The first sentence of Pat McCaffrey's news summary is both enlightening and puzzling: "Alzheimer disease rages in the brain long before plaques form...." It was not that long ago that the century-old adage "amyloid in plaques is the major problem in AD" was modified to "amyloid in neurons." We have come a long way, and it is satisfying to see, with less than a fortnight apart, two major papers pointing to early amyloid peptide-related defects, that is, a new molecular structure referred to as Aβ*56 (Lesné et al., 2006) and new cell-functional consequences in vivo (Jacobsen et al., 2006).

I disagree with McCaffrey's second sentence that "Most in the field agree that early interventions are the best hope of nipping memory loss and cognitive decline in the bud." I am convinced most in the field actually know that this is the only way forward, instead of trying to treat the late symptoms that actually signal an already irreversibly established pathology. Evidently, current clinical...  Read more

The first sentence of Pat McCaffrey's news summary is both enlightening and puzzling: "Alzheimer disease rages in the brain long before plaques form...." It was not that long ago that the century-old adage "amyloid in plaques is the major problem in AD" was modified to "amyloid in neurons." We have come a long way, and it is satisfying to see, with less than a fortnight apart, two major papers pointing to early amyloid peptide-related defects, that is, a new molecular structure referred to as Aβ*56 (Lesné et al., 2006) and new cell-functional consequences in vivo (Jacobsen et al., 2006).

I disagree with McCaffrey's second sentence that "Most in the field agree that early interventions are the best hope of nipping memory loss and cognitive decline in the bud." I am convinced most in the field actually know that this is the only way forward, instead of trying to treat the late symptoms that actually signal an already irreversibly established pathology. Evidently, current clinical practice of treatment must be continued and efforts to improve on them even stepped up as fast and as far as possible. But the grand aim of research must be identifying the most "early defects" as the tell-tale signs of upcoming, and hopefully reversible, pathology.

We were the first to approach those early signs in our APP mouse models, even when the field was still concentrating on amyloid plaques (Moechars et al., 1996, 1999). The second publication is better known and cited than its predecessor from 1996, but their message was and is the same: Behavioral and cognitive functional defects occur in the absence of any amyloid plaques, on which note we converge on McCaffrey's question: "…when exactly, and where, does AD start?"

Bloom and colleagues analyzed two APP transgenic models and come to the conclusion that LTP is impaired at age 4-6 months concomitant with decreased spine density in the outer molecular layer of the dentate gyrus. This parallel is an important new piece of evidence; all other defects in LTP and in cognition, followed by increased Aβ peptides and amyloid plaque load, were demonstrated and are well known in these and other APP models (for review see Van Dooren et al., 2006).

McCaffrey then rightfully states: "The cause of these synaptic problems remains to be found." This is her cue to switch to the other publication I referred to above: The detailed analysis of soluble Aβ species by Dr. Karen Ashe and colleagues, identifying dodecameric Aβ as cause of memory problems in APP mice (Lesné et al., 2006). The Aβ*56 species is a stable molecular complex of Aβ peptides in brain, which appears together with memory defects in APP transgenic mice and causes memory defects when injected in brain of young rats. Thereby, a strong piece of evidence is provided for Aβ*56 to take up center stage in efforts to nail down the real culprit in AD.

One cannot escape thinking that this might not be "the" but "a" cause of synaptic problems, since several other types of Aβ complexes, isolated or synthesized, prove pathologically active. The precise structural relationship of Aβ dimers, Aβ oligomers, globular oligomers, ADDLs, Aβ*56, etc., remains to be established, but these species point without a doubt to a series of interconvertible molecular forms of Aβ. If they also equilibrate with each other in vivo, it may remain impossible to define which of these forms is the more or most potent in wrecking synaptic functions in the hippocampus. In this respect, I maintain my comparison of amyloid peptides to the ancient dodecaeder, on display in the Gallo-Roman museum in Tongeren, Belgium (see ARF comment). I referred to the dodecaeder as an object with a definite shape, structure, even beauty, but of which we don't know the function or purpose. I humbly confess I never intended to predict the actual dodecamer form of Aβ*56 as the cause of AD!

