The search for the toxic species responsible for the neurodegeneration observed in Alzheimer disease has become this field’s Holy Grail. And much like that mythical search, the search for the toxic species has been full of false leads, dead ends, and even a couple of conspiracy theories. One thing that most of the field will agree on is that Aβ aggregation is a central element to the generation of the toxic species, with most of the recent focus being on the formation of smaller oligomeric forms. However, due to limitations of many methods, studying aggregating proteins and peptides has proved to be an inexact science. For this reason the work by Bernstein et al. using mass spectrometry coupled with ion mobility to characterize the early aggregation pathway of both Aβ40 and 42 is a technical tour de force. The approach is very elegant. It elucidates many of the intermediates on the aggregation pathway and clearly shows that Aβ40 and 42 behave differently. The major difference is that Aβ42 forms a meta-stable dodecamer structure, a species that has previously...  Read more

The search for the toxic species responsible for the neurodegeneration observed in Alzheimer disease has become this field’s Holy Grail. And much like that mythical search, the search for the toxic species has been full of false leads, dead ends, and even a couple of conspiracy theories. One thing that most of the field will agree on is that Aβ aggregation is a central element to the generation of the toxic species, with most of the recent focus being on the formation of smaller oligomeric forms. However, due to limitations of many methods, studying aggregating proteins and peptides has proved to be an inexact science. For this reason the work by Bernstein et al. using mass spectrometry coupled with ion mobility to characterize the early aggregation pathway of both Aβ40 and 42 is a technical tour de force. The approach is very elegant. It elucidates many of the intermediates on the aggregation pathway and clearly shows that Aβ40 and 42 behave differently. The major difference is that Aβ42 forms a meta-stable dodecamer structure, a species that has previously been identified as a candidate for the toxic species (Lesne et al., 2006).

Is this dodecamer the toxic species? We don’t know. While the dodecamer may be the most stable species in a “test tube,” we also know that Aβ is capable of interacting with a range of biological substrates, all of which could stabilize other structural forms of Aβ. These binding partners include lipid membranes (e.g., Kayed et al., 2004; Tickler et al., 2005; Hung et al., 2008; Martins et al., 2008), protein receptors (e.g., Deshpande et al., 2009) and metal ions (Bush, 2003). All of these factors have been implicated in the generation of the toxic species.

The existence of the meta-stable dodecamer structure raises an interesting conundrum with respect to the many efforts that are currently ongoing to isolate the toxic Aβ species from various tissues. Function (toxic or otherwise) is related to structure; Aβ is a pleiomorphic molecule and its structure depends on its context—change the context and you change the structure. The findings by Bernstein et al. suggest that if you have isolated Aβ42, then the default structure that this peptide will most likely adopt is the dodecamer. Whether this is the structure the peptide originally had in the brain is an unanswered question.

The work by Harmeier et al. reminds us that there is more to Aβ toxicity than peptide aggregation. We have previously shown that mutations to the GxxxG motif can alter the rate of Aβ aggregation affecting the formation of smaller oligomers of Aβ and their interactions with lipid membranes (Hung et al., 2008). Harmeier et al. have extended this work into an in-vivo fly model and show that G33 is a key residue in regulating Aβ aggregation and toxicity. Substitutions that increase the hydrophobicity at this residue increase aggregation and in the process decrease the toxicity.

Similarly, we have previously published that a single substitution to Y10 of Aβ inhibited toxicity, even though this peptide formed dimers, trimers, and other oligomeric forms including a 56 KDa species (Barnham et al., 2004). These data show that Aβ aggregation is not the only requirement for toxicity. Other factors do indeed play a role, and it is likely to be a quite specific conformation of Aβ oligomer that is toxic. The requirement for a specific conformation for Aβ toxicity would be consistent with the concept that toxicity is mediated through a specific receptor interaction, e.g., such as with the NMDA receptor (Deshpande et al., 2009).

The work by Bernstein et al. and Harmeier et al. sheds more light on the search for the Holy Grail, but the quest will continue.

References:
Barnham KJ, Haeffner F, Ciccotosto GD, Curtain CC, Tew D, Mavros C, Beyreuther K, Carrington D, Masters CL, Cherny RA, Cappai R, Bush AI. Tyrosine gated electron transfer is key to the toxic mechanism of Alzheimer's disease beta-amyloid. FASEB J. 2004 Sep;18(12):1427-9. Abstract

Bush AI. The metallobiology of Alzheimer's disease. Trends Neurosci. 2003 Apr;26(4):207-14. Abstract

Deshpande A, Kawai H, Metherate R, Glabe CG, Busciglio J. A role for synaptic zinc in activity-dependent Abeta oligomer formation and accumulation at excitatory synapses. J Neurosci. 2009 Apr 1;29(13):4004-15. Abstract

Hung LW, Ciccotosto GD, Giannakis E, Tew DJ, Perez K, Masters CL, Cappai R, Wade JD, Barnham KJ. Amyloid-beta peptide (Abeta) neurotoxicity is modulated by the rate of peptide aggregation: Abeta dimers and trimers correlate with neurotoxicity. J Neurosci. 2008 Nov 12;28(46):11950-8. Abstract

Kayed R, Sokolov Y, Edmonds B, McIntire TM, Milton SC, Hall JE, Glabe CG. Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases. J Biol Chem. 2004 Nov 5;279(45):46363-6. Abstract

Lesné S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH. A specific amyloid-beta protein assembly in the brain impairs memory. Nature. 2006 Mar 16;440(7082):352-7. Abstract

Martins IC, Kuperstein I, Wilkinson H, Maes E, Vanbrabant M, Jonckheere W, Van Gelder P, Hartmann D, D'Hooge R, De Strooper B, Schymkowitz J, Rousseau F. Lipids revert inert Abeta amyloid fibrils to neurotoxic protofibrils that affect learning in mice. EMBO J. 2008 Jan 9;27(1):224-33. Abstract

Tickler AK, Smith DG, Ciccotosto GD, Tew DJ, Curtain CC, Carrington D, Masters CL, Bush AI, Cherny RA, Cappai R, Wade JD, Barnham KJ. Methylation of the imidazole side chains of the Alzheimer disease amyloid-beta peptide results in abolition of superoxide dismutase-like structures and inhibition of neurotoxicity. J Biol Chem. 2005 Apr 8;280(14):13355-63. Abstract

View all comments by Kevin Barnham

We readily agree with some of the data and interpretations given in the interesting paper of Bernstein et al. Moreover, the study shows ESI-MS to be a useful method to analyze non-covalently linked oligomers in the gas phase. In my respectful opinion, some parts of the paper seem to focus too much on the Aβ*56 (12-mer).

There is no doubt that the dimer and tetramer of Aβ42 are important. Quite a while ago, we were able to show why, since engineered dimers have a twofold increased β-sheet content (Schmechel et al., 2003). This was the first report to show that covalently linked dimers of Aβ can serve as a nidus to start fibril growth and that homodimers of Aβ are a risk factor for the formation of higher oligomers.

