This work adds to increasing evidence implicating proteins aggregates in the pathophysiology of neurodegenerative diseases.

View all comments by Benjamin Wolozin

Please do not miss the following comments related to this article: Alzheimer's disease and amyloid beta protein Koudinov AR et al. Science online, Published 25 June 2002 [ Full Text ] ; Amyloid hypothesis: summer 2002 and 8th International Conference on Alzheimer’s disease update. Koudinov and Koudinova BMJ 31 July 2002 [ Full Text ] ; The state versus amyloid-beta: the trial of the most wanted criminal in Alzheimer disease. Rottkamp CA et al. Peptides 2002 Jul;23(7):1333-41 [ PubMed ].

View all comments by Alexei R. Koudinov

It was a pleasure to read this work from the Masliah/Lee duo on the cell-to-cell transmission of α-synuclein (Desplats et al., 2009). This work shows that α-synuclein can be released from cells and is taken up by the neighboring cell, thereby aiding in a progressive spread of the protein. This work continues Seung-Jae Lee’s previous work showing that α-synuclein could be released (Lee et al., 2005) and taken up in neurons (Lee et al., 2008; Lee et al., 2008). While the exact mechanism of the release is currently not well defined, this group has done elegant cell biology work to study the internalization mechanism. They show that fluorescently labeled, recombinant α-synuclein is internalized from the extracellular lumen via a dynamin-1-dependent pathway in vitro. This also occurred in vivo, where injection of GFP-labeled mouse cortical neuronal stem cells into the hippocampus of α-synuclein-transgenic mice led to the...  Read more

It was a pleasure to read this work from the Masliah/Lee duo on the cell-to-cell transmission of α-synuclein (Desplats et al., 2009). This work shows that α-synuclein can be released from cells and is taken up by the neighboring cell, thereby aiding in a progressive spread of the protein. This work continues Seung-Jae Lee’s previous work showing that α-synuclein could be released (Lee et al., 2005) and taken up in neurons (Lee et al., 2008; Lee et al., 2008). While the exact mechanism of the release is currently not well defined, this group has done elegant cell biology work to study the internalization mechanism. They show that fluorescently labeled, recombinant α-synuclein is internalized from the extracellular lumen via a dynamin-1-dependent pathway in vitro. This also occurred in vivo, where injection of GFP-labeled mouse cortical neuronal stem cells into the hippocampus of α-synuclein-transgenic mice led to the efficient uptake of the host α-synuclein into the grafted stem cells after just four weeks. These data add yet another case to the growing list of host-graft relationships in amyloid disorders.

Desplats et al. go on to demonstrate that the internalized α-synuclein promotes inclusion body-like structures in the cytosol of the acceptor cells that are positive for ubiquitin and thioflavin S. This somehow depends on the integrity of lysosomes, as lysosomal pH disruption using v-ATPase inhibitors aggravates the inclusion body formation. How lysosomal function is coupled to a process that occurs in the cytosol is not clear, but the authors rule out membrane leakage. The last figure in the paper gives the take-home message, where the authors demonstrate neurodegeneration in both primary neurons and the mouse cortical stem cells that were exposed to neuronal cell-derived extracellular α-synuclein. This suggests that the released and internalized α-synuclein could be responsible for progression and pathology.

Seeding and spreading are not new features of neurodegenerative diseases (Aβ, e.g., Meyer-Luehmann. et al., 2006; prions, e.g., Bolton et al., 1982). But this time it is different. Three cytoplasmic proteins, as opposed to lumenally exposed membrane proteins, have been recently documented to show this effect. Work from Lee’s group has shown that α-synuclein can be exocytosed from cells. And recently, tau, another cytoplasmic protein of immediate interest to this community because of its involvement in AD and FTD, was shown also to act from outside (Clavaguera et al., 2009). Moreover, polyglutamine aggregates, which are relevant to Huntington disease, are internalized into the cytosol as well (Ren et al., 2009). Several features in this work open up new questions:

1. How are these cytosolic proteins released to the extracellular space (“cytosol to lumen” paradox)? Is the release regulated? Is it only the aggregated versions of the protein, or could the monomeric proteins also be secreted?

2. How does internalized extracellular α-synuclein end up in the cytoplasm (“lumen to cytosol” paradox)? Desplats et al. show that the internalization occurs via endocytosis, but how does the protein enter the cytosol from the endosomes? The authors show that the internalized α-synuclein initiates a Lewy body-like inclusion in the cytosol.

3. In the case of the α-synucleinopathies, the pathology spreads progressively to remote areas of the brain. This work could contribute to the direct understanding of this spreading. On the other hand, in tau pathology, similar spreading occurs (from the transentorhinal cortex to the hippocampus), and again a cytoplasmic protein is released or injected that contributes to the spread of the pathology.

4. How does the spreading occur? Nanotubes could be one way. Prions hijack tunneling nanotubes during their intercellular spreading (Gousset et al., 2009). Though Desplats et al. show that recombinant α-synuclein (and also Ren et al. in the case of polyQ) could enter cells from the extracellular space, nanotubes may aid in the spreading and progression.