The increase with aging of amyloid peptide oligomers of sorts, including dimers, trimers, and dodecamers, correlating with decreased spine density in the dentate gyrus and with the most early defects in cognition in the mouse models, has to be shown to mimic the early memory defects in humans. If so, the mice and tools described will be valuable for testing early treatments, in full agreement with McCaffrey once again!

References:
Moechars D, Lorent K, De Strooper B, Dewachter I, Van Leuven F. Expression in brain of amyloid precursor protein mutated in the alpha-secretase site causes disturbed behavior, neuronal degeneration and premature death in transgenic mice. EMBO J. 1996 Mar 15;15(6):1265-74. Abstract Moechars D, Dewachter I, Lorent K, Reverse D, Baekelandt V, Naidu A, Tesseur I, Spittaels K, Haute CV, Checler F, Godaux E, Cordell B, Van Leuven F. Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain. J Biol Chem. 1999 Mar 5;274(10):6483-92. Abstract van Dooren T, Dewachter I, Borghgraef P, van Leuven F. Transgenic mouse models for APP processing and Alzheimer's disease: early and late defects. Subcell Biochem. 2005;38:45-63. Review. Abstract

View all comments by Fred Van Leuven

I think we in this field have to be careful with overinterpreting phenomena in our APP mouse models. Transgenic mouse models expressing AD-associated mutant forms of the amyloid-β precursor protein (APP), or both mutant APP and mutant presenilin-1 (PS1), develop robust amyloid pathology with abundant neurotic plaques that recapitulate many of the features of the Aβ deposits found in humans with AD. As they age, they also show other AD-like features including decreased synaptic density, reactive astro- and microgliosis, and the presence of plaque-associated inflammatory proteins. However, these transgenic models show little evidence of overt neuronal loss and do not, without additional genetic manipulation, develop NFT pathology.

The APP and APP/PS1 mice also develop cognitive deficits. In most studies, these deficits are observed coincident with the earliest biochemical signs of Aβ accumulation, consistent with early aggregation events, yet the cognitive deficits show limited progression as the mice age and are not tightly linked to the degree of amyloid pathology. Such...  Read more

I think we in this field have to be careful with overinterpreting phenomena in our APP mouse models. Transgenic mouse models expressing AD-associated mutant forms of the amyloid-β precursor protein (APP), or both mutant APP and mutant presenilin-1 (PS1), develop robust amyloid pathology with abundant neurotic plaques that recapitulate many of the features of the Aβ deposits found in humans with AD. As they age, they also show other AD-like features including decreased synaptic density, reactive astro- and microgliosis, and the presence of plaque-associated inflammatory proteins. However, these transgenic models show little evidence of overt neuronal loss and do not, without additional genetic manipulation, develop NFT pathology.

The APP and APP/PS1 mice also develop cognitive deficits. In most studies, these deficits are observed coincident with the earliest biochemical signs of Aβ accumulation, consistent with early aggregation events, yet the cognitive deficits show limited progression as the mice age and are not tightly linked to the degree of amyloid pathology. Such deficits also appear highly reversible as Aβ immunotherapies rapidly reverse the cognitive deficits even when they have little effects on overall Aβ load. As a result, it seems likely that the APP mice recapitulate only part of the cognitive decline that is seen in AD patients.

The absence of a more complete recapitulation of AD-type pathology in APP mouse models has been used to argue against a primary role of Aβ accumulation in the development of AD. Although such models do demonstrate that Aβ accumulation and amyloid deposition alone are not sufficient to cause overt neuronal loss in mice, it is inappropriate to conclude, based on such data, that Aβ does not drive these changes in humans. The APP and PS mutations included in the transgenes used to generate these mice do cause AD; therefore, the lack of a complete pathological phenotype in these models simply demonstrates that current transgenic mice are not capable of producing all the features of the human disease.

The inability to develop a more complete animal model of AD has been a significant problem because it imposes limits on the ability to dissect the pathogenic cascade, which hinders therapeutic studies aimed at downstream targets. It is, therefore, important to understand why APP mice fail to develop the complete spectrum of AD pathology. Further insight into the phenotype of the mice, such as is found in the current manuscript, is of interest, but still does not address the fundamental difference between our APP mouse models and humans with AD, namely the profound neuronal loss.

