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...  Read more

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

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...  Read more

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

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

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

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...  Read more

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

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

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

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

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

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

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

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

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

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

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

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

The current paper from Brad Hyman´s group very nicely shows that transgenic mice overexpressing FAD-mutated APP have reduced ocular dominance plasticity in the visual cortex. The data are very convincing as the study is carefully performed on two independent transgenic lines, applying two complementary methods assessing synaptic reorganisation after visual deprivation. Confounding effects of transgene expression on the basic spatial extent and laminar distribution of the visual cortex response to light or the overall responsiveness of the visual cortex have been ruled out, indicating that baseline functional organization of visual responses most unlikely account for the observed effects.

In line with recent evidence that NMDA signalling, a mechanism required for synaptic plasticity, can be affected by Aβ (e.g. Hsieh et al. Neuron 2006;52:831), it is very tempting to assume a causative role for Aβ in disrupting synaptic plasticity. Still, other explanations might be possible, and it would be interesting to compare those strains analysed in the present study with transgenic...  Read more

The current paper from Brad Hyman´s group very nicely shows that transgenic mice overexpressing FAD-mutated APP have reduced ocular dominance plasticity in the visual cortex. The data are very convincing as the study is carefully performed on two independent transgenic lines, applying two complementary methods assessing synaptic reorganisation after visual deprivation. Confounding effects of transgene expression on the basic spatial extent and laminar distribution of the visual cortex response to light or the overall responsiveness of the visual cortex have been ruled out, indicating that baseline functional organization of visual responses most unlikely account for the observed effects.

In line with recent evidence that NMDA signalling, a mechanism required for synaptic plasticity, can be affected by Aβ (e.g. Hsieh et al. Neuron 2006;52:831), it is very tempting to assume a causative role for Aβ in disrupting synaptic plasticity. Still, other explanations might be possible, and it would be interesting to compare those strains analysed in the present study with transgenic mice expressing human wild-type APP at a comparable level. This also might shed light on previous discrepant findings reporting decreased (Wegenast-Braun et al. 2009) or increased (Grinevich et al. 2009; Perez-Cruz et al. 2011) Arc expression in different APP mouse strains. Accordingly, a recent study by Seeger et al. (Neurobiol.Dis. 2009;35:258) has shown a synaptotrophic effect for transgenic wild-type APP, which is lost when FAD-mutated APP is overexpressed instead.

Irrespectively of the precise molecular mechanisms that account for the observed changes, the present study adds an important piece of evidence to the concept (e.g. Arendt; Neuroscience 2001;102:723) that a failure of synaptic reorganisation is of utmost importance in the AD pathomechanism. Realizing that Aβ and perhaps other fragments of APP might have an intrinsic role in making and reshaping our brain, the size of the challenge to interfere with these mechanisms with a therapeutic intention immediately becomes clear.

References:

Hsieh H, Boehm J, Sato C, Iwatsubo T, Tomita T, Sisodia S, Malinow R. AMPAR removal underlies Abeta-induced synaptic depression and dendritic spine loss. Neuron. 2006 Dec 7;52(5):831-43.

Wegenast-Braun BM, Fulgencio Maisch A, Eicke D, Radde R, Herzig MC, Staufenbiel M, Jucker M, Calhoun ME. Independent effects of intra- and extracellular Abeta on learning-related gene expression. Am J Pathol. 2009 Jul;175(1):271-82.

Grinevich V, Kolleker A, Eliava M, Takada N, Takuma H, Fukazawa Y, Shigemoto R, Kuhl D, Waters J, Seeburg PH, Osten P. Fluorescent Arc/Arg3.1 indicator mice: a versatile tool to study brain activity changes in vitro and in vivo. J Neurosci Methods. 2009 Oct 30;184(1):25-36. Epub 2009 Jul 21.

Perez-Cruz C, Nolte MW, van Gaalen MM, Rustay NR, Termont A, Tanghe A, Kirchhoff F, Ebert U. Reduced spine density in specific regions of CA1 pyramidal neurons in two transgenic mouse models of Alzheimer's disease. J Neurosci. 2011 Mar 9;31(10):3926-34.

Seeger G, Gärtner U, Ueberham U, Rohn S, Arendt T. FAD-mutation of APP is associated with a loss of its synaptotrophic activity. Neurobiol Dis. 2009 Aug;35(2):258-63. Epub 2009 May 18.