5. That a toxic protein would instruct amyloid formation in the wild-type counterpart (be it in the host or graft) on one hand is alarming as it raises caution about stem cell therapies. But on the other, it unfolds a new paradigm in the amyloid pathogenesis. An insightful commentary by Adriano Aguzzi (2009) suggests that many proteins could act like prions; Aguzzi calls them prionoids. Certainly a body of recent work points in this direction. Such studies could be extended to other neurodegenerative amyloid disorders to see if this could be generalized.

One thing is clear. The gap between cell biology and neuroscience is narrowing. Exciting times are to come where we will use cell biology not just for basic understanding of these diseases, but, as shown in the current work, also for therapeutic deliberations.

View all comments by Lawrence Rajendran

Comment posted on PD Online Research by Markus Zweckstetter, "What species of alpha-synuclein should be targeted therapeutically?"

View all comments by June Kinoshita

The ability of many cell types, both prokaryotic and eukaryotic, to disseminate and retrieve biological material is increasingly apparent. The purpose of such exchange in many instances remains unclear, and in the case of shared pathogenic protein aggregates, even seems counterproductive. Is one cell’s trash another’s (Trojan) treasure? Depending on the mechanism, this exchange involves varying levels of specificity, and an effective but relatively non-specific means that is beginning to garner needed attention in neurodegenerative diseases is via exosomes, tiny vesicles formed from the endocytosis of a small segment of invaginated cell membrane, which are eventually released into the extracellular space. The ability of exosomes to transport numerous macromolecules over long distances suggests that they could serve as vectors for the prion-like spread of proteopathies (Aguzzi and Rajendran, 2009).

Emmanouilidou and colleagues, in a comprehensive set of experiments, provide evidence for a role or exosomes in the spread of...  Read more

The ability of many cell types, both prokaryotic and eukaryotic, to disseminate and retrieve biological material is increasingly apparent. The purpose of such exchange in many instances remains unclear, and in the case of shared pathogenic protein aggregates, even seems counterproductive. Is one cell’s trash another’s (Trojan) treasure? Depending on the mechanism, this exchange involves varying levels of specificity, and an effective but relatively non-specific means that is beginning to garner needed attention in neurodegenerative diseases is via exosomes, tiny vesicles formed from the endocytosis of a small segment of invaginated cell membrane, which are eventually released into the extracellular space. The ability of exosomes to transport numerous macromolecules over long distances suggests that they could serve as vectors for the prion-like spread of proteopathies (Aguzzi and Rajendran, 2009).

Emmanouilidou and colleagues, in a comprehensive set of experiments, provide evidence for a role or exosomes in the spread of cytotoxic forms of cellularly generated α-synuclein from cell to cell in vitro. The secretion of vesicles is calcium-dependent, and the cytotoxicity is mitigated by immunodepletion of α-synuclein or interference with oligomers. The findings support the idea that Parkinson’s-type synucleinopathy can be transferred among cells (e.g., Desplats et al., 2009; Luk et al., 2009; Kordower and Brundin, 2009). The uptake of secreted α-synuclein by cycling SH-SY5Y cells, but not (non-cycling) neurons, remains an important issue for additional work, as the authors note, inasmuch as the state of multimerization of the protein could be a factor. However, this nice study, in the context of recent work on other disorders, reinforces the view (Miller, 2009) that the prion-like seeding of protein aggregation is a common feature of several neurodegenerative diseases, including Alzheimer disease. The potential role of exosomes in this process clearly deserves further exploration.

View all comments by Lary Walker

Cytosolic Amyloids: Being Out Is In
In the last few months, the neurodegeneration community has witnessed a paradigm shift in the way we understand the spread of amyloids in the brain. Several reports suggested a prion-like behavior of amyloid proteins such as α-synuclein, tau, and huntingtin. [Editor’s note: see ARF Live Discussion.] These amyloids indeed seem to be released from cells and then effect the conversion of their monomeric counterparts in the neighboring cells/grafts. At the same time, there are two major reasons why these amyloids are fundamentally different from prions. First, prions are transmissible between humans/animals; second, they are confined to the lumenal side of the cell, whereas α-synuclein, tau, and huntingtin amyloids are cytoplasmic in nature. Therefore, a puzzling question arises: how do these amyloids get released from the cell and re-enter the neighboring cell (or the target graft as in the case of the Parkinson’s stem cell transplants)?

One...  Read more

Cytosolic Amyloids: Being Out Is In
In the last few months, the neurodegeneration community has witnessed a paradigm shift in the way we understand the spread of amyloids in the brain. Several reports suggested a prion-like behavior of amyloid proteins such as α-synuclein, tau, and huntingtin. [Editor’s note: see ARF Live Discussion.] These amyloids indeed seem to be released from cells and then effect the conversion of their monomeric counterparts in the neighboring cells/grafts. At the same time, there are two major reasons why these amyloids are fundamentally different from prions. First, prions are transmissible between humans/animals; second, they are confined to the lumenal side of the cell, whereas α-synuclein, tau, and huntingtin amyloids are cytoplasmic in nature. Therefore, a puzzling question arises: how do these amyloids get released from the cell and re-enter the neighboring cell (or the target graft as in the case of the Parkinson’s stem cell transplants)?

