In this study, we report the establishment of a cell culture system in which central nervous system-derived cells produce and accumulate large amounts of Aβ peptide from endogenous APP within their neurites. This process is remarkably similar to what is now believed to occur within the neurites of affected neurons in the initial phases of AD. Using this system, we confirm that significant accumulation of insoluble Aβ begins within neurites, in particular at their terminals, long before Aβ deposits are detected extracellularly. Accumulation of Aβ within neurites is normally restricted to a small population of neighboring cells that also show redistribution of BACE1 to the processes, where it colocalizes with Aβ. These events occur exclusively in differentiated cells, prior to any detectable signs of neurodegeneration or abnormal transport of vesicular cargoes into neurites. Taken together, these results suggest that the process of conversion of Aβ to a detergent-resistant form and its neuritic accumulation is initiated in a small number of neurons by intracellular determinants that alter APP metabolism.
Animal models and cell culture systems manipulated to express human versions of mutant, disease-prone APP or components of the APP processing machinery (e.g., presenilin-1, BACE1) remain important tools for the identification of molecular mechanisms that operate in familial forms of AD. In contrast, the availability of neuronal lines that show a propensity to form intraneuritic Aβ deposits at endogenous levels of APP and secretases may provide insight into the pathogenic mechanisms that alter Aβ metabolism in sporadic AD forms that may not have a genetic basis. Such cell lines are the previously reported human NT2N neurons (46
) and the mouse cell line CAD, used in this study. Initially established by targeted oncogenesis in transgenic mice, CAD cells have spontaneously lost the original oncogene, that encoding simian virus 40 T antigen. They are diploid, chromosomally stable cells that exhibit biochemical and morphological characteristics of primary neurons (40
). In previous work, we demonstrated that the resolution achieved by fluorescence microscopy in the thin processes of CAD cells allows the resolution of individual transport packages even without the use of confocal microscopy (39
). CAD cells are a promising model system for studying neurodegenerative diseases, including torsion dystonia (20
), Parkinson's disease (2
), and AD (29
). As we show here, CAD cells are particularly suited for the investigation of APP metabolism in conjunction with its transport into processes. This is important, in view of the recent evidence that transport and processing of APP are intimately entangled (24
). CAD cells, which are likely derived from the locus coeruleus in the brain stem (40
), are relevant to the neuropathology of AD. Indeed, neurons in the locus coeruleus aberrantly express cell cycle proteins (8
) and are largely affected by cell death in AD (6
), in spite of the fact that this brain region lacks high densities of neuritic plaques and tangles.
The first important result of this study is that CAD cells, expressing endogenous levels of APP, normally develop deposits of Aβ that are detergent resistant, indicative of oligomerized Aβ. Results of immunocytochemistry done with an antioligomer antibody that does not recognize monomeric or fibrillar Aβ confirm that at least a fraction of the Aβ detected in CAD cells is in an oligomeric form. We also note that the Aβ accumulations reported in this study are best detected with antibody 6E10, which binds to Aβ oligomers and fibrils, in addition to the monomers (26
; see also the BioSource International product data sheet for the antioligomer antibody AHB0052). This result is in line with the known fact that antibodies that recognize amino-terminal regions in Aβ (e.g., 6E10) detect oligomerized Aβ more efficiently than antibodies that bind to internal regions (such as 4G8) (4
). The fact that under our experimental conditions the 6E10 antibody indeed detects Aβ deposits was confirmed by colocalization with two antibodies to rodent Aβ (Fig. and our unpublished results) and the absence of immunoreactivity to antibodies to APP regions outside Aβ (Fig. ). Finally, experiments using Aβ carboxy-terminal-end antibodies (Fig. ) indicate that both Aβ40 and Aβ42 are present—to various extents—in CAD cells. While these deposits likely also contain CTFs, in addition to Aβ, these may either be below the detection limit of our assays or have their carboxy-terminal epitopes masked. In this respect, we note that the exact composition and the aggregation state of the Aβ polypeptides present in these accumulations cannot be revealed by immunocytochemistry and biochemistry alone. Indeed, the accessibility of antibodies to the various Aβ epitopes is certainly differentially affected by the aggregation state of the Aβ polypeptide. For example, some epitopes that are accessible in Aβ oligomers might not be accessible in Aβ fibrils. This likely explains why double-labeling experiments with antibodies to distinct epitopes in Aβ showed—in many cases—only partial colocalization. Studies to further characterize these deposits with regard to composition and aggregation state of the polypeptides are under way.
The second important result is that Aβ accumulates in the distal portion of CAD cell neurites, in particular at their terminals, a process thought to occur in early stages of AD (5
). Oligomerization of Aβ within processes and synapses of cultured neurons expressing human APP with the Swedish mutation (derived from Tg2576 mice) has been recently described (49
). However, unlike Tg2576 neurons, where Aβ gradually accumulates within processes over time in culture (49
), CAD cells show preferential neuritic deposits of Aβ starting very early during differentiation. Eventually, all processes of affected CAD cells become filled with deposits, irrespective of neurite length and neurite number per cell. Whether and how these intracellular Aβ accumulations can become extracellular (likely by cell disintegration or a form of exocytosis) remains to be established in future studies.