Our data published last week in the Journal of Neuroscience (Harmeier et al., 2009) show that toxicity also requires a specific conformation of Aβ42 variants. The G33I substitution and mutant in Drosophila shows that it might be important to compare the conformations of Aβ42...  Read more

We readily agree with some of the data and interpretations given in the interesting paper of Bernstein et al. Moreover, the study shows ESI-MS to be a useful method to analyze non-covalently linked oligomers in the gas phase. In my respectful opinion, some parts of the paper seem to focus too much on the Aβ*56 (12-mer).

There is no doubt that the dimer and tetramer of Aβ42 are important. Quite a while ago, we were able to show why, since engineered dimers have a twofold increased β-sheet content (Schmechel et al., 2003). This was the first report to show that covalently linked dimers of Aβ can serve as a nidus to start fibril growth and that homodimers of Aβ are a risk factor for the formation of higher oligomers.

Our data published last week in the Journal of Neuroscience (Harmeier et al., 2009) show that toxicity also requires a specific conformation of Aβ42 variants. The G33I substitution and mutant in Drosophila shows that it might be important to compare the conformations of Aβ42 toxic and non-toxic variants to learn which receptors might mediate the toxicity. This view is supported by Bernstein and colleagues, who show that tetramers of Aβ42 matter but not of Aβ40. In this regard, the S26C-linked dimer published last year by Tom Kukar and colleagues shows that engineered Aβ40 dimers can inhibit LTP and might adopt a toxic conformation, although it should be added that data of this paper are being intensely discussed in the field (Kukar et al., 2008).

We agree that intrinsic factors matter, independent of the length of the peptide (i.e., 42 or 40 residues) and that the specific conformation is important. One also has to be mindful that LTP and cell toxicity are relevant assays, but their link to the AD pathology is still missing; in other words, are LTP inhibition or cell toxicity really the cause of degrading synapses and neuronal loss leading to memory loss in the end?

In our view, another important point is that a therapeutic approach that aims to arrest aggregation at the stage of tetramers might be dangerous since tetramer and dimers were described as the most toxic species. It is possible that if a therapeutic approach attempts to arrest oligomer growth, the level of (toxic) forms might be unwittingly increased from the reservoir of non-toxic oligomers which might be converted into toxic forms by changing the conformation, e.g., by binding to a specific receptor? Thus, one has to make sure that a conversion of non-toxic into toxic oligomers will not happen.

Fibril growth might not be a problem as long as those fibrils (non-toxic per se) are not a source of dimers or tetramers that are brought back into solution. It may sound crazy to some, but I believe it might be better to quickly deposit Aβ oligomers as fibrils that are then not harmful any longer, or at least less harmful. Finally, it could turn out that the longer dimers and tetramers are available over time, i.e., the longer their half-life, the higher the likelihood that damage accrues.

View all comments by Gerd Multhaup

Elusive oligomerization-mediated amyloid-β-protein toxicity: Where have all the trimers gone?
Evidence that the pathogenesis of Alzheimer disease (AD) is strongly associated with occurrence of oligomeric assemblies of Aβ is strongly challenging researchers who wish to identify suitable therapeutic strategies for prevention and cure of this debilitating illness. It is well known that of the two dominant alloforms of Aβ—Aβ40, and Aβ42—AD is more correlated with the latter, longer alloform. How does a small difference in the primary structure so critically affect the pathology? Within this past week, two inspiring papers addressed Aβ40 and Aβ42 oligomer formation and toxicity from unique angles.

Application of ion-mobility mass spectroscopy to resolve the oligomer sizes of Aβ40 and Aβ42 performed in Michael Bowers’ group, in collaboration with experimental labs of Gal Bitan and Dave Teplow and the computational group of Joan-Emma Shea, yielded both expected and unexpected results (Bernstein et al.,...  Read more

Elusive oligomerization-mediated amyloid-β-protein toxicity: Where have all the trimers gone?
Evidence that the pathogenesis of Alzheimer disease (AD) is strongly associated with occurrence of oligomeric assemblies of Aβ is strongly challenging researchers who wish to identify suitable therapeutic strategies for prevention and cure of this debilitating illness. It is well known that of the two dominant alloforms of Aβ—Aβ40, and Aβ42—AD is more correlated with the latter, longer alloform. How does a small difference in the primary structure so critically affect the pathology? Within this past week, two inspiring papers addressed Aβ40 and Aβ42 oligomer formation and toxicity from unique angles.

Application of ion-mobility mass spectroscopy to resolve the oligomer sizes of Aβ40 and Aβ42 performed in Michael Bowers’ group, in collaboration with experimental labs of Gal Bitan and Dave Teplow and the computational group of Joan-Emma Shea, yielded both expected and unexpected results (Bernstein et al., 2009). Expected was the fact that Aβ40 and Aβ42 oligomerized through distinct pathways, as has been experimentally demonstrated in 2003 by using photo-induced cross-linking of unmodified proteins (PICUP) (3). The present study by Bernstein and collaborators provides an independent confirmation of oligomerization differences between Aβ40 and Aβ42 and concurs that their differences are not induced by a particular chemistry of PICUP (5).

Unexpected was the lack of trimers of both Aβ40 and Aβ42. Trimers represented a significant oligomeric species of Aβ40 oligomer population in the original study by Bitan and collaborators (3). Bernstein and collaborators found trimers to be present in oligomeric states of the two Aβ40 mutants, one with F19P substitution and the other with oxidized M35, demonstrating that the lack of trimers was not due to limitations of the experimental setup.

Of all peptides under study, only Aβ42 formed oligomers larger than tetramers and was thus uniquely characterized by hexamers and dodecamers.

Experimental data by Gerd Multhaup's group (Harmeier et al., 2009) show that a single-point mutation at G33 but not G29 can offset the oligomerization pathway of Aβ42. Both single-point mutations of Aβ42 under investigation, G33A and G33I, resulted in an enhanced oligomerization characterized by larger oligomers as compared to Aβ42 wild-type. Surprisingly, despite enhanced oligomerization, the resulting mutant oligomers were less toxic than Aβ42 wild-type oligomers! In the old days, the amyloid hypothesis of AD was associating neuronal loss and toxicity with amyloid plaques until a paradigm shift from amyloid fibrils to oligomers occurred. The present work by Harmeier and collaborators now demonstrates that toxicity is not necessarily correlated with the oligomerization propensity either (Harmeier et al., 2009), suggesting that specificity of the oligomer structure plays a crucial role in mediating toxicity.

Both studies identified as the low molecular mass Aβ42 oligomeric species (in addition to monomers) also dimers, tetramers, and hexamers. The common feature between the two studies was also the fact that Aβ42 oligomer size distribution was multi-modal in contrast to the Aβ40 oligomer size distribution, in agreement with the prior work by Bitan et al. (3) , with additional peak at dodecamers (Bernstein et al., 2009) or 16- to 20-mers (Harmeier et al., 2009). No trimers were detected in either of the two studies. The two studies differ in their interpretation of Aβ42 toxicity. Bernstein et al. associated Aβ42 toxicity with occurrence of Aβ42 dodecamers with a proposed annular structure resembling ion channels, which if inserted into the membrane bilayer would cause ion leakage and thereby induce cell death. Harmeier et al. identified the key amino acid responsible for Aβ42 toxicity, G33, which if substituted by alanine or isoleucine would result in decreased toxicity of Aβ42(G33A) or Aβ42(G33I) assemblies despite increased oligomerization.