On another note, more and more evidence is emerging that amyloid deposition precedes the onset of clinical symptoms perhaps by more than a decade. If this is true, then we have to be careful about statements regarding cause and effect.

View all comments by Todd E. Golde

This is an impressive and important contribution. It links the appearance of a particular multimeric species of the amyloid-β peptide—Aβ*56—to a specific behavioral perturbation, and induces the same perturbation in naïve rats by reintroducing the Aβ*56 species purified from Tg2576 brains. It begins to address the conundrum that Aβ levels, soluble or insoluble, do not correlate with the onset and severity of behavioral changes in these animals.

Expectedly, this report stimulates a raft of questions, not with the work itself, but in teasing out more of the details and in stimulating new approaches. It will also energize corroboration of their findings in other Tg mouse models, as well as a search for correlates in Alzheimer disease brain. The findings from those studies will either further validate the animal model or set limits on its interpretation, both of which will be valuable. To begin to understand this complex paper, you must also study the supplementary information...  Read more

This is an impressive and important contribution. It links the appearance of a particular multimeric species of the amyloid-β peptide—Aβ*56—to a specific behavioral perturbation, and induces the same perturbation in naïve rats by reintroducing the Aβ*56 species purified from Tg2576 brains. It begins to address the conundrum that Aβ levels, soluble or insoluble, do not correlate with the onset and severity of behavioral changes in these animals.

Expectedly, this report stimulates a raft of questions, not with the work itself, but in teasing out more of the details and in stimulating new approaches. It will also energize corroboration of their findings in other Tg mouse models, as well as a search for correlates in Alzheimer disease brain. The findings from those studies will either further validate the animal model or set limits on its interpretation, both of which will be valuable. To begin to understand this complex paper, you must also study the supplementary information provided online.

Hypothesizing that a particular species of Aβ was responsible for deficits in memory retention that progress in a specific pattern with age in Tg 2576 mice, the authors developed a sequential extraction method to distinguish pools of Aβ peptide in the brains of these mice. They were able to empirically quasi-separate “extracellular-enriched” and “intracellular-enriched” fractions, although it is not clear how detergent-containing extractions can avoid solubilizing membranes. Perhaps a specific detergent/protein ratio limits the effect of the detergent.

Combined with primary neuronal and astrocyte culture work, this fractionation allowed them to conclude that oligomers of Aβ larger than trimers assemble extracellularly. Interestingly, cultured cells contained only trimers, while the extracellular species were trimers and tetramers, but not hexamers, nonamers, or Aβ*56 (dodecamers), which are observed in the whole aged animals. This could be due to the fact that the cultured cells are embryonic, not mature neurons, and they are not in a tissue environment. What is apparent is that oligomer formation in cells yields a different size spectrum of products from those reported in the literature for synthetic peptides and from those observed in aged (>6 months) animals (this article). This may be due to clearance mechanisms and/or modulating intra- and extracellular processes.

The importance of these observations is that they direct attention to extracellular events as being important in the maturation of trimers and tetramers into Aβ*56. This has not previously been appreciated. It also suggests that the change occurring at 6 months of age in Tg 2576 mice may be in the cellular environment. This will undoubtedly launch new lines of research focusing on that aspect. An extracellular target is also an easier one to reach with therapeutics.

In the studies reported here, Aβ*56 is purified by immunoprecipitation. Along with the specificity of that method for Aβ peptides comes the risk of missing cryptic epitopes. As an example, in Lesne et al., hexamers do not seem to IP well, yet they can be readily seen in non-IPed material. Antigen recovery by boiling of the blots before immunodetection affects the relative intensity of the species observed, as noted by the authors.

The authors are careful not to claim that Aβ*56 is the only species capable of inducing behavioral effects; indeed, they do not report testing the other SEC-separated species they isolate.

The story, of course, is far from complete with the identification of an Aβ*56 species that reproduces particular behavioral deficits in rats that are seen in the transgenic APP mice. Transgenic mice are at best a partial model of the uniquely human Alzheimer disease. There is minimal neuronal cell death in mice and in most cases (including Tg 2576), only moderate synapse loss is apparent. While more detailed study may reveal subtle changes in synaptic architecture/function, the devastation in the Alzheimer brain is not recapitulated. Thus, mice may represent a model of the earliest stages of a process that becomes Alzheimer disease in humans. That is a particularly important issue, because it is precisely at that stage at which intervention is likely to be most effective and detection of pathology first possible.