One could envision several possibilities. Amyloids might acquire a property of interacting with membrane lipids to mediate their translocation; they might leach out of a part of the membrane, or make transient pores and get released out of the cell. There is no convincing evidence for any such mechanism. Non-classical secretion of cytokines and growth factors has been observed, for example, fibroblast growth factor 2, IL-1β, annexins, migration inhibitory factor1, galectins, but at the moment we do not understand the precise mechanism behind this phenomenon. Several possibilities exist (Aguzzi and Rajendran, 2009), one mechanism being through the exosomal pathway. This paper from the group of Kostas Vekrellis in Athens now suggests that α-synuclein takes this route.

First author Evangelia Emmanouilidou and colleagues used an inducible cell system to study the controlled release of α-synuclein. When induced, these cells produced and released α-synuclein, a finding that confirms previous observations (Desplats et al., 2009; Luk et al., 2009). [Editor’s note: see also ARF SfN story.] But Vekrellis’s group asked: how? Through a series of simple but necessary cell-based experiments, they establish that:

1. α-synuclein doesn’t pass the lipid bilayer;
2. overexpression was not the reason for the release;
3. serum factors shown to affect non-classical secretion of proteins did affect α-synuclein release; and
4. heat shock increased the release of α-synuclein.

To understand the mechanisms by which α-synuclein is externalized, the Athens group set out a series of rigorous cell biological experiments. They show that neither scaffolding (actin) nor signaling proteins (protein kinases) played a major role in the release. Instead, they found two familiar players to be crucially involved in the exocytosis of α-synuclein. Increasing cytosolic calcium boosted release of α-synuclein, as did blocking acidification of endocytic compartments. Since both endocytosis and calcium have been previously linked to the release of exosomes—small vesicles that are released from the cells through the endocytic pathway—the authors suspected (rightfully so!) that perhaps α-synuclein utilizes the exosomal pathway to get released via this route.

It is worth mentioning that Seung-Jae Lee and colleagues have previously demonstrated that interfering with lysosomal acidification indeed affected the release of α-synuclein (Desplats et al., 2009), but now Vekrellis and colleagues show that lysosomal acidification affects α-synuclein release presumably via release of exosomes (also see Vingtdeux et al., 2007).

How do exosomes release cytosolic proteins such as α-synuclein? We know more about endocytic mechanisms. They have largely been associated with internalization and release of lumenal contents (i.e., coming from the extracellular space) and membrane proteins. What’s less well known is that the exosomal pathway also releases a considerable number of cytosolic proteins into the extracellular space. During endocytosis, plasma membrane invagination (outside-in) gives rise to early endosomes. Their limiting membrane undergoes another round of invagination (which is now inside-out) to form the intralumenal vesicles, giving the endosome a multivesicular appearance. During this invagination, these intralumenal vesicles encapsulate a significant amount of cytosol with them. Multivesicular bodies harboring such intralumenal vesicles can now fuse with the plasma membrane to release these ILVs as exosomes. This also explains the topology of exosomes being identical to that of the plasma membrane (outside-out; inside-in) with the cytosol encapsulated within them (Aguzzi and Rajendran, 2009).

The current work from Vekrellis’s group shows that this is indeed the case. Through a battery of biochemical experiments, the authors show that α-synuclein is released via exosomes. Since α-synuclein could associate with lipid membranes, the authors looked at whether α-synuclein in exosomal vesicles is membrane-associated (to the inner leaflet of the vesicle) or in the soluble part, and they found that it is associated with both fractions. This is an interesting finding because lipid-mediated oligomerization of α-synuclein has been found to be important for its toxicity and the amyloid conformation (also in other amyloids: see Wang et al., 2010 on lipids and PrP, and Yuyama et al., 2008; Yuyama and Yanagisawa, 2009).

Next, the Greek authors looked at whether the released α-synuclein is toxic to cells. Conditioned media from α-synuclein expressing cells induced toxicity that was rescued when pre-incubated with α-synuclein antibody. Though both monomeric and oligomeric α-synuclein were found in the extracellular medium, this toxicity seems, at least partially, to be mediated by α-synuclein oligomers (of the high- and low-molecular-weight kind). Proliferating cells, not differentiated cells, were particularly vulnerable to the toxicity. Perhaps remodeling of the cell membrane during cell division makes cells particularly vulnerable for oligomer-induced toxicity. Moreover, compounds that interfered with oligomerization inhibited the toxicity.

In summary, this study is important in many aspects. For the first time, it elegantly demonstrates that cytosolic amyloids can be released on exosomes, and it suggests a cellular mechanism for their release. I believe exosomal release could be a key mechanism for the spread of amyloids. That may well have implications for cell-based therapy. It would be interesting to know if exosome-associated α-synuclein was indeed the agent that was responsible for the reported seeding effect in the grafted tissues (Brundin et al., 2008). Perhaps blocking exosome release could become one way to inhibit the spread. Currently, there are no means or tools available that selectively inhibit the release of exosomes.

