Using a newly developed conformation-dependent, fibril-specific, polyclonal antibody [35
] that recognizes a generic epitope associated with fibrils and soluble fibrillar oligomers we have characterized AD and age-associated changes in Aβ fibril accumulation. Several technical aspects to the use of the OC antibody were identified in the current study. First, formic acid pretreatment, typically used to enhance Aβ immunostaining [39
] does not significantly improve OC labeling. This suggests that the epitope/conformation recognized by the OC antibody is not obscured when fibrils form and have not adopted a full beta-pleated sheet assembly state. Second, based on our studies in DS brain, post mortem interval may be a key factor to consider when using this antibody. Longer postmortem intervals (>10 h) may be associated with a degradation of the signal, possibly related to protease activity. Whether this represents a loss of tissue morphology, a spontaneous disassembly of fibrils or oligomeric protofibrils or the opposite, a conversion into beta-pleated sheet fibrils post mortem, is difficult to determine. Further, a PMI effect on OC immunolabeling may potentially distinguish sporadic AD deposits from DS with AD deposits and there is some evidence that the properties of Aβ in these two groups may behave differently [1
OC-positive fibrillar deposits accumulate in human brain as overlapping deposits with thioflavine-S plaques but are also found in deposits that are diffuse, i.e. thioflavine S-negative. By examining the brains of individuals with and without dementia, we show increased fibrillar accumulation in AD but when compared to controls. However, CIND/MCI cases showed similar OC immunolabeling as controls. Further, the extent of fibril accumulation in AD, CIND/MCI and control brain correlates with the severity of cognitive decline measured by MMSE scores. Similarly, in individuals with DS, we observe fibril accumulation that is both age- and AD neuropathology-associated but with a rapid rise in levels after the age of 40 years. Finally, in one of the most commonly used animal models of Aβ pathogenesis, we find a rapid age-dependent accumulation of fibrils that is first detectable at 6 months of age but dramatically increases after 12 months of age after overexpression of mutant human APP in the Tg2576 model system. Thus, OC-positive deposits may be visible histologically at an earlier age than Aβ-positive deposits reported previously [34
] and may represent an early neuropathological feature more consistent with the onset of behavioral dysfunction [31
OC-positive deposits that are thioflavine S-negative may reflect fibrillar oligomers. This is consistent with reports that protofibrillar forms of Aβ have low thioflavine-T response [75
]. The morphology of the OC-positive, thioflavine S-negative deposits are distinct and appear to be shorter and wider than thioflavine S-positive fibrils. In addition, we frequently find OC-positive deposits at the periphery of thioflavine S-positive plaques providing further evidence that soluble fibrillar oligomers may represent a reservoir for fibril formation [35
]. OC, interestingly, was also able to detect diffuse plaques characterized as being thioflavine S-negative but also containing intact neurons. This may suggest that the pathogenesis of diffuse plaques involves the accumulation of thioflavine S-negative fibrils and soluble fibrillar oligomers. Interestingly, a similar conclusion was made in an earlier study by Davies and Mann [16
] based on electron microscopy in biopsy samples from AD cases showing diffuse plaques contained fibrillar material. Parallel studies in the canine model, that only develop diffuse Aβ plaques also suggests that these early deposits are fibrillar in nature [68
]. Indeed, diffuse plaques, though to be one of the earlier plaque subtypes accumulating Aβ pathology [27
], contain primarily the longer toxic Aβ1-42 peptide [15
], are observed at the threshold of detectable dementia [56
], can occur after traumatic brain injury leaving affected patients at higher risk for developing AD [17
] and may reflect synaptic activity induced Aβ production and release into the interstitial space [10
]. Further, individuals with early onset AD caused by a missense mutation in APP (T714I), develop primarily diffuse Aβ deposits and an aggressive form of dementia [40
]. In combination, diffuse plaques may not be entirely benign as has been suggested previously [51
] although they may be “clinically silent”.
When OC is detected in more compact plaques, we observe dystrophic neurites, microglial and astrocyte involvement similar to previous reports of associated neurodegeneration in these more mature plaque subtypes [19
]. Further, microglial cells may engulf OC-positive deposits for degradation as we observe OC-positive material within phagocytic vacuoles within HLA-DR expressing microglial cells. We have observed a similar phenomenon with oxidatively modified Aβ [29
] and others have reported Aβ within microglial cells in vitro and within AD brain previously [21
]. A critical role for microglial cells in clearing Aβ from the brain has been established in transgenic mice [3
] and the results from this study also suggest efforts by microglial cells to clear soluble and insoluble fibrils.
