Following these original studies, soluble Aβ molecules became the specific focus of subsequent studies in attempt to shed light on their possible contribution to AD pathophysiology. To try to clarify the fast accumulating data on this topic, we classified these reports based on the putative Aβ oligomers directly involved. These endogenous assemblies included dimers, trimers, Aβ*56, species immunoreactive to amyloid-beta derived diffusible ligands (ADDLs)/globulomers antibodies, and annular protofibrils.
Aβ dimers are probably the most studied oligomeric species at the present time. Multiple reasons account for the latter: (i) Aβ dimers are SDS-stable (McLean et al. 1999), (ii) Aβ dimers brain levels are elevated in AD mouse models and in subjects with AD (Kawarabayashi et al. 2004; Shankar et al. 2008, 2009; Mc Donald et al. 2010), (iii) size-exclusion chromatography (SEC) segregated Aβ dimers from AD brain extracts impair long-term potentiation (Shankar et al. 2008). While the potency of Aβ dimers appears to be remarkable (Reed et al. 2009), the question relative to the origin of dimers remains nevertheless speculative.
In animal models of AD such as Tg2576 (Kawarabayashi et al. 2004) and J20 (Meilandt et al. 2009; Shankar et al. 2009), Aβ dimers can be found in soluble protein extracts at 10–12 and 10–14 months of age, respectively. Interestingly, these ages correspond to amyloid plaques depositing in cortical areas (Mucke et al. 2000; Kawarabayashi et al. 2001), suggesting that Aβ dimers and fibrillar Aβ are related to each other. Figure 2 illustrates that the relative abundance of Aβ dimers and monomers detected in the formic-acid (FA) soluble fraction (containing fibrillar proteins) sharply increased starting at 12 months of age in Tg2576. Specifically, FA-soluble Aβ monomers followed a sigmoidal profile similar to the one previously described (Kawarabayashi et al. 2001). In contrast, dimeric Aβ levels showed an exponential profile in Tg2576 (Fig. 2b). As Aβ monomers reached a plateau prior than Aβ dimers in this model (Fig. 2b and c), it is tempting to hypothesize that, dimeric Aβ levels appear to follow plaque accumulation in vivo in this model (Kawarabayashi et al. 2001). In plaque-free brains of young transgenic mice, Aβ dimers have yet to be found. Earlier work (Kawarabayashi et al. 2004) reported the presence of Aβ dimers in lipid raft preparations from 6-month-old Tg2576, but later studies revealed that specific lipid enrichment could trigger Aβ oligomerization artificially (Yu et al. 2005; Kim et al. 2006), questioning the intrinsic existence of these species in very young Tg2576 mice.
Figure 2. Temporal accumulation of endogenous Aβ dimers in brain tissue of Tg2576 mice. (a) Western blots showing the relative levels of Aβ dimers detected following immunoprecipitation using formic acid extracts. Dimeric Aβ levels increase in parallel to plaque accumulation in Tg2576, suggesting that dimers are closely related to deposited amyloid. (b) Densitometry analysis of formic acid-soluble Aβ monomers and dimers in Tg2576. Empty circles represent monomeric Aβ while filled black circles correspond to Aβ dimers (n = 3/group). (c) Relative ratio between Aβ monomers and Aβ dimers in Tg2576. *Non-specific band caused by the immunoprecipitation. For notes and abbreviations refer to Figure 1.
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Similarly in human studies, apparent endogenous Aβ dimers were only found in brains of subjects with AD (Shankar et al. 2008; Mc Donald et al. 2010) when comparing individuals with extensive amyloid and tau pathology and subjects devoid of amyloid accumulation. In the former study (Shankar et al. 2008), proteins were extracted from subjects without AD (mean age = 65.6 ± 12.1 years; n = 3) or with severe AD (mean age = 78.1 ± 5.8 years; n = 7). Aβ dimers were identified only in the AD group, even though these groups were not normalized for age or amyloid burden. Thus, Aβ dimers appear to be closely linked to fibrillar amyloid in humans as well.