The accompanying commentary on the work by Bernstein et al. was given by Clemmer and Valentine, who discussed whether the specific experimental set-up used in ion-mobility mass spectroscopy, involving peptides in solution to be sprayed into droplets, followed by solvent evaporation resulting in peptide assemblies immersed in a gas, alters the structure of resulting Aβ oligomers or not. If the answer was yes, then the resulting conformers might be less relevant to the etiology of AD, unless gaseous phase could be considered a model of a water-free membrane environment. If the answer is no, then one can assume that the resulting conformers had structure characteristic of an aqueous solution. The paper by Bernstein and collaborators discusses their experimental results using a modeling approach where individual spherical monomer conformers of Aβ42 form a planar hexamer ring, resulting in a close conformation that would not allow for a further addition of monomers and would, upon two-ring hexamer merge, form a potentially toxic dodecamer (Bernstein et al., 2009).

Considering the possible Aβ oligomer structure in an aqueous environment from the viewpoint of a computational biophysicist whose work revolves around Aβ structural predictions (2), I see an alternative explanation of the observed structural differences between Aβ40 and Aβ42 oligomers and resulting oligomer-mediated toxicity. The sequence of either Aβ can be viewed as ~1/3 hydrophilic in the N-terminal region and ~2/3 hydrophobic in the central and the C-terminal regions. In water, the hydrophobic regions will be adjusted to achieve the least contact with water. Because the majority of the peptide is hydrophobic, a folded monomer will have many hydrophobic residues exposed to solvent. However, as oligomers form, the central and C-terminal regions will be more efficiently screened from water molecules by the hydrophilic N-terminal regions. The simulations which captured the oligomer size distribution differences as reported by previous work of Gal Bitan and Dave Teplow (3) revealed globular structures of all oligomers with a most intriguing structural difference between Aβ40 and Aβ42 oligomers at the N-terminal region (2). Aβ40 oligomers were characterized by a β-strand structure involving A2-F4 region, rendering more ordered and restricted N-termini, compared to Aβ42 oligomers with random-coil-like and spatially more extended structure at the N-termini (2), suggesting an increased cross-section for Aβ42 oligomers as compared to Aβ40 oligomers and thereby providing an alternative model for explaining the differences in the cross-sections reported by Bernstein and collaborators (Bernstein et al., 2009).

A 5 percent difference in the primary structure at the C-terminus (Aβ40 versus Aβ42) results in an alteration of oligomer pathway as well as structural differences in the N-terminal region. Moreover, structural differences between alloforms appear already at the stage of folding. Our computational study exploring folded structures of Aβ(1-40), Aβ(1-42), and their Arctic mutants demonstrated a lack of the β-strand structure at the N-terminal regions of both Arctic mutants, rendering the folded structure of the Arctic mutants proximate to the more toxic Aβ(1-42) (1). If sequence differences at the C-terminus (Aβ40 and Aβ42) or a single-point mutation in the central hydrophobic region effectively changes the structure at the N-terminal region, then generalizations of any experimental or theoretical results from one alloform to another should be questioned and applied with great caution. Considering such long-range structural effects of a single-point mutation, the conclusion of Harmeier et al. that the amino acid G33 is the key amino acid that mediates toxicity should be slightly rephrased. It is possible that the substitution of G33 by alanine or isoleucine changes not only the local but also the global structure of the resulting oligomers and thus the key structural change that directly impacts the toxicity might be remote from the position G33.

Substantial experimental and computational evidence on Aβ folding and oligomer formation demonstrates that we are dealing with a protein very sensitive to subtle changes in preparation and environment required for application of a specific experimental technique. Perhaps this very disordered nature of Aβ is key to understanding its oligomer formation and the highly selective toxicity of the resulting structures.

References:
1. Lam AR, Teplow DB, Stanley HE, Urbanc B. Effects of the Arctic (E22-->G) mutation on amyloid beta-protein folding: discrete molecular dynamics study. J. Am. Chem. Soc. 130, 17413-17422 (2008). Abstract

2. Urbanc B, Cruz L, Yun S, Buldyrev SUV, Bitan G, Teplow DB, Stanley HE. In silico study of amyloid beta-protein folding and oligomerization. Proc. Natl. Acad. Sci. USA 101, 17345-17350 (2004). Abstract

3. Bitan G, Kirkitadze MD, Lomakin A, Volles SS, Benedek GB, Teplow DB. Amyloid beta-protein (Abeta) assembly: Abeta 40 and Abeta 42 oligomerize through distinct pathways. Proc. Natl. Acad. Sci. USA 100, 330-335 (2003). Abstract

4. Bitan G, Volles SS, Teplow DB. Elucidation of primary structure elements controlling early amyloid beta-protein oligomerization. J. Biol. Chem. 278, 34882-34889 (2003). Abstract

5. Bitan G. Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins. Methods Enzyme. 413, 217-236 (2006). Abstract

View all comments by Brigita Urbanc

I am concerned about drawing firm conclusions about what happens in the human brain from pure synthetic oligomers. My lab prefers to work with naturally produced oligomers, even though they have obvious experimental limitations, and biophysical measures unfortunately cannot be applied due to their small quantities. I do believe from my own work that there will not turn out to be one predominant synaptotoxic oligomer form in the human brain but several assembly forms that are in dynamic equilibrium in vivo.

View all comments by Dennis Selkoe

Sex and the Single Amino Acid—A Devilish Problem
In an article published in the 10 June issue of Journal of Neuroscience (1), Harmeier et al. report results of structure-activity studies of the 42-residue form of the amyloid-β protein, Aβ42. The work seeks to understand the role of single amino acids within Aβ on the folding dynamics, structure, and cellular activity of the peptide. The work reveals that glycine 33 (Gly33) may have a particularly significant role. (Actually, this role has nothing to do with sex, but the title apparently did induce you to read the commentary!)

First, the stipulations of fact: 1) the Multhaup group has done, and continues to do, beautiful, interesting, and significant work; 2) the work of Harmeier et al. continues this tradition; 3) notwithstanding these facts, this commentator believes that it is fun, stimulating, and valuable for the field to play “Devil’s advocate” at times (this being one of them).

The experimental work of Harmeier et al. is quite compelling. Substitution of the hydrophobic...  Read more

Sex and the Single Amino Acid—A Devilish Problem
In an article published in the 10 June issue of Journal of Neuroscience (1), Harmeier et al. report results of structure-activity studies of the 42-residue form of the amyloid-β protein, Aβ42. The work seeks to understand the role of single amino acids within Aβ on the folding dynamics, structure, and cellular activity of the peptide. The work reveals that glycine 33 (Gly33) may have a particularly significant role. (Actually, this role has nothing to do with sex, but the title apparently did induce you to read the commentary!)