As is true with any good study, certain limitations exist and new questions emerge:

1. How and why was α-synuclein toxic to the cells? Is the membrane-associated α-synuclein or the free soluble α-synuclein the culprit? In all likelihood, it was the soluble oligomeric α-synuclein. That’s because immunodepletion of α-synuclein from the conditioned media reduced the toxicity and exosome-encapsulated α-synuclein would be inaccessible to the antibody. Then, what is the role of exosome-associated α-synuclein?

2. How is soluble, non-exosomal α-synuclein secreted? This seems to parallel the Aβ case. We showed that even though Aβ is generated by β/γ-secretase-mediated cleavage of APP in early endosomes and released via the exosomal pathway (Rajendran et al., 2006), actually only a small fraction of this Aβ is associated with exosomes. Several explanations exist for this puzzle. For example, Aβ peptides might become unstable and lose their affinity to lipid membranes after being released from the cell, due to differences in pH or ionic concentrations. Thereby, they might contribute to the soluble pool. Perhaps this could explain the soluble fraction of the released α-synuclein, as well.

3. Beyond this intriguing finding, the presence of oligomers is worth pondering. While the authors do not show if oligomers are associated with exosomal vesicles, lipid-mediated oligomerization seems to be an important issue in amyloid formation. In fact, high-resolution lipidomic analysis of exosomes shows enrichment of lipids involved in amyloid formation (our unpublished data). Thus, exosomes could mediate on one hand the release of cytosolic α-synuclein and, on the other, provide an environment that is conducive for oligomerization processes.

4. This study merely showed that α-synuclein is released via exosomes. It did not show specifically if exosome-associated α-synuclein is toxic to the cells, nor did it show if exosome-associated α-synuclein is then taken up by neighboring cells to mediate the seeding effect. Perhaps it is only the soluble fraction that is toxic. In a way, that would be a good thing. It would imply that diffusion-limited spread represents a main cause for the seeding, not a vesicle-mediated process. Besides the fact that these amyloids are entirely different from prions in that they are incapable of transmission from person to person, this study suggests that there is no danger that long-range transmission by these cytosolic amyloids could occur.

View all comments by Lawrence Rajendran

Extracellular α-Synuclein: Multiple roles for the same protein

Without doubt the role of secreted α-synuclein needs to be characterized further. Our data suggests that synuclein may be exerting its effects extracellularly either by entering proliferating cells or acting solely on the cell membrane as is the case with neurons. Whether these effects are mediated via a still-unidentified receptor remains to be examined. We failed to observe synuclein internalization by neuronal cells; however, we cannot rule out the possibility that specific oligomeric species may be internalized by neuronal cells but are too minute in amount to be detected by our labeling assay.

Our study further points toward “free” and exosome-associated alpha-synuclein having different roles in the extracellular space. However, in our study we did not attempt to establish a toxic role for exosome-associated synuclein. This is indeed a question that remains to be answered, especially in light of the observed increase of secreted synuclein levels after treatment of our cells with acidotropic...  Read more

Extracellular α-Synuclein: Multiple roles for the same protein

Without doubt the role of secreted α-synuclein needs to be characterized further. Our data suggests that synuclein may be exerting its effects extracellularly either by entering proliferating cells or acting solely on the cell membrane as is the case with neurons. Whether these effects are mediated via a still-unidentified receptor remains to be examined. We failed to observe synuclein internalization by neuronal cells; however, we cannot rule out the possibility that specific oligomeric species may be internalized by neuronal cells but are too minute in amount to be detected by our labeling assay.

Our study further points toward “free” and exosome-associated alpha-synuclein having different roles in the extracellular space. However, in our study we did not attempt to establish a toxic role for exosome-associated synuclein. This is indeed a question that remains to be answered, especially in light of the observed increase of secreted synuclein levels after treatment of our cells with acidotropic agents that affect the endocytic pathway. In this respect it would be also interesting to examine how (Macro) autophagy may influence the synthesis and release of exosomes.

It is also possible that exosomes could be a mechanism of confinement and removal of the toxic intracellular synuclein species; as such exosomal release of synuclein could be a way of cell defense against the toxic agent. Our data that synuclein presumably “toxic” oligomers are readily detectable in exosomes is in favor of this idea. In this respect, the involvement of microglia in this clearance would also be worth investigating. Alternatively, exosomes may be a way of modifying synuclein (since exosomes–as all other vesicular compartments-are thought to have a specific environment).

View all comments by Evangelia Emmanouilidou

The same has been previously described with respect to tau protein. Thus, extracellular tau protein is toxic for SH-SY5Y (Gomez-Ramos et al., 2006). Aggregated and phosphorylated tau are less toxic than dephosphorylated tau. In addition, tau increases intracellular calcium likely through muscarinic receptors (Gomez-Ramos et al., 2008, 2009). Thus, the extracellular toxicity of tau protein, and now α-synuclein, suggest a common mechanism to explain propagation in those diseases.