Although these in vivo studies suggest that dimeric Aβ and plaques may play a deleterious and mysterious tango in the brain, the origin of dimers still remains elusive. Previous research revealed that primary cortical neurons from Tg2576 produce monomers and trimers; Aβ dimers were not detected under the experimental conditions used (Lesne et al. 2006) indicating that neurons preferentially produce and secrete monomers and trimers but not dimers. More recently, our own group observed that Aβ dimers are in fact produced and secreted by primary neurons but at very low amounts (1 : 20 and 1 : 15 ratio to monomers and trimers respectively; Fig. 3a). These results are in sharp contrast to the relative abundance of dimers in the conditioned medium of 7PA2 cells (Podlisny et al. 1998; Walsh et al. 2002). The reason why dimers are produced and secreted so prominently in this cell line remains unknown, but the profile observed by biochemical analyses performed by our own group clearly differs from those from primary neurons (Fig. 3b). Lastly, dimers are found as what appears to be the primary oligomeric species formed in mice over-expressing the E693Δ APP mutant (Tomiyama et al. 2010). Identified in 2008, this deletion within the mid-region of Aβ engages Aβ peptides to form preferentially oligomeric species instead of forming fibrils (Tomiyama et al. 2008, 2010). Of interest, these Aβ dimers are accumulating intracellularly and co-segregated within the insoluble protein fraction extracted with formic acid but not within soluble protein fractions (Tomiyama et al. 2010). These independent findings further support the hypothesis that endogenous Aβ dimers are the molecular brick for fibrillar Aβin vivo. Identifying what might favor dimeric Aβ formation in absence of mutation may shed light on the origin of these assemblies.
Figure 3. Comparison between endogenous Aβ species secreted by mature Tg2576 primary cortical neurons and 7PA2 cells. (a, b) Western blots showing the relative levels of Aβ oligomers detected with 6E10 following immunoprecipitation using 1 mL of conditioned medium. While dimeric Aβ levels is preferentially secreted by 7PA2 cells (b), Aβ trimers are produced and secreted by primary neurons derived from Tg2576 mice (a). The observed profile in neurons (a) suggests a trimer-based oligomerization process under physiological conditions. For notes and abbreviations refer to Figure 1.
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Several lines of evidence indicate that Aβ trimers might constitute the molecular brick for non-fibrillar assemblies of Aβ. First, as mentioned in the above section, trimers are the preferential, most abundant species produced and secreted by primary neurons in vitro (Lesne et al. 2006), even though the origin of trimeric Aβ remains elusive. It is possible that this preference might be due to lower energetic requirement to form trimers or due to the absence of a chaperone protein X allowing the conversion to dimers. Second, trimers are present in brain tissue of Tg2576 as early as embryonic day 14 and its expression persists throughout life (Lesne et al. 2006). Analyzing extracellular- and intracellular-enriched protein fractions in these mice indicated that brain levels of Aβ trimers steadily but very slowly rise with aging (Lesne et al. 2006). These findings were mirrored in human studies where trimers are found as early as 1 year of age, contrasting with brain Aβ dimers which were not detected in subjects until 50–60 years of age in Tris-buffered saline extracts (S. Lesne, M. A. Sherman, M. Kuskowski, J. A. Schneider K. R. Zahs, D. A. Bennett and K. H. Ashe, unpublished data).
Importantly, when comparing Tg5469 and Tg2576 lines, which express equal amounts of human APP but vastly different levels of Aβ caused by the presence of the Swedish mutation in the latter line, Aβ trimers could not be readily observed in Tg5469 brains (Ma et al. 2007). This suggests that the formation of trimers in vivo appears to be dependent on the levels of Aβ production. Cumulatively, these findings suggest that trimers are the earliest endogenous oligomeric Aβ species produced and secreted by neurons.
Based on the finding that neurons preferentially secrete Aβ trimers, it was likely that these assemblies could form larger oligomeric species based on multiples of 3, that is, hexamers (6-), nonamers (9-) and dodecamers (12-), in absence of deposited Aβ. Examining young Tg2576 mice prior to plaque formation, apparent trimers (13 kDa), hexamers (27 kDa), nonamers (40 kDa) and dodecamers (56 kDa) were detected using antibodies targeting the N-terminus and central domain of Aβ, such as 6E10, and using the A11 antibody detecting pre-fibrillar oligomeric proteins (Kayed et al. 2003; Lesne et al. 2006). The discovery of Aβ*56, a putative dodecameric Aβ assembly, derived from the observation that cognitive decline in Tg2576 starts at 6 months of age, when Aβ*56 first appears, and remains relatively stable for months (up to 15 months of age) when numerous endogenous Aβ species (soluble monomers, insoluble monomers, dimers, trimers) accumulate in brain tissues many fold (Lesne et al. 2006). Aβ*56 can be detected with multiple antibodies, which contribute to establishing the antibody reactivity profile of this Aβ species: A11+ , OC−, 6E10+, 4G8+, Aβx-40+, Aβx-42+, 82E1−. This epitope specificity allows to categorize Aβ*56 as a non-fibrillar Aβ oligomer (Fig. 4).