First, the stipulations of fact: 1) the Multhaup group has done, and continues to do, beautiful, interesting, and significant work; 2) the work of Harmeier et al. continues this tradition; 3) notwithstanding these facts, this commentator believes that it is fun, stimulating, and valuable for the field to play “Devil’s advocate” at times (this being one of them).

The experimental work of Harmeier et al. is quite compelling. Substitution of the hydrophobic amino acids Ala or Ile for Gly33 produces a peptide that has a high propensity to self-associate, as determined by size exclusion chromatography (SEC). In contrast, substitution of Ala for Gly29, has little effect. Interestingly, both glycines participate in the formation of GxxxG motifs that have been found to mediate APP self-association and affect Aβ production (2). The data thus show how single amino acids in different contexts (APP or Aβ) may produce disparate structural effects. Interestingly, the corresponding doubly substituted peptide, [Gly29Ala/Gly33Ala]Aβ42, displayed aggregation properties (SEC) intermediate between those of the singly substituted peptides. One might predict, based on the aggregation characteristics of the singly substituted peptides, that [Gly29Ala/Gly33Ala]Aβ42 would assemble like [Gly33Ala]Aβ42. This was not the case, an observation again emphasizing the context-behavior relationship, namely the effect of a Gly33Ala substitution alone (i.e., in wild-type Aβ42) versus its behavior in the context of [Gly29Ala]Aβ42.

Similar results were observed in experiments designed to probe the structural dynamics of Aβ42 and its “mutants.” In these experiments, limited trypsin proteolysis was used to evaluate the protease accessibility of the Lys peptide bonds (Lys16 and Lys28) in Aβ42. Conformers in which monomer folding, or monomer self-association, sequester these susceptible peptide bonds show increased protease resistance. In contrast, destabilizing substitutions increase proteolysis rates (e.g., see 3,4). Both Gly33 substitutions significantly decreased proteolysis, whereas the presence of Ala29, whether alone or with Ala33, increased protease susceptibility.

Mechanistic explanations for the conformational dynamics data illustrate the complexity of the Aβ system and emphasize the difficulty in establishing simple models of peptide behavior. Harmeier et al. suggest that the data illustrate how the Gly33 substitution “further enhances the stability of the folding nucleus around this lysine residue with the turn region (Lazo et al., 2005; Grant et al., 2007)” (3,4). However, the data also could be interpreted to show that while substitution at Gly29 decreases turn stability, homologous substitution at Gly33 does not affect turn stability per se but rather facilitates peptide monomer self-association, which itself sequesters the Lys28 peptide bond, a bond that otherwise would be just as labile as it is within Aβ42.

With respect to efforts to elucidate atomic-resolution characteristics of the system, one must suggest caution in the use of computational modeling of substituted Aβ42 peptides, especially when they involve framework structures from fibrils. There is no reason, a priori, to assume that identical structures exist within monomers, oligomers, and fibrils. In fact, much evidence exists that substantial conformational change occurs in the monomer→oligomer, oligomer→protofibril, and even the nascent fibril→mature fibril transitions. Simulations of monomer conformational dynamics and early oligomerization steps reveal a constellation of structures without a predominant conformer. This is a critical observation, one that must be kept in mind when considering Aβ assembly—the Aβ system should be considered a statistical distribution, not a linear assembly pathway. The authors’ modeling may be correct, but experimental confirmation of the ideas is necessary.

The “activity” portion of the structure-activity analyses was very compelling. The authors show clearly that the Gly33 “mutants” have diminished, or no, toxic activity in assays on SH-SY5Y neuroblastoma cells, primary hippocampal neurons, LTP, and Drosophila eye development. These data were beautiful! Now comes interpretation.

Is Gly33 “the key amino acid in the toxic activity of Aβ”? How does one know unless the substitution strategy executed by the authors is applied to each amino acid in Aβ? If Gly33 is the key amino acid, why is its activity, both with respect to conformational dynamics and toxicity, altered significantly by sister substitutions at Gly29? I bet one triple Americano at Starbucks (very meaningful to this commentator) that if these experiments are done, an interesting distribution of conformational and toxicity effects will be observed. Gly33 will not be the sole site at which structural changes produce large effects. Rather, this amino acid will be one of a number that have the potential to affect both assembly and toxicity.

A distribution of toxic activity versus oligomer order also is expected. Harmeier et al. argue that among “different SEC fractions, tetramers of Aβ42 WT [and [Gly29Ala]Aβ42] exhibit the highest toxicity.” In part, these conclusions are based on Western blots of SEC fractions. This technique does not provide accurate assessments of oligomer state within a population of non-covalently associated assemblies (5). The SEC itself, especially for assemblies larger than dimer, cannot produce pure oligomer populations (e.g., see Fig. S1) and the toxicities of oligomers of different orders sometimes were similar, depending on the assay (MTT, MTS, or Live-Dead). The determination of “the most toxic” Aβ oligomer, akin to the “most infectious prion” oligomer (6), thus remains an open question.

Moot is also the notion that “aggregation of Aβ42 peptides is uncoupled from toxicity.” My bet (the same as above) is that some oligomers will be toxic and others will not. Why? Because one must consider in great detail the question of structure-activity. A change of a single atom in Aβ, e.g., as in the Iowa Asp23Asn substitution that replaces an oxygen atom with a nitrogen atom, can cause disease (in this case, the Iowa form of cerebral amyloid angiopathy). However, other substitutions have little or no effects. Does one say, based on the latter observation, that primary structure is uncoupled from effect? I think not. Rather, one must consider each change, and the effects of that change, on an ad hoc basis.

Finally, no evidence exists for Aβ “strains.” Strains are microbiological constructs that have to do with organismal characteristics that are perpetuated during organism replication. Although used appropriately in the prion case, no evidence exists for the existence of strains in AD.

In conclusion, the work of Harmeier et al. should stimulate the field to dig deeper into what may be the Holy Grail of AD research, the determination of the frequency distributions of oligomer order and toxicity. Through this process, it is hoped that key therapeutic targets can be identified.

