassar Statins Cause Intracellular Accumulation of Amyloid Precursor Protein, {beta}-Secretase-cleaved Fragments, and Amyloid {beta}-Peptide via an Isoprenoid-dependent Mechanism J. Biol. Chem., May 13, 2005; 280(19): 18755 - 18770. [Abstract] [Full Text] [PDF]
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Y.-w. Zhang, W.-j. Luo, H. Wang, P. Lin, K. S. Vetrivel, F. Liao, F. Li, P. C. Wong, M. G. Farquhar, G. Thinakaran, et al. Nicastrin Is Critical for Stability and Trafficking but Not Association of Other Presenilin/{gamma}-Secretase Components J. Biol. Chem., April 29, 2005; 280(17): 17020 - 17026. [Abstract] [Full Text] [PDF]
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R. A. Christensen, A. Shtifman, P. D. Allen, J. R. Lopez, and H. W. Querfurth Calcium Dyshomeostasis in {beta}-Amyloid and Tau-bearing Skeletal Myotubes J. Biol. Chem., December 17, 2004; 279(51): 53524 - 53532. [Abstract] [Full Text] [PDF]
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D. Langui, N. Girardot, K. H. El Hachimi, B. Allinquant, V. Blanchard, L. Pradier, and C. Duyckaerts Subcellular Topography of Neuronal A{beta} Peptide in APPxPS1 Transgenic Mice Am. J. Pathol., November 1, 2004; 165(5): 1465 - 1477. [Abstract] [Full Text] [PDF]
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K. H. Gylys, J. A. Fein, F. Yang, D. J. Wiley, C. A. Miller, and G. M. Cole Synaptic Changes in Alzheimer's Disease: Increased Amyloid-{beta} and Gliosis in Surviving Terminals Is Accompanied by Decreased PSD-95 Fluorescence Am. J. Pathol., November 1, 2004; 165(5): 1809 - 1817. [Abstract] [Full Text] [PDF]
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K. S. Vetrivel, H. Cheng, W. Lin, T. Sakurai, T. Li, N. Nukina, P. C. Wong, H. Xu, and G. Thinakaran Association of {gamma}-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes J. Biol. Chem., October 22, 2004; 279(43): 44945 - 44954. [Abstract] [Full Text] [PDF]
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H. Wang, W.-j. Luo, Y.-w. Zhang, Y.-M. Li, G. Thinakaran, P. Greengard, and H. Xu Presenilins and {gamma}-Secretase Inhibitors Affect Intracellular Trafficking and Cell Surface Localization of the {gamma}-Secretase Complex Components J. Biol. Chem., September 24, 2004; 279(39): 40560 - 40566. [Abstract] [Full Text] [PDF]
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Y. Zhang, N. Champagne, L. K. Beitel, C. G. Goodyer, M. Trifiro, and A. LeBlanc Estrogen and Androgen Protection of Human Neurons against Intracellular Amyloid {beta}1-42 Toxicity through Heat Shock Protein 70 J. Neurosci., June 9, 2004; 24(23): 5315 - 5321. [Abstract] [Full Text] [PDF]
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R. H. Takahashi, C. G. Almeida, P. F. Kearney, F. Yu, M. T. Lin, T. A. Milner, and G. K. Gouras Oligomerization of Alzheimer's {beta}-Amyloid within Processes and Synapses of Cultured Neurons and Brain J. Neurosci., April 7, 2004; 24(14): 3592 - 3599. [Abstract] [Full Text] [PDF]
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L.-W. Jin, I. Maezawa, I. Vincent, and T. Bird Intracellular Accumulation of Amyloidogenic Fragments of Amyloid-{beta} Precursor Protein in Neurons with Niemann-Pick Type C Defects Is Associated with Endosomal Abnormalities Am. J. Pathol., March 1, 2004; 164(3): 975 - 985. [Abstract] [Full Text] [PDF]
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J. Magrane, R. C. Smith, K. Walsh, and H. W. Querfurth Heat Shock Protein 70 Participates in the Neuroprotective Response to Intracellularly Expressed {beta}-Amyloid in Neurons J. Neurosci., February 18, 2004; 24(7): 1700 - 1706. [Abstract] [Full Text] [PDF]
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S. H. Pasternak, R. D. Bagshaw, M. Guiral, S. Zhang, C. A. Ackerley, B. J. Pak, J. W. Callahan, and D. J. Mahuran Presenilin-1, Nicastrin, Amyloid Precursor Protein, and {gamma}-Secretase Activity Are Co-localized in the Lysosomal Membrane J. Biol. Chem., July 11, 2003; 278(29): 26687 - 26694. [Abstract] [Full Text] [PDF]
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W.-j. Luo, H. Wang, H. Li, B. S. Kim, S. Shah, H.-J. Lee, G. Thinakaran, T.-W. Kim, G. Yu, and H. Xu PEN-2 and APH-1 Coordinately Regulate Proteolytic Processing of Presenilin 1 J. Biol. Chem., February 28, 2003; 278(10): 7850 - 7854. [Abstract] [Full Text] [PDF]
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