Brain accumulation of Aβ is thought to be central to AD, and it is likely to be responsible for the neurodegeneration that underlies dementia; however, the mechanistic role of Aβ and the form in which it is toxic remain controversial. Support for the hypothesis that classic fibrillar amyloid plaques are deleterious to the brain comes from numerous studies, showing that the subpopulation of dense-core Aβ plaques in particular, the so-called neuritic plaques, are intimately associated with local dendritic spine loss, changes in neurites, and gliosis in AD and mouse models (35
). On the other hand, total numbers of amyloid plaques do not correlate well with the severity of illness (38
) or with loss of neurons (3
), arguing against a direct causal effect of plaques on cognition or neuronal cell death in AD. A second hypothesis that has increasing support centers on the idea that the toxic component is within the soluble fraction, more specifically that soluble Aβ oligomers cause the damage and are responsible for the toxicity. Data showing that soluble forms of Aβ correlate more closely with dementia severity than fibrillar Aβ (9
) and that Aβ oligomers alter dendritic spine density and affect hippocampal synaptic plasticity in vivo support this alternate hypothesis (12
We tested these competing, although not necessarily exclusive, hypotheses by measuring brain Aβ oligomers and amyloid plaques and evaluated the form of Aβ associated with cognitive dysfunction and neuropathologic hallmarks of AD in APPsw
mice. We found that brain accumulation of soluble small oligomeric species of Aβ (dimers and trimers) is an early event that predates by months the classic fibrillar amyloid plaque deposition, neuronal cell loss, and memory impairment, suggesting that accumulation of soluble Aβ oligomers may act as the primary trigger event in the cascade leading to neurodegeneration and impaired cognition. Interestingly, in rats, the 2-molecule soluble form of the peptide (dimers) directly isolated from AD brains is the smallest synaptotoxic species of soluble oligomeric Aβ, and it inhibits long-term potentiation (17
). More recently, it has also been shown that Aβ dimers in the blood and cortical extracts from AD patients strongly correlate with cognitive impairment (10
). The oligomeric conformational epitope-specific antibody Nab61 immunolabeled a subset of plaques in the brains of APPsw
mice and revealed an anatomical distribution of oligomeric Aβ deposits that partially overlapped but did not completely match that of classic amyloid plaques. Oligomeric Aβ deposits selectively appeared in the EC and CA1 hippocampal subfield of APPsw
mice at 9 months, but only became consistently present and abundant at a substantially later age in the cingulate cortex. Because identical time course of appearance and comparable or even higher amounts of 4G8-immunostained amyloid plaques and densecore ThioS-reactive plaques were observed in the cingulate cortex when compared with those in the EC and CA1, this was not an artifact of APP and tau transgene expression (43
) or the result of increased Aβ. Thus, it is possible that specific mechanisms responsible for Aβ oligomerization and/or clearance of Aβ oligomers may differ across different brain areas in these mice.
One of our most interesting observations is that the appearance of oligomeric Aβ deposits (i.e. EC and CA1 early and cingulate cortex later) faithfully predicted and matched the selective regional vulnerability of the EC and CA1 to neuronal cell loss and the resilience of neurons in the cingulate cortex through 17 months, which supports the idea that oligomeric AA plays a larger role than amyloid plaques in neuronal damage in this model. Further support comes from the observation that oligomeric AA load and the subpopulation of dense-core ThioS-reactive AA deposits, but not total amyloid plaque burden, correlated with the amount of neuronal cell loss.