OC fibrils in AD
We next used the OC antibody in studies to quantify the extent of fibril formation in individuals with and without AD. OC-positive deposits were ~7-fold higher in AD brain relative to controls. However, based on previous studies in the brains of individuals with MCI [57
], we also predicted that subjects with MCI or with a cognitive impairment although not demented, may be either equivalent or intermediate with respect to extent of OC-positive deposits. However, we found that OC fibrils were similar in MCI/CIND as compared to nondemented aged controls. There are two possible interpretations of these data. The first is that given our sample consisted primarily of CIND subjects; there is a less clear relationship between CIND and progression to dementia as in MCI [62
]. Second, fibrils may be more closely associated with full-blown dementia rather than a possible pro-dromal selective clinical impairment. By immunohistochemistry, we may also be detecting primarily fibrils as opposed to soluble oligomeric fibrils, the latter may be optimally detected using biochemical methods [35
]. Measures of soluble fibrillar oligomers from protein extracts of frozen tissue in these cases may be more sensitive and reveal higher levels of OC in MCI/CIND cases relative to controls because they have the potential of distinguishing insoluble fibrils and soluble fibrillar oligomers; both of which react with OC. Another important consideration is that the current study focused on plaque pathology in the frontal cortex and had we sampled regions in other vulnerable areas, particularly the entorhinal cortex (which was not available for all cases), we may have observed OC levels that are more consistent between MCI/CIND cases and AD cases.
In AD brain, there are various reports of a correlation between the extent of Aβ deposition in plaques and the severity of cognitive decline. For example, several studies show a significant inverse correlation with MMSE scores, suggesting that lower scores associated with dementia are linked to more extensive plaque formation [2
]. In contrast, additional studies do not show this relationship and/or find that neurofibrillary tangles or synapse protein loss, other key pathological features of AD, are also tightly linked to cognition [12
]. Various interpretations of these results have been suggested but one hypothesis is that assembly state of Aβ may be a critical factor in determining its toxicity and thus the link to impaired cognition. The current study suggests a significant correlation between OC-positive fibrillar deposits and MMSE scores with higher scores associated with intact cognition linked to lower levels of OC. Interestingly, we may be observing a threshold effect as OC loads increase significantly when MMSE scores drop below 20 but this should be confirmed in a larger cohort of cases.
OC fibrils in Down syndrome
In the most common form of DS, trisomy 21, chromosome 21 is present in triplicate and leads to lifelong overexpression of the APP gene (21q21.2) [64
]. Despite this life-long overexpression of APP in brain and in peripheral lymphocytes [59
], Aβ accumulation in plaques does not typically begin until after the age of 30 years [50
], although there have been reports of plaques in younger individuals [43
]. Interestingly, the incidence of dementia typically does not increase until adults with DS are over the age of 50 years [41
], suggesting the possibility that either the brain can compensate for significant Aβ accumulation or that Aβ must adopt a specific conformation to become neurotoxic.
To test this hypothesis, we measured OC loads in individuals with DS with a range of ages. In the second experiment, we observed an age-associated increase in OC in DS that was not observed in similarly aged non-DS subjects without dementia. In DS, it is possible to differentiate initiation from acceleration phases of AD pathogenesis. Between the ages of 30–40 years, the first signs of Aβ plaques appear as diffuse deposits [47
]. Between 40 and 50 years, neurofibrillary tangles also form and neuritic plaques appear suggesting an acceleration phase. We may also consider from 50 years and older as when functional declines may occur [41
]. If we classify adults with DS into these groups, OC-positive fibrils are found as early as the initiation phase suggesting a key role in Aβ pathogenesis in DS. Despite life-long overexpression of APP, the brains of individuals with DS under the age of 30 years were negative for fibrils, however, a larger sample of individuals between 20 and 30 years would extend these findings. Thus, OC-positive deposits appear prior to the age at which dementia prevalence rises in DS and may play a role in AD pathogenesis in DS.
OC fibrils in Tg2576
Similar to individuals with DS, transgenic mouse models that overexpress mutant human APP develop Aβ pathology in a progressive age-dependent manner [31
]. In a systematic study of the different types and assembly states of Aβ that accumulate in Tg2576 mice, Kawarabayashi and colleagues [34
] showed that Aβ in the form of plaques appear as early as 7–8 months whereas diffuse plaques appear between 12 and 15 months. However, Aβ extracted with formic acid can be detected using biochemical approaches younger ages between 6 and 8 months. Further, Aβ oligomers are detected in older animals ~12 months of age as variably being increased [34
] or decreased [44
]. However, behavioral impairments may appear prior to significant Aβ plaque pathology as early as 3 months [38
], 6 months of age [73
] or older [11
]. The discrepancy between the onset of behavioral dysfunction in Tg2576 mice and the age at which Aβ plaques first appear suggests a critical role for different assembly states of Aβ on neuronal function. The earliest age at which we consistently observed OC-positive deposits in the Tg2576 mice in the current study was 9 months, with one animal at 6 months of age showing pathology, but this does not rule out soluble fibrillar oligomers, which may not be detected by immunohistochemistry, from being a contributor to neuronal dysfunction. Indeed, Aβ*56 oligomers that appear as early as 7 months are A11-positive, indicating that they are prefibrillar oligomers that would not be expected to react with OC [44
In summary, using a novel antibody that detects amyloid fibrils and soluble oligomeric protofibrils we show a differential distribution from beta-pleated sheet fibrils and association with AD in both the general population and in adults with DS. Further, OC-positive fibrils also accumulate in Tg2576 mice suggesting that several of the dynamics involved with production and accumulation of fibrils may be modeled in this animal. It would be interesting to selectively reduce OC-positive fibrils through a vaccination approach to determine their functional impact.