A third important result of our study is that cells that accumulate Aβ can still differentiate and function normally, at least for some time in culture. Importantly, these cells still appear to normally transport to neurite terminals vesicular cargoes, such as those containing pAPP and JIP-1. Moreover, cells with Aβ accumulations do not show signs of abnormal cytoskeleton, neurodegeneration, or cell death. These results suggest that while a deficient axonal transport may cause neurodegeneration (41
), accumulation of Aβ within neurites can certainly occur in the absence of any detectable abnormal intraneuritic transport. We are currently conducting a more detailed investigation of vesicle transport in CAD cells that accumulate Aβ.
A fourth important result is that cells that contain Aβ accumulations within neurites also contain BACE1, the major β-secretase, at the same location, coincident with Aβ. The extent of BACE1 accumulation within the neuronal processes of these cells is abnormal, since this enzyme is normally localized to Golgi compartments and early endosomes in the cell body (27
). This result suggests that mislocalization of BACE1 may cause the production and accumulation of Aβ within processes and synaptic regions. This result is in line with a recent report that proposes that BACE1 localization—in addition to its expression level (13
)—determines the amount of generated Aβ and its accumulation in plaques (28
The colocalization of Aβ with secretases within neurites and at terminals suggests that Aβ may be generated during transport through the processes. This does not necessarily mean that this Aβ is contained in Golgi-derived secretory vesicles, particularly because BACE1 is active only in the acidic environment provided by endosomes. Our results of colocalization with early endosomal markers and uptake experiments clearly show that the neuritic Aβ does not accumulate in early endosomes, which are concentrated in the cell body region. It is likely that the Aβ is contained in late endosomes, such as multivesicular bodies, that may originate by maturation from early endosomes generated in the cell body and are then transported anterogradely, down the processes. This hypothesis is supported by our data on colocalization with late endosomal markers and is consistent with recent reports that identify the intraneuritic compartments that contain Aβ in neurons as multivesicular bodies (50
). Alternatively, the Aβ could be contained in autophagic vesicles that form within the neurites. Indeed, a recent study showed that macroautophagy may be an important pathway for Aβ generation in AD (58
). Although not yet detected in CAD cells, macroautophagy might be selectively triggered in some of these cells, ultimately leading to the abnormal generation and accumulation of Aβ. Further studies are required to identify the exact pathway of vesicular transport that results in accumulation of Aβ at the distal end of neurites.
An important result of our study is that cells that accumulate Aβ within neurites appear to originate by division from a small number of cells, present in the culture, that possess the propensity to form Aβ accumulations. Thus, intracellular determinants conferring the Aβ phenotype would be clonally transmitted to the progenitors of a few cells that exhibit the biochemical and neuropathological features of degenerating neurons present in the brains of AD patients. These intracellular determinants are not necessarily genetically inherited. As recently shown, expression of neuronal genes can be spontaneously altered by retrotransposition, resulting in neuronal somatic mosaicism, a phenomenon seen both in cultured neurons and in the brains of adult mice, in vivo (33
). Thus, events triggering Aβ oligomerization and accumulation may be initiated randomly within single cells (16
). Overall, our data suggest that intracellular determinants present in a small number of neurons may contribute to the onset of Aβ deposition in AD.
It is intriguing that while other studies done with cultured cortical and hippocampal neurons from APP transgenic mice occasionally identified neuritic staining for Aβ (49
), preferential accumulation at neurite terminals—as found by us in CAD cells—has not been reported. We think that this may be due to differences between neurons from different brain regions. In this respect, neurons derived from the brain stem, such as the CAD cells, may hold the key to explaining the onset of plaque formation in AD. The locus coeruleus neurons innervate many brain regions, including the cerebral cortex and the hippocampus (3
), where neuritic plaques are abundant in AD. Plaque formation in the cerebral cortex and hippocampus could be seeded by oligomerized Aβ that accumulates at the terminals of projections of neurons with their cell bodies in the brain stem. As our results clearly show, the locus coeruleus-derived CAD cells accumulate oligomerized Aβ primarily at the terminals of their processes, which become swollen and contain varicosities. Similarly, tyrosine hydroxylase-containing nerve terminals—extending from the locus coeruleus—are markedly enlarged in the proximity of neuritic plaques in AD brains and in mouse models of AD (15
). We propose that spontaneous accumulations of Aβ at the terminals of brain stem neurons that project into the cortex and hippocampus may nucleate the formation of the neuritic deposits. Thus, the neuropathology of AD may actually begin in subcortical regions and then spread to the cortex and hippocampus. This hypothesis remains to be tested.
In conclusion, we propose a novel cell culture system for the study of AD-like early events in the generation and accumulation of Aβ in neuronal cells. We show how this system can be used to address questions of processing and transport of APP and to potentially identify molecular determinants relevant to AD pathology. Finally, we provide support for the hypothesis that redistribution of BACE1, which may spontaneously occur in a small population of neurons, may lead to abnormal generation and regional accumulation of Aβ. These accumulations may in time become extracellular and serve as seeds for development of neuritic plaques. Our results thus suggest a clonal origin of abnormal Aβ metabolism and plaque formation. Further studies, possibly using CAD cell cultures enriched in cells that accumulate Aβ, are required to test this hypothesis.