References:
Gomez-Ramos A, Diaz-Hernandez M, Cuadros R, Hernandez F and Avila J: Extracellular tau is toxic to neuronal cells. FEBS Lett 580: 4842-50, 2006. Abstract

Gomez-Ramos A, Diaz-Hernandez M, Rubio A, Diaz-Hernandez JI, Miras-Portugal MT and Avila J: Characteristics and consequences of muscarinic receptor activation by tau protein. Eur Neuropsychopharmacol 19: 708-17, 2009. Abstract

Gomez-Ramos A, Diaz-Hernandez M, Rubio A, Miras-Portugal MT and Avila J: Extracellular tau promotes intracellular calcium increase through M1 and M3 muscarinic receptors in neuronal cells. Mol Cell Neurosci 37: 673-81, 2008. Abstract

View all comments by Felix Hernandez

I'd like to submit a technical question for clarification. I was very excited to see this paper. But when I saw the sequence in Figure 3E, I was surprised, because although the sequence looked familiar, two amino acids seemed out of place. To make sure that I was not remembering the sequence wrong, I performed a blast search using the published peptide and found that this peptide is from β-synuclein.

Here are the sequences of the two tryptic peptides:

EGVV_Q_GVA_S_VAEK is β-synuclein
EGVV_h_GVA_t_VAEK for α-synuclein

β-synuclein sequence is:

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPep t&RID=102SVDAU01N&log%24=protalign&blast_rank=2&list_uids=4507111

α-synuclein sequence is:

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPep t&RID=102SVDAU01N&log%24=protalign&blast_rank=3&list_uids=1230575

This is a little troubling, since it cast into question in my mind if the protein that was transfected into the cells was actually α-synuclein? The α and β...  Read more

I'd like to submit a technical question for clarification. I was very excited to see this paper. But when I saw the sequence in Figure 3E, I was surprised, because although the sequence looked familiar, two amino acids seemed out of place. To make sure that I was not remembering the sequence wrong, I performed a blast search using the published peptide and found that this peptide is from β-synuclein.

Here are the sequences of the two tryptic peptides:

EGVV_Q_GVA_S_VAEK is β-synuclein
EGVV_h_GVA_t_VAEK for α-synuclein

β-synuclein sequence is:

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPep t&RID=102SVDAU01N&log%24=protalign&blast_rank=2&list_uids=4507111

α-synuclein sequence is:

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein&dopt=GenPep t&RID=102SVDAU01N&log%24=protalign&blast_rank=3&list_uids=1230575

This is a little troubling, since it cast into question in my mind if the protein that was transfected into the cells was actually α-synuclein? The α and β isoforms are largely similar, so I would expect the antibodies used in this study to pick up both isoforms. In addition, the size difference on an SDS page gel would be small enough that it would be difficult to distinguish the α and β isoforms based on their size. Would the authors kindly want to address this point?

View all comments by Tim West

Reply to comment by Tim West
I am happy to clarify this question. First, I would like to point out that the antibodies used for the detection of α-synuclein in our cell-system are specific to α-synuclein (see also Vekrellis et al., 2009). Indeed, the correct sequence for the α-synuclein tryptic peptide under question is:

EGVVHGVATVAEK.

From this study, a total of two peptides were detected that collectively corroborate the α-synuclein identification.

The tandem mass spectrum shown in our publication was chosen on the basis of a better signal-to-noise ratio. However, this tandem mass spectrum suggests a Glu>pyro-Glu modification at the N-terminus and exhibits a low peptide sequence coverage. The additional tandem mass spectrum detected in this study translated to the amino acid sequence (-)TKEQVTNVGGAVVTGVTAVAQK(-) (observed with one miscleavage at 95 percent ID confidence in concordance to the Mascot software and validated with the Scaffold software program and further verified with manual...  Read more

Reply to comment by Tim West
I am happy to clarify this question. First, I would like to point out that the antibodies used for the detection of α-synuclein in our cell-system are specific to α-synuclein (see also Vekrellis et al., 2009). Indeed, the correct sequence for the α-synuclein tryptic peptide under question is:

EGVVHGVATVAEK.

From this study, a total of two peptides were detected that collectively corroborate the α-synuclein identification.

The tandem mass spectrum shown in our publication was chosen on the basis of a better signal-to-noise ratio. However, this tandem mass spectrum suggests a Glu>pyro-Glu modification at the N-terminus and exhibits a low peptide sequence coverage. The additional tandem mass spectrum detected in this study translated to the amino acid sequence (-)TKEQVTNVGGAVVTGVTAVAQK(-) (observed with one miscleavage at 95 percent ID confidence in concordance to the Mascot software and validated with the Scaffold software program and further verified with manual de novo sequencing interpretation) is uniquely surrogate to α-synuclein. Collectively, the proteomics evidence strongly favors the α-synuclein protein identification over that of β-synuclein.