References:
1. Harmeier A, Wozny C, Rost BR, Munter LM, Hua H, Georgiev O, Beyermann M, Hildebrand PW, Weise C, Schaffner W, Schmitz D, Multhaup G. Role of amyloid-beta glycine 33 in oligomerization, toxicity, and neuronal plasticity. J Neurosci. 2009 Jun 10;29(23):7582-90. Abstract

2. Munter LM, Voigt P, Harmeier A, Kaden D, Gottschalk KE, Weise C, Pipkorn R, Schaefer M, Langosch D, Multhaup G. GxxxG motifs within the amyloid precursor protein transmembrane sequence are critical for the etiology of Abeta42. EMBO J. 2007 Mar 21;26(6):1702-12. Abstract

3. Lazo ND, Grant MA, Condron MC, Rigby AC, Teplow DB. On the nucleation of amyloid beta-protein monomer folding. Protein Sci. 2005 Jun;14(6):1581-96. Abstract

4. Grant MA, Lazo ND, Lomakin A, Condron MM, Arai H, Yamin G, Rigby AC, Teplow DB. Familial Alzheimer's disease mutations alter the stability of the amyloid beta-protein monomer folding nucleus. Proc Natl Acad Sci U S A. 2007 Oct 16;104(42):16522-7. Abstract

5. Bitan G, Fradinger EA, Spring SM, Teplow DB. Neurotoxic protein oligomers--what you see is not always what you get. Amyloid. 2005 Jun;12(2):88-95. Abstract

6. Silveira JR, Raymond GJ, Hughson AG, Race RE, Sim VL, Hayes SF, Caughey B. The most infectious prion protein particles. Nature. 2005 Sep 8;437(7056):257-61. Abstract

View all comments by David Teplow

Two New Articles Use Synthetic Aβ to Study Oligomerization
The first article by Bernstein et al. uses ion mobility coupled with mass spectrometry to study how Aβ40 and Aβ42 oligomerize in vitro. The measurements of arrival time distributions show that Aβ40 oligomers are restricted to low-n species (i.e., dimers and tetramers) while Aβ42-derived oligomers self-assemble into two additional structures, hexamers and dodecamers. The collision cross-sections for each Aβ42 oligomer led them to propose that Aβ42 tetramers are folded in an open structure able to accept one additional dimer to form hexamers, and that Aβ42 hexamers form a planar hexagon which can then stack with one more hexamer to create the largest oligomeric assembly, Aβ42 dodecamers (whose estimated mass was 55.2 kDa).

These new findings complement the observations reported by our group in Tg2576 APP transgenic mice (Lesne et al., 2006). We identified and isolated a putative dodecameric Aβ...  Read more

Two New Articles Use Synthetic Aβ to Study Oligomerization
The first article by Bernstein et al. uses ion mobility coupled with mass spectrometry to study how Aβ40 and Aβ42 oligomerize in vitro. The measurements of arrival time distributions show that Aβ40 oligomers are restricted to low-n species (i.e., dimers and tetramers) while Aβ42-derived oligomers self-assemble into two additional structures, hexamers and dodecamers. The collision cross-sections for each Aβ42 oligomer led them to propose that Aβ42 tetramers are folded in an open structure able to accept one additional dimer to form hexamers, and that Aβ42 hexamers form a planar hexagon which can then stack with one more hexamer to create the largest oligomeric assembly, Aβ42 dodecamers (whose estimated mass was 55.2 kDa).

These new findings complement the observations reported by our group in Tg2576 APP transgenic mice (Lesne et al., 2006). We identified and isolated a putative dodecameric Aβ assembly, Aβ*56, a ~56 kDa assembly that correlates with memory dysfunction in Tg2576 and J20 mice (Lesne et al., 2006; Cheng et al., 2007), and disrupts memory consolidation when applied to young, healthy rats. Although we found putative hexamers migrating at ~27 kDa on SDS-PAGE (also confirmed by non-denaturing SEC) preceding the age at which Aβ*56 appears, we found trimers but no dimers or tetramers in the brains of these “middle-aged” mice (6-11 months), and therefore proposed that trimers, not dimers or tetramers, are the basic building block of hexamers and Aβ*56. In vitro analyses performed on Tg2576 primary neurons supported trimers as the basic unit synthesized within neurons (Lesne et al., 2006; Supplementary data). Not until the mice were “old” (>13 months) and showed dense-core plaques did tetramers and dimers appear, suggesting an alternate pathway leading to Aβ fibrils that uses dimers as the principle building element.

Another element of similarity between our two studies is the observation that, in both cases, endogenous Aβ*56 and in vitro Aβ dodecamers are the terminal soluble species.

Bernstein et al. begin to address the quaternary structure of Aβ oligomers. However, until the quaternary structures of endogenous Aβ oligomers can be ascertained, we cannot know whether the pathways leading to Aβ oligomerization in vitro result in the same structures as those leading to oligomers in vivo. Hopefully, the same methodologies applied to synthetic Aβ oligomers can soon be utilized to examine purified endogenous Aβ oligomers, in order to answer this question.

The second article by Harmeier et al. reports the role of Aβ glycine 33 in oligomerization, toxicity and neuronal plasticity. These authors show that single residue substitutions at position 33 (G33A or G33I) drastically affect Aβ oligomerization, resulting in increased high-n oligomers (10-, 16- and 20-mers) at the expense of the low-n Aβ species, dimers and tetramers. Using MS/MS spectrometry analyses, the authors demonstrate that the subtle folding change induced by G33A/G33I alters the intermolecular peptide interactions which control the assembly of Aβ to form larger Aβ oligomers. With these artificially created peptides favoring high-ordered Aβ42 oligomers, the authors then performed toxicity assays of the respective oligomers separated by size exclusion chromatography using neuronal cell lines and 10-day-old primary neurons, and generated transgenic drosophila expressing the various glycine 33 variants. They also examined how well the different glycine 33 mutants inhibited LTP. All results point to tetramers inducing cell death and inhibiting LTP more effectively than high-n oligomers.

Based upon our earlier conclusion, that fibrils and plaques are derived from dimers, this work may be more relevant to the plaque / fibril pathway than to the Aβ*56 pathway. It is noteworthy that Tg2576 mice and J20 mice producing Aβ*56 for nearly a lifetime do not develop neuronal loss, indicating that Aβ*56 does not induce neurotoxicity leading to cell death, unlike the low-n species studied here.

In summary, the diversity of Aβ-induced effects suggest that multiple oligomeric Aβ species trigger specific mechanisms at different stages of AD. We believe that Aβ*56 initiates AD pathogenesis, perhaps during the asymptomatic or pre-dementia phase of disease. In contrast, low-n oligomers, associated with dense core plaques, may play a role in the progression of AD at later stages.

View all comments by Karen Hsiao Ashe
View all comments by Sylvain Lesne

The paper by Lesne et al. is interesting. It would be more convincing if it had included additional controls/information relating to the Aβ oligomer.

For example, do the authors have evidence for this oligomer from in-vitro preparations of Aβ42? If not, why not? If they do, is it ThT-reactive?

Could the authors present TEM evidence of the oligomer, either generated via the transgene or from in-vitro preparations?

If, as I have assumed, the oligomer is only formed in vivo, perhaps only in transgenes, and has not been identified in in-vitro preparations, then some speculation as to why this should be so would be pertinent. It is apparently quite stable, as the authors were able to isolate it for subsequent injection into rats.

In relation to the final experiments in which the isolated oligomer was injected into rat brains, a control consisting of "the vehicle" is surely not sufficient to demonstrate activity of this particular oligomer. We are all aware that injections of Aβ cause behavioral changes in the rat. The authors could have used a positive...  Read more

The paper by Lesne et al. is interesting. It would be more convincing if it had included additional controls/information relating to the Aβ oligomer.

For example, do the authors have evidence for this oligomer from in-vitro preparations of Aβ42? If not, why not? If they do, is it ThT-reactive?

Could the authors present TEM evidence of the oligomer, either generated via the transgene or from in-vitro preparations?