Figure 4. Epitope mapping for Aβ*56 in Tg2576 soluble extracts. Immunoprecipitation/western blots showing the detection profile of Aβ*56 present in Tg2576 mouse brains. A comparison between 6E10, A11 and OC antibodies (kind gifts from Rakez Kayed and Charles Glabe) is presented. The 6E10+/A11+/OC− profile characterizes Aβ*56 as a non-fibrillar Aβ oligomer. *Non-specific bands generated by the amplification process. For notes and abbreviations refer to Figure 1.
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Importantly, the presence of Aβ*56 was also reported in other APP transgenic mouse models of AD, for example, J20 (Cheng et al. 2007; Meilandt et al. 2009), Arc6/48 (Cheng et al. 2007), 3xTgAD (Oddo et al. 2006) and APP23 (S. Lesne, M. Sherman and K. H. Ashe, unpublished data), clearly arguing against the possibility that it is an artifact intrinsic of one isolated mouse model.
Contrary to dimers and trimers, which can be detected in vitro, Aβ*56 is not detected in cellular protein extracts of cultured neurons or in conditioned media indicating that it is not produced and secreted by primary neurons (Lesne et al. 2006). This finding suggests that this assembly requires a co-factor X, present in brain tissue, to promote its formation. Furthermore, aging must regulate the expression of this unidentified co-factor X explain the sudden accumulation of Aβ*56 in 6-month-old Tg2576 brains.
In mouse models, Aβ*56 is present in two soluble protein fractions, extracellular-enriched (Lesne et al. 2006, 2008) and membrane-enriched extracts (Cheng et al. 2007), indicating that this assembly is formed extracellularly and may interact with surface receptors at neuronal membranes. Similar segregation was observed in studies with a large human cohort (n = 135; S. Lesne, M. A. Sherman, M. Kuskowski, J. A. Schneider, K. R. Zahs, D. A. Bennett and K. H. Ashe, unpublished data). When comparing across clinical diagnosis groups, Aβ*56 brain levels seem to peak during the pre-clinical stage of AD and decrease in mild cognitive impairment and AD subjects (S. Lesne, M. A. Sherman, M. Kuskowski, J. A. Schneider, K. R. Zahs, D. A. Bennett and K. H. Ashe, unpublished data). The data are in agreement with evidence gathered in Tg2576 suggesting that Aβ*56 may form in the aging brain prior to the formation of amyloid plaques.
Of interest, the relative levels of soluble Aβ*56 in the extracellular protein fraction of human brain tissue showed no relationship with Aβ dimers (Fig. 5a). In contrast, brain levels of human Aβ*56 were positively correlated to trimeric soluble Aβ levels in the same fraction (Fig. 5b). This observation further supports our hypothesis that trimeric Aβ might constitute the building block for larger oligomers in the non-fibrillar pathway.
Figure 5. Relationship between endogenous low molecular weight Aβ oligomers and Aβ*56 in human brain tissue. (a, b) Linear regression analyses between the relative levels of soluble Aβ dimers (a) or trimers (b) and Aβ*56 detected in extracellular-enriched protein fractions of human brain tissue from the Religious Order Study (n = 88). Dimeric Aβ levels showed no association with Aβ*56 whereas the abundance of Aβ trimers was positively correlated with Aβ*56 brain levels (Rho is indicated next to the p-value for Spearman Rank Correlation. Correlations were performed using the whole cohort, n = 88). For notes and abbreviations refer to Figure 1.
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ADDLs and globulomers
Even though this review aims at discussing accumulated findings about endogenous oligomeric Aβ species, tools initially developed to target synthetic soluble Aβ molecules (e.g. Aβ-derived diffusible ligands or ADDLs and globulomers) appear to detect endogenous Aβ entities, even though the exact nature of these molecules needs further studying (Gong et al. 2003; Barghorn et al. 2005; Georganopoulou et al. 2005; Hillen et al. 2010). The antibodies termed NU-x and anti-globulomer antibodies (5598, 8F5, A-887755), developed by Klein’s group at Northwestern and Abbott respectively, seem able to identify 35–60 kDa oligomeric Aβ species recognized by these groups as dodecamers (Barghorn et al. 2005; Lambert et al. 2007; Hillen et al. 2010).