The annotated tandem mass spectral evidence for: (-)TKEQVTNVGGAVVTGVTAVAQK(-) with over 80 percent peptide sequence coverage is as follows:

(A) Observed and interpreted tandem mass spectrum (see PDF A).

(B) Tabulation of the observed product B (red) and Y (blue) ions from theabove spectrum and concordant amino acid sequence interpretation (see PDF B).

View all comments by Kostas Vekrellis

In their discussion, Emmanouilidou et al. consider the possibility that the degenerative effects of extracellular aggregates of α-synuclein on differentiated SH-SY5Y cells and primary cortical neurons are mediated by a specific receptor or by the formation of membrane pores. The neurotrophin receptor p75 is a suitable candidate for such a receptor. According to evidence provided in the Aβ-crosslinker-hypothesis [.pdf], aggregates of NAC, a natural fragment of α-synuclein, can activate p75 and induce neurite budding and apoptosis via p75, and these effects can be prevented by administration of a juxtamembrane stalk fragment of p75 that is part of the stalk binding site of Aβ on p75. The hypothesis argues that Aβ, which is known to interact with α-synuclein, crosslinks p75 with α-synuclein species and thereby mediates certain protective and deleterious effects of p75 and α-synuclein.

View all comments by Rudolf Bloechl

Our study identifies a specific form of naturally produced human amyloid beta protein, namely stable low-n oligomers, as directly interrupting a key correlate of memory and learning in a living animal. Previous research by many scientists had linked Ab in general to interruption of neural function, but precisely which form of the protein and how that occurred under natural conditions remained obscure. We now identify a specific form of naturally secreted Ab and show directly in living, anesthetized rats that it blocks long-term potentiation in the absence of monomers, protofibrils and fibrils. Thus soluble, diffusible Ab oligomers can interrupt memory circuits in the brain.

Finally, we use a chemical compound that inhibits the production of Aβ to lower the oligomers enough to completely prevent the synaptic interruption, while still leaving appreciable monomer levels (60 percent of normal). This supports the potential utility of modest doses of β- or γ-secretase inhibitors in the disease.

View all comments by Dennis Selkoe

Already back in the early 1990s, a research team led by Dr. Saitoh co-purified a novel molecule from amyloid-rich AD brain tissue. The so-called NACP, or non-Aβ component of Alzheimer’s disease amyloid plaques, was cloned and found to be a human homolog of the Torpedo ray synuclein, later known as α-synuclein. In the late 1990s the issue of coexistence between Aβ and α-synuclein was reinvestigated by Drs. Masters and Li, who co-stained against the two molecules on several AD and DLB/AD brains but did not find any immunohistochemical evidence for such complexes.

Eliezer Masliah and colleagues have now adopted new strategies to answer an old question. By beautifully combining different biochemical and modeling approaches, the authors have shed more light on the issue of possible seeding and co-aggregation between Aβ and α-synuclein. Interestingly, an interaction between the two molecules seems to occur only, or at least predominantly, in diseased human and transgenic mice brains. Given the fact that coexisting pathologies are...  Read more

Already back in the early 1990s, a research team led by Dr. Saitoh co-purified a novel molecule from amyloid-rich AD brain tissue. The so-called NACP, or non-Aβ component of Alzheimer’s disease amyloid plaques, was cloned and found to be a human homolog of the Torpedo ray synuclein, later known as α-synuclein. In the late 1990s the issue of coexistence between Aβ and α-synuclein was reinvestigated by Drs. Masters and Li, who co-stained against the two molecules on several AD and DLB/AD brains but did not find any immunohistochemical evidence for such complexes.

Eliezer Masliah and colleagues have now adopted new strategies to answer an old question. By beautifully combining different biochemical and modeling approaches, the authors have shed more light on the issue of possible seeding and co-aggregation between Aβ and α-synuclein. Interestingly, an interaction between the two molecules seems to occur only, or at least predominantly, in diseased human and transgenic mice brains. Given the fact that coexisting pathologies are commonplace—approximately 50 percent of DLB brains also display AD changes—such an interaction could have a true pathogenic significance. Moreover, the authors find support for the notion that the interaction is taking place on the oligomeric level. Several lines of evidence are indeed suggesting that intermediate species in the formation of both plaques and Lewy bodies are particularly noxious to the brain. However, even though the in silico- and in vitro-based methods adopted in this report are pointing towards the involvement of oligomers, it remains unclear which Aβ and α-synuclein species form such potentially toxic protein hybrids in the affected brain. The development of additional tools, such as conformation-specific antibodies against various Aβ and α-synuclein species in the aggregation process, could help to clarify this issue. Moreover, it remains to be determined under which circumstances that potential seeding effects are taking place in vivo and how strong the respective proteins are affecting their presumed molecular partners. Although both proteins have a strong propensity to aggregate, in vitro data indicate that Aβ may have a greater impact on α-synuclein aggregation than vice versa.