If, as I have assumed, the oligomer is only formed in vivo, perhaps only in transgenes, and has not been identified in in-vitro preparations, then some speculation as to why this should be so would be pertinent. It is apparently quite stable, as the authors were able to isolate it for subsequent injection into rats.

In relation to the final experiments in which the isolated oligomer was injected into rat brains, a control consisting of "the vehicle" is surely not sufficient to demonstrate activity of this particular oligomer. We are all aware that injections of Aβ cause behavioral changes in the rat. The authors could have used a positive control, for example, aggregated Aβ, to try to demonstrate that it was not simply the injection of Aβ, in any form, that produced the behavioral differences. In addition, the authors might have tried to demonstrate that the oligomer was actually present in the rat brain. Upon injection, it might have immediately aggregated or dissolved; we have no way of knowing.

The authors may be correct in their assertion that this oligomer causes the behavioral changes seen in both transgenic mice and rats, though the research as presented does not appear to do more than suggest a relationship. Given the weight afforded to research published in Nature, it is surprising that the lack of suitable controls was not commented upon in the accompanying News and Views.

View all comments by Chris Exley

Does this paper provide a new model of memory loss? No, but it advances our understanding of the basis of memory loss in a well-known transgenic mouse model of Alzheimer disease. Above all, the paper offers us a concrete biochemical entity to study and compare against other Aβ oligomer species that various groups have themselves found in recent years.

The paper fits nicely with prior studies that address the major question of what brain changes account for the deficits in memory and cognition in AD. Here is some historical context of this work: In the early 1990s, DeKosky and Scheff, 1990, as well as Robert Terry and Robert Katzman (Terry et al., 1991), showed that loss of synapses was the best correlate of the declines of memory and cognition in AD. Plaques did not correlate with memory and cognition, and tangles correlated slightly. But in these studies of the early 1990s, loss of synapses only accounted for about half the losses of memory and cognition in AD. Where...  Read more

Does this paper provide a new model of memory loss? No, but it advances our understanding of the basis of memory loss in a well-known transgenic mouse model of Alzheimer disease. Above all, the paper offers us a concrete biochemical entity to study and compare against other Aβ oligomer species that various groups have themselves found in recent years.

The paper fits nicely with prior studies that address the major question of what brain changes account for the deficits in memory and cognition in AD. Here is some historical context of this work: In the early 1990s, DeKosky and Scheff, 1990, as well as Robert Terry and Robert Katzman (Terry et al., 1991), showed that loss of synapses was the best correlate of the declines of memory and cognition in AD. Plaques did not correlate with memory and cognition, and tangles correlated slightly. But in these studies of the early 1990s, loss of synapses only accounted for about half the losses of memory and cognition in AD. Where might the missing 50 percent be?

In 2003, our group showed that the brains of AD patients were deficient in a protein, called dynamin 1, that is crucial to the functioning of synapses and, hence, for memory formation and information processing in the brain (Yao et al., 2003; Coleman and Yao, 2003). More specifically, dynamin 1 is a key protein in the trafficking of presynaptic vesicles that contain neurotransmitters. In 2005, Brent Kelly, Robert Vassar, and Adriana Ferreira showed that Aβ peptide caused depletion of dynamin 1, and they confirmed our major finding by showing depletion of dynamin 1 in a mouse model of AD (Kelly et al., 2005).

The current paper by Lesne et al. specifies the form of Aβ that probably was responsible for the loss of dynamin 1 described by Kelly et al. in the Tg2576 mouse model of AD, and by Yao et al. in human AD cases.

These papers all fit together when one posits that a major part of the missing 50 percent in DeKosky’s, Scheff’s, and Terry’s earlier observations lies in defective functioning of synapses that remain structurally present but are unable to function optimally due to deficient expression of dynamin 1 (and other molecules related to synaptic function), and, further, that this deficient expression of dynamin 1 is caused by a specific form of Aβ, which Lesne et al. have now identified. At present, more attention is being paid to Aβ effects on specific transmitters than on vesicle recycling. I believe the latter deserves focused exploration, as well. For one, it would be interesting to know whether Aβ effects on synaptic vesicle trafficking are selective.

One of the major questions unanswered by Lesne et al. lies in the fact that the mouse model they used contains a mutated form of the human APP molecule that is found in only a small percent of AD patients. The work of Kelly et al. apparently used the wild-type form of Aβ. Would Lesne et al. have obtained similar results with the wild-type form of Aβ?

View all comments by Paul Coleman

This study is impressive both for the breadth and detail of the experiments undertaken. Using the well-characterized Tg2576 APP transgenic mouse line, the authors searched for the appearance of an Aβ species that coincided with the first observed changes in spatial memory. Starting at 6 months, the time when cognitive changes are first apparent, the authors detected Aβ species that migrated on SDS-PAGE as nonamers and dodecamers. Aβ monomer, trimer, and hexamer were seen at earlier time points and were therefore not considered to have a deleterious effect on cognition. Indeed, comparison of spatial memory and the levels of Aβ monomer, trimer, hexamer, nonamer, and dodecamer revealed that only nonamer and dodecamer levels correlated with memory impairment.

The authenticity of these various Aβ species as discrete assemblies was confirmed using a gel filtration paradigm previously employed to fractionate cell culture-derived low-n oligomers (Walsh et al., 2005), and was combined with immunoaffinity...  Read more

This study is impressive both for the breadth and detail of the experiments undertaken. Using the well-characterized Tg2576 APP transgenic mouse line, the authors searched for the appearance of an Aβ species that coincided with the first observed changes in spatial memory. Starting at 6 months, the time when cognitive changes are first apparent, the authors detected Aβ species that migrated on SDS-PAGE as nonamers and dodecamers. Aβ monomer, trimer, and hexamer were seen at earlier time points and were therefore not considered to have a deleterious effect on cognition. Indeed, comparison of spatial memory and the levels of Aβ monomer, trimer, hexamer, nonamer, and dodecamer revealed that only nonamer and dodecamer levels correlated with memory impairment.

The authenticity of these various Aβ species as discrete assemblies was confirmed using a gel filtration paradigm previously employed to fractionate cell culture-derived low-n oligomers (Walsh et al., 2005), and was combined with immunoaffinity chromatography to achieve purification of the dodecamer.

The authors then conducted the most important and compelling experiment of their study: They injected purified dodecamer into the ventricle of normal pre-trained rats and tested if the injected dodecamer could alter spatial memory. Rats given dodecamer showed a dramatic fall-off in performance; thus, the dodecamer shown to correlate with decreased cognition in Tg2576 mice was also capable of directly mediating impairment of memory in normal rats.