View all comments by Harry LeVine III

Amyloid-β protein dodecamer in the brain impairs memory in the Tg2576 mouse
The experience from genetic findings in the early 1990s strongly point to Aβ as the culprit in Alzheimer disease. However, we still do not understand how Aβ confers cognitive dysfunction and neuronal atrophy. Recent years have witnessed an increased interest in soluble Aβ oligomers as being the important pathogenic form of Aβ. This article is a significant contribution to the field. Most impressive is perhaps the author’s ability to isolate a soluble Aβ species from the brain and prove that it affects cognition. The research team, headed by Karen Ashe, has for a long time sought the elusive Aβ species responsible for cognitive decline in their transgenic mouse model Tg2576, which harbors the Swedish APP mutation.

Tg2576 lack neuropathology and are cognitively unimpaired until 6 months of age, when spatial memory declines but then remains stable for another 7-8 months. Animals aged more than 14 months develop neuropathology including neuritic plaques containing amyloid-β peptides and...  Read more

Amyloid-β protein dodecamer in the brain impairs memory in the Tg2576 mouse
The experience from genetic findings in the early 1990s strongly point to Aβ as the culprit in Alzheimer disease. However, we still do not understand how Aβ confers cognitive dysfunction and neuronal atrophy. Recent years have witnessed an increased interest in soluble Aβ oligomers as being the important pathogenic form of Aβ. This article is a significant contribution to the field. Most impressive is perhaps the author’s ability to isolate a soluble Aβ species from the brain and prove that it affects cognition. The research team, headed by Karen Ashe, has for a long time sought the elusive Aβ species responsible for cognitive decline in their transgenic mouse model Tg2576, which harbors the Swedish APP mutation.

Tg2576 lack neuropathology and are cognitively unimpaired until 6 months of age, when spatial memory declines but then remains stable for another 7-8 months. Animals aged more than 14 months develop neuropathology including neuritic plaques containing amyloid-β peptides and further cognitive deficits. The authors posited the existence of an Aβ oligomeric form, designated Aβ*, responsible for early cognitive decline in Tg2576. Two criteria were used: Aβ* should appear at 6 months of age and remain stable between 6-14 months of age. The best correlation was found between Aβ 12-mers and spatial memory.

Curiously, non-transgenic mice also show a trend toward impaired spatial memory at 6 months of age (Figure 1a). It would be interesting to investigate whether increase in Aβ* is coincident with cognitive deficits also in other APP mouse models, since cognitive dysfunction is known to be highly dependent upon strain background.

Most surprisingly, levels of Aβ*56 in brain do not increase upon onset of senile plaque deposition when total Aβ levels increase 100-fold (Kawarabayashi et al. 2001). This would tend to suggest a dichotomous model of Aβ amyloidosis in the brain, where Aβ* formation is unrelated to senile plaque formation. It would be interesting to determine turnover of endogenous Aβ* in the brain, especially since Aβ* confers a transient effect on memory retention. What would happen with levels of Aβ* in Tg2576 following acute or chronic treatment with a potent γ-secretase inhibitor or in a TET-off APP transgenic model?

Most important would be, of course, to investigate if Aβ* exists in the brain or CSF of Alzheimer disease patients, and if Aβ* levels are linked to mild cognitive impairment (MCI) and further cognitive decline in the human disease.

View all comments by Lars Lannfelt
View all comments by Lars Nilsson

Star-struck by Amyloid
Lesne and colleagues show that Aβ*56 is found in cognitively impaired Tg2576 animals without Aβ plaques, but not in unimpaired animals, and that it correlates to early declines in memory but not later ones. Notably, when isolated and injected into rats, Aβ*56 leads to reversible cognitive deficits. This is an interesting study and will definitely appeal to supporters of the amyloid hypothesis. However, before we get ahead of ourselves, a few salient aspects bear remembrance.