View all comments by Joakim Bergstrom
View all comments by Martin Ingelsson

This is a very important paper illustrating for the first time at high resolution the relation between Abeta oligomers and the condition of dendritic spines in a highly significant animal model of AD. Obviously, several points remain to be addressed, for instance the presence of AMPA and NMDA receptors in the neurite membranes immediately surrounded by the oligomers (they could reveal a distribution similar to that imaged for the dendritic spines with respect to Abeta oligomer gradient) and the levels of free calcium in neurons contacted by Abeta oligomers. However I trust that, when provided, those results will confirm the direct effect of Abeta oligomers on the neuritic membrane.

Another point that must still be clarified is the following: if Abeta oligomers leak from mature fibrils found in the plaques, why in many cases do people bearing plaques not suffer the symptoms of AD? I think that a possible explanation can be searched in several recent papers indicating that Abeta and other proteins can polymerize into fibrils with different structural features, and hence...  Read more

This is a very important paper illustrating for the first time at high resolution the relation between Abeta oligomers and the condition of dendritic spines in a highly significant animal model of AD. Obviously, several points remain to be addressed, for instance the presence of AMPA and NMDA receptors in the neurite membranes immediately surrounded by the oligomers (they could reveal a distribution similar to that imaged for the dendritic spines with respect to Abeta oligomer gradient) and the levels of free calcium in neurons contacted by Abeta oligomers. However I trust that, when provided, those results will confirm the direct effect of Abeta oligomers on the neuritic membrane.

Another point that must still be clarified is the following: if Abeta oligomers leak from mature fibrils found in the plaques, why in many cases do people bearing plaques not suffer the symptoms of AD? I think that a possible explanation can be searched in several recent papers indicating that Abeta and other proteins can polymerize into fibrils with different structural features, and hence stabilities, depending on several factors including environmental conditions. It could well be that those plaque-bearing people who do not suffer AD have highly stable Abeta fibrils that actively and unidirectionally recruit Abeta monomers and oligomers before they can damage neurons. It would be interesting if the authors check for the presence of Abeta oligomers around the plaques in samples from plaque-bearing asymptomatic people.

View all comments by Massimo Stefani

The paper by Koffie et al., by showing correlation between oligomeric Aβ and PSD loss, adds significantly to our appreciation of mechanisms by which flavors of APP, especially of Aβ, attack synapses in Alzheimer disease. There are now publications that demonstrate Aβ induced decrements not only of postsynaptic sites (Koffie, et al., 2009; Lacor et al., 2004) but also of presynaptic entities (e.g., Kelly, et al., 2005; Yao et al., 2003; Callahan et al., 1999). But what is the contribution of synaptic deficits to the cognitive declines of AD? The early studies of DeKosky and Scheff (1990) and Terry et al. (1991) agree in finding a correlation of about 0.70 between postmortem measures of synapse density and antemortem scores on cognitive tests. However, a correlation of 0.7 yields an R2 of about 0.50 which leaves 50 percent of the variance in cognitive scores unaccounted for by synapse density. Where might the missing 50 percent lie? Of course, it is presumptuous to assert that synapse density in one small tissue block from a single brain region should explain a...  Read more

The paper by Koffie et al., by showing correlation between oligomeric Aβ and PSD loss, adds significantly to our appreciation of mechanisms by which flavors of APP, especially of Aβ, attack synapses in Alzheimer disease. There are now publications that demonstrate Aβ induced decrements not only of postsynaptic sites (Koffie, et al., 2009; Lacor et al., 2004) but also of presynaptic entities (e.g., Kelly, et al., 2005; Yao et al., 2003; Callahan et al., 1999). But what is the contribution of synaptic deficits to the cognitive declines of AD? The early studies of DeKosky and Scheff (1990) and Terry et al. (1991) agree in finding a correlation of about 0.70 between postmortem measures of synapse density and antemortem scores on cognitive tests. However, a correlation of 0.7 yields an R2 of about 0.50 which leaves 50 percent of the variance in cognitive scores unaccounted for by synapse density. Where might the missing 50 percent lie? Of course, it is presumptuous to assert that synapse density in one small tissue block from a single brain region should explain a phenomenon as complex as cognition. However, let us proceed. May part of the missing 50 percent be attributed to the fact that reduced synapse density in a shrinking cortex means an even greater loss of synapses than that indicated by density data alone? May part of the missing 50 percent be attributed to data indicating that even synapses that are structurally present may not be optimally functional—as evidenced by data showing reduced expression of dynamin 1 in AD (Kelly et al., 2005; Yao et al. 2003), by reduced expression of synaptophysin by single neurons in association with tau phosphorylation or tangles (Callahan et al., 1999), as well as by reductions in transmitter systems (e.g., Lanari et al., 2006 for recent, brief review)? And, to what extent might decrements in these indices of synaptic functional capacity be a consequence of or a cause of synaptic loss?

Data from imaging studies suggest that we need to look more broadly than at the synapse alone in attempting to correlate cognitive deficits of AD to neurobiological variables. For example, such studies demonstrate the importance of metabolic function in cognitive decline (e.g., Villain et al., 2008; Alexander et al., 2002). Gene expression array studies also indicate that transcripts that are not directly synaptic play significant roles in the pathophysiology of AD. These include transcripts related to metabolism, transport, oxidative phosphorylation, protein localization, myelin and inflammation, as examples (e.g., Liang et al., 2008; Miller et al., 2008; Wilmot et al., 2008; Blalock et al., 2004; Colangelo et al., 2002).