These studies demonstrate for the first time that a soluble, brain-derived form of Aβ can directly mediate brain dysfunction in the absence of neurodegeneration. They open up new avenues of investigation, and yet, as with all scientific advances, the Lesne study raises more questions than it answers. Going forward it will be vitally important to validate the human relevance of the Tg2576 dodecamer—is it present in human brain or CSF? Can it be detected in other animal models of AD? While there is no doubt that Aβ dodecamer present in Tg2576 brain is capable of impairing memory, it is not clear if this species also exists in human brain. Based on the novel homogenization protocol used by Lesne et al., one would predict that Aβ dodecamer should be present in the interstitial fluid and by extension should be readily detectable in CSF. To my knowledge, no such Aβ assembly has been detected in human CSF to date. Indeed, in prior studies, high-molecular-weight Aβ oligomers were not detected in human CSF, whereas Aβ monomer, dimers and trimers were consistently detected (Ida et al., 1996; Walsh et al., 2000).

Of course, the Tg2576 line is a model for AD, and, like all models, it may differ from the human condition. For instance, the authors demonstrate a time-dependent increase in "extracellular-enriched" Aβ monomer, yet in humans it is well documented that CSF Aβ falls with increasing disease severity (Nitsch et al., 1995; Andreasen et al., 1999; Lewczuk et al., 2003). Moreover, recent studies indicate that the fall in Aβ monomer (it has been previously demonstrated that the Takeda ELISA does not readily detect Aβ oligomers; see Morishima and Ihara, 1998) is due to sequestration into senile plaques (Fagan et al., 2005). Together, these results suggest that the overall economy of Aβ in the Tg2576 may be different from that of human brain, and raise the possibility that Aβ assemblies other than or in addition to Aβ dodecamer underlie the memory loss that characterizes AD.

View all comments by Dominic Walsh

To their credit, the authors have attempted to look for early changes in the TG 2576 mouse model, which are more likely to deal with pathogenesis than pathogenic consequences. Lesne et al. have identified an unusual, high molecular-weight component in the brains of these mice that contains Abeta determinants and is only present before amyloid deposits accumulate. The claim that this material is necessarily all derived from extracellular spaces is questionable, since it was isolated from detergent-solubilized brain tissue. It is also not clear how much of the 56K band is made up of Abeta peptides. The authors describe an Abeta-derived peptide as representing the "core" of the material, but careful mass spec analysis should have revealed how much and what else was present in the sample. Until this is done, it is premature to declare this a special form of Abeta. I also agree that the biological activity of this material has not yet been studied adequately.

View all comments by Vincent Marchesi

I would just like to comment on the questions/remarks that followed our article. First and foremost, I would like to point out that we did not write in the article that Aβ*56 is an assembly composed of 12 units of Aβ. We did not include any hard data that would directly demonstrate this statement. What we did mention, however, is the possibility that Aβ*56 could represent a 12-mer because of the following observations: 1) Aβ trimers are formed intracellularly and are secreted by neurons in vivo and in vitro; 2) Aβ-immunoreactive species of high molecular weights (above 20 kDa) migrate at molecular weights that match theoretical migrations for 6-mer, 9-mer, and 12-mers of Aβ1-42. It remains to be determined whether these proteins/assemblies are only composed of Aβ, but we postulated so due to the fact that trimers are predominant in vitro and in vivo and only multiples of three monomers appear to form these Aβ-immunoreactive larger structures in vivo. Further analyses are underway to confirm our hypothesis.

View all comments by Sylvain Lesne

Normally, soluble Aβ molecule (39-43 amino acids) undergoes conformational changes in disease and is deposited in the brain as insoluble fibrils, oligomers and protofibrills. Previously it was demonstrated that Aβ neurotoxicity required insoluble fibril formation (mainly Aβ42 and to lesser degree Aβ40) (Lorenzo, 1994) and the fibrils served as inducers of neuronal apoptosis (Loo, 1993). Recently, emphasis has shifted to smaller soluble Aβ. Aβ42 dimers and trimers naturally secreted from a 7PA2 cell line were suggested to be responsible for the disruption of cognitive functions (Cleary, 2005). Importantly, intraventricular injection of such Aβ42 small oligomers inhibited long-term potentiation (LTP) in rat hippocampus and an anti-Aβ monoclonal antibody (6E10) that binds to N-terminal region of Aβ42 prevented this inhibition (Klyubin, 2005). It has also...  Read more

Normally, soluble Aβ molecule (39-43 amino acids) undergoes conformational changes in disease and is deposited in the brain as insoluble fibrils, oligomers and protofibrills. Previously it was demonstrated that Aβ neurotoxicity required insoluble fibril formation (mainly Aβ42 and to lesser degree Aβ40) (Lorenzo, 1994) and the fibrils served as inducers of neuronal apoptosis (Loo, 1993). Recently, emphasis has shifted to smaller soluble Aβ. Aβ42 dimers and trimers naturally secreted from a 7PA2 cell line were suggested to be responsible for the disruption of cognitive functions (Cleary, 2005). Importantly, intraventricular injection of such Aβ42 small oligomers inhibited long-term potentiation (LTP) in rat hippocampus and an anti-Aβ monoclonal antibody (6E10) that binds to N-terminal region of Aβ42 prevented this inhibition (Klyubin, 2005). It has also been demonstrated that passive immunization with monoclonal antibodies (NAB61), that specifically recognizes a pathologic conformation present in Aβ dimers, soluble oligomers and higher order species of Aβ, resulted in rapid improvement in spatial learning and memory (Lee, 2006). Other authors showed that 12-mer oligomers of Aβ42, also known as Aβ-derived diffusible ligands (ADDLs) increased about 70-fold in AD patient’s brains over controls (Gong, 2003; Klein, 2006). Collectively, these data suggest that the Aβ oligomers of various sizes are the most pathologic substrate responsible for disrupting neuronal functions and cognitive decline in AD.

The current paper showed that although different forms of Aβ42 are deposited in the brains of aged APP/Tg2576 mice, the memory deficits are induced in 6-month-old or older mice by that accumulated 12-mer oligomer, termed Aβ*56. This is an interesting paper that indicates that levels of soluble or insoluble Aβ do not fully correlate with behavioral changes in this mouse model of AD. Instead only levels of 9-mer (p = 0.0169; r2 = 0.4505) and 12-mer (p = 0.0014; r2 = 0.6556) oligomers showed an inverse correlation with spatial memory. These data indicates that AD therapy should target particular species of Aβ that are responsible for AD like pathology and memory deficits. Thus, if Aβ*56 is a major player in AD that is implicated in memory deficits in middle-aged Tg2576 mice, then learning about misfolding of human Aβ peptide is an important task. As we gain more knowledge of the mechanisms of assembly of Aβ peptides more potent AD therapies will be developed.

One important conclusion from this paper is that such AD therapy should be based on the prevention of accumulation of oligomers, rather than on clearing already formed toxic forms of Aβ. In other words AD therapy that can block oligomerization of Aβ should be started earlier, before the accumulation of monomers in the brains. This can be done by the prototype AD vaccine that will be able to generate antibodies that can bind all forms of Aβ and block oligomerization of the monomeric peptide. Of course such a vaccine should be used in middle-aged healthy people to prevent generation of AD-like pathology, rather than for vaccination of elderly AD patients with immunosenescence (therapeutic vaccine strategy). Such a vaccine would have to be safe and should be able to generate high titers of antibodies that can block oligomerization of β amyloid peptide, although they can be specific to any form of Aβ (monomers, oligomers, and fibrils).