First, different groups have reported that knockout of PS1 (i.e., no Aβ and probably no Aβ*56, either), while attenuating Aβ pathology in APP mutant transgenic mice, does not cure cognitive deficits (Dewachter et al., 2002; Saura et al., 2005). Therefore, cognitive deficits do not relate to Aβ (in any guise, even *). Second, mitochondrial, apoptotic, and oxidative events all precede frank Aβ deposition and are linked to cognitive decline in APP transgenic mice (Pratico et al., 2001; Reddy et al., 2004). Since oxidative stress leads to increases in Aβ (Yan et al., 1995;...  Read more

Star-struck by Amyloid
Lesne and colleagues show that Aβ*56 is found in cognitively impaired Tg2576 animals without Aβ plaques, but not in unimpaired animals, and that it correlates to early declines in memory but not later ones. Notably, when isolated and injected into rats, Aβ*56 leads to reversible cognitive deficits. This is an interesting study and will definitely appeal to supporters of the amyloid hypothesis. However, before we get ahead of ourselves, a few salient aspects bear remembrance.

First, different groups have reported that knockout of PS1 (i.e., no Aβ and probably no Aβ*56, either), while attenuating Aβ pathology in APP mutant transgenic mice, does not cure cognitive deficits (Dewachter et al., 2002; Saura et al., 2005). Therefore, cognitive deficits do not relate to Aβ (in any guise, even *). Second, mitochondrial, apoptotic, and oxidative events all precede frank Aβ deposition and are linked to cognitive decline in APP transgenic mice (Pratico et al., 2001; Reddy et al., 2004). Since oxidative stress leads to increases in Aβ (Yan et al., 1995; Li et al., 2004), we suspect this is the true star. Third, related to these issues, mutations in APP cause increases in oxidative stress (Yamatsuji et al., 1996; Hashimoto et al., 2000).

In sum, Aβ*56 may be the brightest star, but, as any amateur astronomer can attest, “stars that burn brightest burn fastest and thus have the shortest lifetimes.”

References:
Dewachter I, Reverse D, Caluwaerts N, Ris L, Kuiperi C, Van den Haute C, Spittaels K, Umans L, Serneels L, Thiry E, Moechars D, Mercken M, Godaux E, Van Leuven F. Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice. J Neurosci. 2002 May 1;22(9):3445-53. Abstract

Hashimoto Y, Niikura T, Ito Y, Nishimoto I. Multiple mechanisms underlie neurotoxicity by different types of Alzheimer's disease mutations of amyloid precursor protein. J Biol Chem. 2000 Nov 3;275(44):34541-51. Abstract

Li F, Calingasan NY, Yu F, Mauck WM, Toidze M, Almeida CG, Takahashi RH, Carlson GA, Flint Beal M, Lin MT, Gouras GK. Increased plaque burden in brains of APP mutant MnSOD heterozygous knockout mice. J Neurochem. 2004 Jun;89(5):1308-12. Abstract

Pratico D, Uryu K, Leight S, Trojanoswki JQ, Lee VM. Increased lipid peroxidation precedes amyloid plaque formation in an animal model of Alzheimer amyloidosis. J Neurosci. 2001 Jun 15;21(12):4183-7. Abstract

Reddy PH, McWeeney S, Park BS, Manczak M, Gutala RV, Partovi D, Jung Y, Yau V, Searles R, Mori M, Quinn J. Gene expression profiles of transcripts in amyloid precursor protein transgenic mice: up-regulation of mitochondrial metabolism and apoptotic genes is an early cellular change in Alzheimer's disease. Hum Mol Genet. 2004 Jun 15;13(12):1225-40. Epub 2004 Apr 28. Abstract

Saura CA, Chen G, Malkani S, Choi SY, Takahashi RH, Zhang D, Gouras GK, Kirkwood A, Morris RG, Shen J. Conditional inactivation of presenilin 1 prevents amyloid accumulation and temporarily rescues contextual and spatial working memory impairments in amyloid precursor protein transgenic mice. J Neurosci. 2005 Jul 20;25(29):6755-64. Abstract

