There is increasing interest in the possibility that soluble Aβ oligomers (ADDLs) could be the proximal neurotoxins in AD [49
]. It therefore is important to determine directly whether neurons exposed to oligomers undergo pathological changes characteristic of AD brain. In the current study, we have verified that a significant pathological consequence of ADDL binding to CNS neurons is tau hyperphosphorylation. ADDL-induced tau hyperphosphorylation was found at epitopes characteristically phosphorylated in AD, namely Ser404, Thr231, Thr 181, Ser 202 and Thr 205 [18
]. Importantly, we show that tau hyperphosphorylation is induced not only by synthetic ADDLs, but also by soluble extracts containing ADDLs obtained from AD brains. These findings provide strong evidence for the role of human Aβ oligomers in the induction of tau hyperphosphorylation in AD.
We have previously shown that not all neurons in culture are targeted by ADDLs [28
]. As shown here, this binding to specific neurons is required for ADDL-induced tau hyperphosphoylation. Image analysis revealed a marked increase in P-tau levels in a subpopulation of neurons that exhibited ADDL binding (), but not in neurons that were ADDL-free. This cell-to-cell specificity in binding and evoked pathology would presumably provide a basis for selective neuronal vulnerability in AD. The molecular basis for this specificity depends on ADDL attachment to trypsin-sensitive sites on neuronal membranes [29
]. The identity of these sites is currently under investigation.
Consistent with the requirement for ADDL binding to neuronal surfaces, tau hyperphosphorylation induced by synthetic ADDLs (, ) or soluble AD brain extracts () was blocked by NU1. This monoclonal antibody targets pathological assemblies of Aβ and blocks their binding to cell surfaces [31
]. Use of monoclonal antibodies that target and neutralize Aβ oligomers remains a promising alternative to active vaccination as a strategy for AD therapeutics. Although Aβ immunization approaches originally aimed to clear amyloid plaques [4
], the mounting evidence suggests that immunotherapy should be directed at blocking the early toxic action of Aβ oligomers, maximizing the chances of slowing or reversing the cognitive and memory deficits in AD. Transgenic mouse models of AD responds to anti-Aβ monoclonal antibodies with improved memory performance prior to any impact on insoluble Aβ deposits [12
]. Recent studies have extended these investigations to pathology. A single intrahippocampal injection of an anti-oligomer antibody is sufficient to clear both Aβ pathology and tau pathology in a triple transgenic mouse model harboring mutant human amyloid precursor protein, presenilin 1 and tau [46
]; related studies showed extracellular Aβ was cleared before intracellular Aβ [45
]. In addition, it has been shown that other antibodies can produce a decline of soluble Aβ oligomers, but not insoluble Aβ, and reduce both glycogen synthase kinase-3β activation and tau phosphorylation both in vivo
and in vitro
]. Along with the present results, these observations indicate that anti-oligomer antibodies may protect against tau pathology and neuritic dysfunction in AD.
We note that the current studies maximized ADDL concentrations in order to investigate effects that might be cumulative and potentially take longer times in vivo
. Most likely, the active species are 12mers, which have been shown to accumulate in AD affected human brain [14
] and more recently in tg-mice models [34
]. The 12mers, which appear to represent only a fraction of the ADDL prep, can be readily detected in Western blots of synthetic preparations because of the sensitivity of the NU1 antibody (), but detection in silver stain requires further concentration [14
]. Recent procedures have been described that enhance formation of SDS-stable 12mers from synthetic Aβ [7
]. The ability of crude AD brain extracts to stimulate tau hyperphosphorylation in an antibody-sensitive manner suggests it would be possible to immunoisolate the neurologically active species, but efforts to recover active toxins has so far been unsuccessful.
With respect to the cellular mechanism, ADDL-induced tau phosphorylation was found to be blocked by the Src family tyrosine kinase inhibitor, PP1, and by the PI3K inhibitor, LY294002 (). In accord with these results, phosphorylation of neuronal proteins including tau [30
] and focal adhesion kinase (FAK) [56
] has been shown to be induced by fibrillar Aβ and blocked by PP1 and LY294002 [55
]. Phosphorylation of FAK is believed to be central in a number of important signaling pathways involving Src family kinases, PI3K and Akt. FAK serves as a regulated adaptor protein, recruiting other proteins by autophosphorylation of Tyr397, a high-affinity binding site for SH2 domains of Src proteins including Fyn [57
] and PI3K [9
]. Binding of these kinases to FAK in turn enables them to phosphorylate tyrosine residues in the C-terminal region of FAK and also other cytoskeletal proteins associated with FAK. Phosphorylation is followed by recruitment of other proteins, resulting in the formation of multiprotein complexes that trigger a number of different signaling pathways. For example, activation of Fyn/FAK/PI3K/MAPK/extracellular signal-regulated kinase (ERK) pathways is associated with protective (e.g., anti-apoptotic, neuritogenic) responses under normal physiological conditions [e.g., 23
]. Although previous studies suggest that activation of PI3K/Akt may protect against neuronal death induced by Aβ [e.g. 3
], those same signaling pathways also have been reported to be activated by Aβ [e.g., 5
]. Therefore, it is possible that ADDL binding to neuronal target receptors leads to aberrant activation of trophic signaling and to an incomplete set of downstream events that lead to tau hyperphosphorylation and neuronal dysfunction. In this regard, recent studies suggest a potential role of aberrant control of Akt and PI3K signaling in AD. For example, increased Akt activation and hyperphosphorylation of critical Akt substrates has been demonstrated in AD brain [17
]. Moreover, it has been suggested that excessive activation of the PI3K/Akt pathway contributes to neuronal degeneration in a mouse model of Niemann-Pick type C [6
], a neurodegenerative disease also characterized by the presence of neurofibrillary tangles. Furthermore, transient PI3K inhibition has been shown to protect neurons from oxidative stress via suppression of ERK activation, suggesting that the PI3K pathway may serve opposing roles, acting at distinct kinetic phases to either promote or limit a slowly developing program of cell death [35
ADDLs have previously been shown to trigger rapid synaptic dysfunction, blocking LTP and the reversal of LTD while inducing aberrant expression of the memory-linked protein Arc [13
]. Recently, ADDLs have also been shown to induce excessive generation of reactive oxygen species [11
]. These effects of ADDLs seem likely to be related to early AD memory failure. Here we show that selective binding of ADDLs to neurons also stimulates tau hyperphosphorylation, a major facet of AD pathology. The properties of ADDLs thus appear capable of accounting for key features of AD pathology and cognitive failure, consistent with the unifying hypothesis that soluble oligomers of Aβ act as proximal neurotoxins in AD.