Thus, the important aim of AD-immunotherapy research must be the identification of the most safe and immunogenic form of the vaccine that can generate therapeutically potent antibodies that can block oligomerization of the peptide. For example, using an epitope vaccine strategy we recently generated polyclonal antibodies specific to the N-terminal region of Aβ that can not only bind all forms of Aβ, but also delay oligomerization of Aβ42 in vitro (paper submitted). In the nearest future it will be important to test the ability of this and other prototype AD vaccines to block generation of Aβ*56 in the brains of immunized APP/Tg 2576 or other APP/Tg mice.

View all comments by Michael G. Agadjanyan

This is an exciting piece of research from Ehud Cohen and coworkers from Andrew Dillin’s group at The Salk Institute (La Jolla, CA), concerning the role of insulin-like growth factor (IGF)-1 receptor signaling in the pathogenesis of Alzheimer disease (AD). Recent data suggest that IGF-1 receptor signaling might be involved in the development of AD, since postmortem studies on brains of AD patients showed decreased insulin receptor and IGF-1 receptor expression (1). However, it is unclear whether these changes in IGF-1 receptor signaling are cause, consequence, or even counter-regulation to neurodegeneration.

The work of Ehud Cohen and coworkers revealed new insights into this highly debated field. In 2006, the same research group showed that reduced daf-2 (ortholog to insulin/IGF-1 receptors in mammals) signaling in C. elegans protects the worms from Aβ toxicity via a heat shock factor 1 (HSF-1)-dependent mechanism, which regulates Aβ disaggregation, and a DAF-16 (ortholog to FOXO in mammals)-dependent mechanism, which facilitates the formation of larger,...  Read more

This is an exciting piece of research from Ehud Cohen and coworkers from Andrew Dillin’s group at The Salk Institute (La Jolla, CA), concerning the role of insulin-like growth factor (IGF)-1 receptor signaling in the pathogenesis of Alzheimer disease (AD). Recent data suggest that IGF-1 receptor signaling might be involved in the development of AD, since postmortem studies on brains of AD patients showed decreased insulin receptor and IGF-1 receptor expression (1). However, it is unclear whether these changes in IGF-1 receptor signaling are cause, consequence, or even counter-regulation to neurodegeneration.

The work of Ehud Cohen and coworkers revealed new insights into this highly debated field. In 2006, the same research group showed that reduced daf-2 (ortholog to insulin/IGF-1 receptors in mammals) signaling in C. elegans protects the worms from Aβ toxicity via a heat shock factor 1 (HSF-1)-dependent mechanism, which regulates Aβ disaggregation, and a DAF-16 (ortholog to FOXO in mammals)-dependent mechanism, which facilitates the formation of larger, less toxic Aβ aggregates (2). Subsequently, this hypothesis is now tested in a mouse model of Alzheimer disease.

Cohen and coworkers present data from an AD mouse model expressing the Swedish mutation of APP and the presenilin-1 ΔE9 variant (AD mice) that were additionally heterozygous for the IGF-1 receptor (IGF-1R). These mice presented partial memory restoration and improved motor skills compared to their AD littermates. Interestingly, reduced IGF-1R signaling protected these animals from neuronal loss. Furthermore, these mice provided higher synaptic density, decreased inflammation, and smaller but more condensed Aβ aggregates compared to AD mice.

These data corroborate the findings in C. elegans and reveal a novel and promising mechanism of Aβ oligomer detoxification via enhanced aggregation in mammals. In line with the current work, there have been two more reports this year suggesting a beneficial effect of reduced insulin receptor or IGF-1R signaling on different aspects of AD pathology (3,4). However, most of the beneficial effects of partial IGF-1 resistance described in Cohen's paper occur in later stages of disease, suggesting a complex interaction between disease progression and transcriptional changes triggered by mild IGF-1 resistance. Interesting candidates for mediating these effects might be the Foxo transcription factors, which are regulated by IGF-1R signaling, as well as "stress" kinases. The current paper might also prompt a reinterpretation of previously published studies suggesting a beneficial effect of IGF-1 treatment on AD pathology (5). In summary, the work of Cohen and colleagues reveals a novel IGF-1-dependent mechanism, conserved from C. elegans to mammals, to reduce Aβ toxicity by facilitating the formation of larger, less toxic Aβ aggregates.

References:
1. Rivera EJ, Goldin A, Fulmer N, Tavares R, Wands JR, de la Monte SM. Insulin and insulin-like growth factor expression and function deteriorate with progression of Alzheimer's disease: link to brain reductions in acetylcholine. J Alzheimers Dis. 2005 Dec;8(3):247-68. Abstract

2. Cohen E, Bieschke J, Perciavalle RM, Kelly JW, Dillin A. Opposing activities protect against age-onset proteotoxicity. Science. 2006 Sep 15;313(5793):1604-10. Abstract

3. Freude S, Hettich MM, Schumann C, Stöhr O, Koch L, Köhler C, Udelhoven M, Leeser U, Müller M, Kubota N, Kadowaki T, Krone W, Schröder H, Brüning JC, Schubert M. Neuronal IGF-1 resistance reduces Abeta accumulation and protects against premature death in a model of Alzheimer's disease. FASEB J. 2009 Oct;23(10):3315-24. Abstract

4. Killick R, Scales G, Leroy K, Causevic M, Hooper C, Irvine EE, Choudhury AI, Drinkwater L, Kerr F, Al-Qassab H, Stephenson J, Yilmaz Z, Giese KP, Brion JP, Withers DJ, Lovestone S. Deletion of Irs2 reduces amyloid deposition and rescues behavioural deficits in APP transgenic mice. Biochem Biophys Res Commun. 2009 Aug 14;386(1):257-62. Abstract

5. Carro E, Trejo JL, Gomez-Isla T, LeRoith D, Torres-Aleman I. Serum insulin-like growth factor I regulates brain amyloid-beta levels. Nat Med. 2002 Dec;8(12):1390-7. Abstract

View all comments by Katharina Schilbach
View all comments by Markus Schubert

Maintaining the Correct Balance of IGF-1R Signaling in the Brain With Age May Protect Against AD
This very interesting study led by Andrew Dillin shows that crossing long-lived heterozygous igf-1r+/- mice (10) with AD APPswe/PS1ΔE9 mice (11) delays age-related Aβ proteotoxicity and protects against several AD-like symptoms. The major finding of the work shows reducing total levels of IGF-1R signaling by 50 percent in the entire mouse is associated with the emergence of more dense, tightly packed Aβ plaques in the brain, which most likely sequester potentially synaptotoxic, oligomeric Aβ. This raises the exciting possibility that diminishing signaling through IGF-1R may enable the formation of more “inert” Aβ plaques and diminish Aβ oligomer synaptic toxicity in patients with AD.

The insulin/insulin-like growth factor-1 (IGF-1) receptor signaling (IIS) pathway has long been a subject of fascination in aging research and in understanding the regulation of lifespan. DAF-2 is the one and only insulin/IGF-1 receptor in Caenorhabditis...  Read more