Yamatsuji T, Matsui T, Okamoto T, Komatsuzaki K, Takeda S, Fukumoto H, Iwatsubo T, Suzuki N, Asami-Odaka A, Ireland S, Kinane TB, Giambarella U, Nishimoto I. G protein-mediated neuronal DNA fragmentation induced by familial Alzheimer's disease-associated mutants of APP. Science. 1996 May 31;272(5266):1349-52. Abstract

Yan SD, Yan SF, Chen X, Fu J, Chen M, Kuppusamy P, Smith MA, Perry G, Godman GC, Nawroth P, et al. Non-enzymatically glycated tau in Alzheimer's disease induces neuronal oxidant stress resulting in cytokine gene expression and release of amyloid beta-peptide. Nat Med. 1995 Jul;1(7):693-9. Abstract

View all comments by Hyoung-gon Lee
View all comments by Akihiko Nunomura
View all comments by George Perry
View all comments by Mark A. Smith
View all comments by Xiongwei Zhu

This exciting paper set outs to define the site and conformation of Aβ in the brain that may be critical for cognitive dysfunction in Tg2576 mice. The co-occurrence of Aβ*56 with behavioral alterations is quite interesting, yet aspects of the study are surprising. Aβ* does not progressively increase, while Alzheimer disease and Tg2576 mice are characterized by progressive synaptic pathology. Aβ* appears at the onset of what seems to be a progressive decline in behavior in Tg2576 mice, were it not for transient improvement at 13 months, which surprisingly also occurs in wild-type mice.

The data used to support that Aβ* accumulates extracellularly in Tg2576 mice are challenging. As suggested in previous comments (LeVine; Marchesi), it would seem difficult to be certain that one is mainly looking at extracellular peptides after detergent treatment (0.01 percent NP-40; 0.1 percent SDS) and homogenization of the intricate mass of neurons and processes of brain by 10 passages through a 20-gauge needle. The authors did provide some data on other intracellular proteins not leaking...  Read more

This exciting paper set outs to define the site and conformation of Aβ in the brain that may be critical for cognitive dysfunction in Tg2576 mice. The co-occurrence of Aβ*56 with behavioral alterations is quite interesting, yet aspects of the study are surprising. Aβ* does not progressively increase, while Alzheimer disease and Tg2576 mice are characterized by progressive synaptic pathology. Aβ* appears at the onset of what seems to be a progressive decline in behavior in Tg2576 mice, were it not for transient improvement at 13 months, which surprisingly also occurs in wild-type mice.

The data used to support that Aβ* accumulates extracellularly in Tg2576 mice are challenging. As suggested in previous comments (LeVine; Marchesi), it would seem difficult to be certain that one is mainly looking at extracellular peptides after detergent treatment (0.01 percent NP-40; 0.1 percent SDS) and homogenization of the intricate mass of neurons and processes of brain by 10 passages through a 20-gauge needle. The authors did provide some data on other intracellular proteins not leaking out in the process, although one might expect cytoskeleton proteins, such as tau and MAP2, to be more readily retained in cells in the presence of detergent compared to a hydrophobic/lipid-associated peptide such as Aβ. A readily releasable marker such as LDH could have been helpful.

In the supplement, the authors compare previous work addressing Aβ increases in Tg2576 mice, including work from our lab on intraneuronal Aβ42 increases. Their interpretation appears inconsistent with our immunoelectron microscopy studies demonstrating pathological intraneuronal Aβ42 increases and oligomerization in Tg2576 mouse brains (Takahashi et al., 2002; 2004). Remarkably, a fascinating report relating Pin1 and amyloid by Lu and colleagues (Pastorino et al., 2006) in the following issue of Nature provided further confirmation of intraneuronal Aβ accumulation in MVBs of Tg2576 mice.

Can the findings of Lesne et al. be reconciled with evidence for intraneuronal Aβ? It is possible that Aβ* is the specific oligomer responsible for the invariable association of intracellular pathology with Aβ oligomers (Takahashi et al., 2004), which could then be released from neurites following destruction of the plasma membrane from within. This is not inconsistent with a subsequent important role for extracellular Aβ* as well. A potentially similar scenario of intracellular amyloid formation is described in a recent article on diabetes, another common amyloid associated age-related disease (Paulsson et al., 2006).

View all comments by Gunnar K. Gouras