In Tg2576, ADDLs are increased ∼5- to ∼100-fold (Chang et al. 2003) at ages during which spatial reference memory remains unchanged. In addition, measurements of ADDLs in brain tissue and in CSF revealed 70-fold increases in AD compared to age-matched controls (Gong et al. 2003; Georganopoulou et al. 2005). These findings suggest that ADDLs levels rise sharply in AD, reminiscent of the marked elevation in Aβ dimers (Shankar et al. 2008).
Similarly to ADDLs, the use of mid-range molecular weight globular synthetic Aβ oligomers migrating at 38–48 kDa, termed globulomers, generated antibodies targeting alleged endogenous Aβ species. Because of the apparent size of the synthetic assemblies detected, Barghorn and coworkers indicated that globulomers corresponded to Aβ dodecamers. In animal studies performed with Tg2576 mice, globulomers were mostly unchanged between 2.5 and 10 months of age prior to plaque formation (26%), but brain levels rose 496% at 12 months of age when amyloid plaques start forming (Kawarabayashi et al. 2001). Furthermore, immunohistochemical analyses using the anti-globulomer 8F5 antibody labeled amyloid plaques in brains of Tg2576 animals and subjects with AD (Barghorn et al. 2005). These findings indicate that, regardless of the exact endogenous Aβ species detected by anti-globulomer antibodies, these molecules are highly associated with amyloid plaques and are elevated in AD.
In both cases of ADDLs and globulomers, there has been some confusion that these ‘dodecamers’ correspond to the endogenous 56 kDa Aβ assembly, Aβ*56 (Wilcox et al. 2011). However, several lines of evidence indicate that Aβ*56 and ADDLs/globulomers are different entities. First, Aβ*56 brain levels are remarkably stable between 6 and 15 months of age in extracellular-enriched fractions of Tg2576 (Lesne et al. 2006) while ADDLs and globulomers are vastly increased during that same time interval. Second, Aβ*56 levels in both extracellular and membrane-enriched protein fractions are lowered in AD compared with non-impaired age-matched controls (S. Lesne, M. A. Sherman, M. Kuskowski, J. A. Schneider, K. R. Zahs, D. A. Bennett and K. H. Ashe, unpublished data) while ADDLs/globulomers increase in AD (Gong et al. 2003; Barghorn et al. 2005; Georganopoulou et al. 2005; Nimmrich et al. 2008). Based on these observations, ADDLs antibodies likely detect in vivo species others than trimers and Aβ*56, possibly fibrillar oligomers or annular protofibrils (Glabe 2008). This subclass of oligomers is characterized by being immunopositive to the antibody OC (OC+) and immunonegative to A11 (A11−). Even though it is not known whether ADDLs and globulomers are OC+/A11−, the fact that ADDLs and globulomer antibodies detect amyloid plaques suggests that ADDLs correspond to fibrillar oligomers.
In addition to the point that ADDLs/globulomers and Aβ*56 represent different entities, the origin(s) of the endogenous Aβ species detected by ADDLs/globulomer antibodies is completely unknown. Further studies are needed to determine how these possible Aβ assemblies are produced and when they do form in the continuum aging-AD.
Annular protofibrils (∼100–150 kDa)
Annular protofibrils (APFs) are pore-like structures that are believed to derive from the circularization of non-fibrillar Aβ assemblies (Kayed et al. 2009). With a molecular size over 90 kDa, they resemble bacterial pore-forming toxins. APFs have been proposed to permeabilize lipidic membranes and induce cell death by altering neuronal homeostasis (Glabe and Kayed 2006; Lashuel and Lansbury 2006). Recent data revealed that APFs might constitute a distinct Aβ assembly from non-fibrillar oligomers (Kayed et al. 2009). Based on this work, APFs originate from pre-existing non-fibrillar oligomers, which upon some conformational change allows the recruitment of additional oligomers to form APFs. In 17- to 20-month-old APP23 mice, ∼2% of examined synapses showed an accumulation of APFs using a specific αAPF antibody (Kokubo et al. 2009). On a follow-up study, these same groups recently demonstrated that APFs are associated with diffuse plaques and intracellular punctate deposits in AD brain sections (Lasagna-Reeves et al. 2011). Despite very small specimen numbers (n = 2/group; N = 4), these findings indicate that APF exist in vivo.
Even though these new studies and conformation-dependent antibodies are allowing us to discriminate among the multiple oligomeric forms of Aβin vitro and in vivo, we know nothing about the age at which APFs first form in animal models and in human brain tissue and CSF.