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View all comments by Thomas Arendt

• The nature of toxic Aβ intermediates can be more accurately controlled by injecting Aβ preparations that are characterized before and after chronic injection, as we did in our paper (Fig. 1 and Fig. 2D).
• Since the intrahippocampal injections are done in awake, freely moving mice, there are no confounding interference effects between any anesthetic agents and the Aβ solution on intracellular pathways.
• To take into account aging—the most robust risk factor associated with AD—the effects of soluble Aβ1-42 oligomers were determined during the process of aging in 12-month-old mice. Chronic Aβ1-42 injections can also be done in younger and older mice to see their effects at different ages.
• The collateral injection of soluble Aβ1-42 oligomers and vehicles permitted the control of any alteration within the same mouse.
• Since Aβ accumulates in a time-dependent manner, the number of injections and the dose of Aβ can be adjusted to obtain more or less severe readouts of Aβ...  Read more
  

• The nature of toxic Aβ intermediates can be more accurately controlled by injecting Aβ preparations that are characterized before and after chronic injection, as we did in our paper (Fig. 1 and Fig. 2D).
• Since the intrahippocampal injections are done in awake, freely moving mice, there are no confounding interference effects between any anesthetic agents and the Aβ solution on intracellular pathways.
• To take into account aging—the most robust risk factor associated with AD—the effects of soluble Aβ1-42 oligomers were determined during the process of aging in 12-month-old mice. Chronic Aβ1-42 injections can also be done in younger and older mice to see their effects at different ages.
• The collateral injection of soluble Aβ1-42 oligomers and vehicles permitted the control of any alteration within the same mouse.
• Since Aβ accumulates in a time-dependent manner, the number of injections and the dose of Aβ can be adjusted to obtain more or less severe readouts of Aβ pathogenicity.
• Because cell death occurs in the proximity of the Aβ injection site, neuronal loss can be induced in various and very localized brain regions.
• The toxic effect of Aβ oligomers on molecular and cellular pathways can also be determined before and after neuronal loss within a reasonably short timeframe.
• Since Aβ species were cleared gradually after injection, the long-term effects of Aβ oligomers after their removal can be analyzed both at the molecular and behavioral levels.
• This new animal model can be used for preclinical validation of agents designed to prevent Aβ neurodegeneration, as shown in our paper using transthyretin (TTR).

As discussed in the manuscript, TTR monomers have previously been shown to bind more extensively to Aβ monomers, impeding the further growth of Aβ aggregates (Du and Murphy, 2010). On the other hand, TTR tetramers interact more with Aβ aggregates than with Aβ monomers, and have been observed disrupting fibril formation (Du and Murphy, 2010). Thus, one could argue that the neuroprotective effect of TTR is mainly caused by the prevention of fibril/protofibril-induced toxicity. Although we cannot completely exclude the possibility that part of the neuroprotective effect of TTR is attributed to this mechanism, we think that the major mechanism for the TTR-mediated protection against Aβ toxicity is the sequestration of discrete toxic species and the arrest of Aβ monomer growth into multimers, since we observed that solutions containing an elevated concentration of small Aβ species were more toxic than Aβ1-42 preparation containing larger oligomers (Fig. 5).

In summary, our novel animal model recapitulated many key neuropathological hallmarks of AD in a time-dependent manner, such as Aβ accumulation, marked neuronal loss, abnormal tau phosphorylation, and memory dysfunction. This in-vivo approach can prove useful in determining the toxicity of Aβ preparations as a function of their temporal profile.

Since current methods of oligomer characterization are very limited and provide only semi-quantitative information, it is difficult to compare different oligomeric preparations in terms of concentration, conformation, and their potential relevance to the disease. In terms of oligomer identification and characterization, "in-vitro" and "ex-vivo" Aβ preparations have their own advantages and pitfalls (for more details, see our critical review, Benilova et al., 2012). In our study, we used recombinant human Aβ42 that was previously assessed under denaturing and non-denaturing conditions using Western blot, transmission electron microscopy (TEM), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), electrospray-ionization mass spectrometry (ESI-MS), and nuclear magnetic resonance spectroscopy (NMR) (Kuperstein et al., 2010; Broersen et al., 2011). In future studies, it will be interesting to determine if Aβ preparations isolated directly from AD brains can induce similar effects using this model.

References:
Benilova I, Karran E, De Strooper B (2012) The toxic Aβ oligomer and Alzheimer's disease: an emperor in need of clothes. Nat Neurosci. 15(3):349-357. Abstract

Broersen K, Jonckheere W, Rozenski J, Vandersteen A, Pauwels K, Pastore A, Rousseau F, Schymkowitz J (2011) A standardized and biocompatible preparation of aggregate-free amyloid β peptide for biophysical and biological studies of Alzheimer's disease. Protein Eng Des Sel. 24:743-750. Abstract

Du J, Murphy RM (2010) Characterization of the interaction of β-amyloid with transthyretin monomers and tetramers. Biochemistry. 49:8276-8289. Abstract

Kuperstein I, Broersen K, Benilova I, Rozenski J, Jonckheere W, Debulpaep M, Vandersteen A, Segers-Nolten I, Van Der WK, Subramaniam V, Braeken D, Callewaert G, Bartic C, D'Hooge R, Martins IC, Rousseau F, Schymkowitz J, De Strooper B (2010) Neurotoxicity of Alzheimer's disease Aβ peptides is induced by small changes in the Aβ42 to Aβ40 ratio. EMBO J. 29:3408-3420. Abstract

View all comments by Jonathan Brouillette