In AD, neuronal loss is to a great extent limited to specific areas of the neocortex, limbic systems, and subcortical ascending projection systems. We reported that even at prodromal clinical stages, AD patients had a 60% of neuron loss in layer II of the entorhinal cortex and 40% loss in layer IV, as compared with controls (45
). Significant neuronal loss has also been reported in AD brains in the CA1 hippocampal subfield (48%) at slightly later clinical stages (46
). Our stereological analyses showed a strikingly similar regional pattern of neuronal vulnerability in the brains of APPsw−
mice, with neuronal cell death starting in the EC at 9 months and extending to CA1 at around 12 months but no significant loss of neurons in the cingulate cortex through 17 months (21
). Interestingly, no overt neuronal loss has been reported in the human cingulate cortex even at advanced stages of the disease, despite an observed early reduction of blood flow and metabolism on single-photon emission computed tomography and fluorodeoxyglucose-positron emission tomography in this area (47
). Therefore, the cingulate cortex may provide a window for studying events linked to neuronal cell death that medial temporal lobe structures (i.e. EC and CA1) pass through decades before death in humans (49
) and several months in APPsw
mice. Our present results indicate that oligomeric Aβ accumulation and activation of astrocytes are the 2 events that most faithfully predict and more closely correlate with the highly selective regional pattern of neuronal vulnerability observed in APPsw
Inflammatory changes, including the presence of abundant activated microglia and astrocytes, and release of proinflammatory mediators are consistently identified in association with dense-core Aβ plaques from early stages of AD and in Tg mouse models, pointing to inflammation as an important pathogenetic component of the disease. Interestingly, in vitro studies have shown differential effects of oligomeric and fibrillar Aβ on astrocyte-mediated inflammation (50
). Oligomeric Aβ induced a profound early inflammatory response, whereas fibrillar Aβ showed less increase of proinflammatory molecules (i.e. interleukin-1β, tumor necrosis factor, and NO), consistent with a more chronic form of inflammation. In the present study we noted increasing amounts of GFAP-positive astrocytes in the EC and CA1 of APPsw
mice that tightly correlated with the amount of oligomeric Aβ deposits and ThioS-positive cored plaques, but not with the total amyloid plaque burden, further suggesting that oligomeric Aβ species along with the subpopulation of “neuritic” plaques likely play a larger role than total amyloid plaque deposition in the brain inflammatory glial cell response in these animals. The numbers of GFAP-positive astrocytes very closely paralleled the amount of neuronal loss in the EC and CA1. Moreover, the total number of GFAP-positive astrocytes in the cingulate cortex was much lower than in the EC and CA1, both in Tg and WT littermates. This intriguing numerical inferiority in the amount of reactive astrocytes in the cingulate cortex (which rendered a much smaller GFAP-positive astrocyte to neuron ratio than in the EC and CA1) might account, at least in part, for the comparatively less neuronal vulnerability than that observed in the medial temporal lobe structures in APPsw
mice and the apparent relative resilience of cingulate cortex neurons. Furthermore, immunostaining with an antibody against NF-κB, a key regulator of proinflammatory genes (e.g. interleukin-1β tumor necrosis factor or cyclooxygenase 2) in glial cells, revealed abundant signal associated with astrocytes surrounding ThioS-positive cored plaques in the EC and CA1 region of Tg APPsw
mice but minimal signal in astrocytes in the cingulate cortex. These results raise the possibility that oligomeric Aβ, alone or in addition to fibrillar Aβ in dense cored plaques, could contribute to neuronal toxicity via astrocytic NF-κB activation. Favoring this possibility, in vitro studies have shown that Aβ exposure induces NF-κB activation and NO production in astrocytes (51
). Moreover, coculture experiments demonstrated that exposure of primary human astroglia to Aβ resulted in NF-κB activation in astrocytes; those Aβ-activated astrocytes induced in turn apoptotic cell death in primary human neurons (52
). Moreover, Aβ exposure can cause alterations in glucose metabolism in astrocytes and this in turn impairs neuronal viability and functionality (53
). Therefore, the possibility exits that oligomeric Aβ-mediated activation of NF-κB in astrocytes may contribute to neuronal cell death in AD. This hypothesis needs now to be further investigated, as it might prove relevant for therapeutic intervention.
Despite the tight correlations observed between oligomeric Aβ load and number of neurons lost and GFAP-positive astrocytes in the brains of APPsw
mice, neither oligomeric Aβ load nor other morphological markers of Aβ deposition correlated well with memory performance. The number of GFAP-positive astrocytes in EC and CA1 hippocampal sub-field was by far the parameter most intimately correlated with the severity of spatial reference and contextual memory impairment in these mice. Based on these data, we suggest that proliferation and activation of astrocytes is very likely a driving force for impaired cognition in this model. This is consistent with our recent data showing that oral chronic treatment with Triflusal, an agent with potent anti-inflammatory effects in the CNS via inhibition of NF-κB activation, significantly decreased the number of reactive astrocytes and microglial cells and rescued memory deficits in Tg2576 mice despite its lack of impact on total amyloid plaque deposition (54
In conclusion, the present work shows that oligomeric Aβ, but not total amyloid plaque accumulation, in the brain of Tg APPsw-tauvlw mice is an early event that predates and faithfully predicts the selective pattern of regional neuronal vulnerability and closely correlates with the extent of neuronal cell loss. Oligomeric Aβ and fibrillar Aβ in dense-core plaques, but not total diffuse plaque burden, are intimately associated with astrocytic proliferation in the brains of these mice. The number of GFAP-positive astrocytes is by far the parameter most tightly correlated with memory impairment and neuronal cell death in these mice, and astrocyte immunoreactivity to NF-κB is markedly increased in the same medial temporal lobe regions that undergo early oligomeric Aβ accumulation and selective neuronal degeneration. Our observations place astrocytes as a target of oligomeric Aβ and support the hypothesis that the astrocyte inflammatory response, likely mediated by an NF-κB-related mechanism, is a driving force for impaired cognition and may also contribute to neuronal cell damage. Further studies are now needed to demonstrate a causal relation that might prove relevant in AD.