Synapses are unquestionably central players in the pathophysiology of AD, and we are starting to understand how various flavors of Aβ, as well as other molecules, act on synapses in AD. But it is clear that there are also other important players. How they relate to each other, which are drivers and which are driven; where they all fit into the pathophysiological cascade of Alzheimer’s disease remains to be established. Paul D. Coleman

References:
Alexander GE. Chen K. Pietrini P. Rapoport SI. Reiman EM. Longitudinal PET Evaluation of Cerebral Metabolic Decline in Dementia: A Potential Outcome Measure in Alzheimer's Disease Treatment Studies. American Journal of Psychiatry. 159(5):738-45, 2002. Abstract

Blalock EM. Geddes JW. Chen KC. Porter NM. Markesbery WR. Landfield PW. Incipient Alzheimer's disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses. Proceedings of the National Academy of Sciences of the United States of America. 101(7):2173-8, 2004. Abstract

Callahan LM. Vaules WA. Coleman PD. Quantitative decrease in synaptophysin message expression and increase in cathepsin D message expression in Alzheimer disease neurons containing neurofibrillary tangles. Journal of Neuropathology & Experimental Neurology. 58(3):275-87, 1999. Abstract

Colangelo V. Schurr J. Ball MJ. Pelaez RP. Bazan NG. Lukiw WJ. Gene expression profiling of 12633 genes in Alzheimer hippocampal CA1: transcription and neurotrophic factor down-regulation and up-regulation of apoptotic and pro-inflammatory signaling. Journal of Neuroscience Research. 70(3):462-73, 2002. Abstract

DeKosky ST. Scheff SW. Synapse loss in frontal cortex biopsies in Alzheimer's disease: correlation with cognitive severity. Annals of Neurology. 27(5):457-64, 1990. Abstract

Kelly BL. Ferreira A. beta-Amyloid-induced dynamin 1 degradation is mediated by N-methyl-D-aspartate receptors in hippocampal neurons. Journal of Biological Chemistry. 281(38):28079-89, 2006. Abstract

Kelly BL. Vassar R. Ferreira A. Beta-amyloid-induced dynamin 1 depletion in hippocampal neurons. A potential mechanism for early cognitive decline in Alzheimer disease. Journal of Biological Chemistry. 280(36):31746-53, 2005. Abstract

Koffie RM, Melanie Meyer-Luehmanna,, Tadafumi Hashimoto, Kenneth W. Adams, Matthew L. Mielke, Monica Garcia-Alloza, Kristina D. Micheva, Stephen J. Smith, M. Leo Kim, Virginia M. Lee, Bradley T. Hyman, and Tara L. Spires-Jones. Oligomeric amyloid associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques Proceedings of the National Academy of Sciences of the United States of America. 2009.

Lacor PN. Buniel MC. Chang L. Fernandez SJ. Gong Y. Viola KL. Lambert MP. Velasco PT. Bigio EH. Finch CE. Krafft GA. Klein WL. Synaptic targeting by Alzheimer's-related amyloid beta oligomers. Journal of Neuroscience. 24(45):10191-200, 2004. Abstract

Lanari A. Amenta F. Silvestrelli G. Tomassoni D. Parnetti L. Neurotransmitter deficits in behavioural and psychological symptoms of Alzheimer's disease. [Review] Mechanisms of Ageing & Development. 127(2):158-65, 2006. Abstract

Liang WS. Reiman EM. Valla J. Dunckley T. Beach TG. Grover A. Niedzielko TL. Schneider LE. Mastroeni D. Caselli R. Kukull W. Morris JC. Hulette CM. Schmechel D. Rogers J. Stephan DA. Alzheimer's disease is associated with reduced expression of energy metabolism genes in posterior cingulate neurons. Proceedings of the National Academy of Sciences of the United States of America. 105(11):4441-6, 2008. Abstract

Miller JA. Oldham MC. Geschwind DH. A systems level analysis of transcriptional changes in Alzheimer's disease and normal aging. Journal of Neuroscience. 28(6):1410-20, 2008. Abstract

Terry RD. Masliah E. Salmon DP. Butters N. DeTeresa R. Hill R. Hansen LA. Katzman R. Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment. Annals of Neurology. 30(4):572-80, 1991. Abstract

Villain N. Desgranges B. Viader F. de la Sayette V. Mezenge F. Landeau B. Baron JC. Eustache F. Chetelat G. Relationships between hippocampal atrophy, white matter disruption, and gray matter hypometabolism in Alzheimer's disease. Journal of Neuroscience. 28(24):6174-81, 2008. Abstract

Wilmot B. McWeeney SK. Nixon RR. Montine TJ. Laut J. Harrington CA. Kaye JA. Kramer PL. Translational gene mapping of cognitive decline. Neurobiology of Aging. 29(4):524-41, 2008. Abstract

Yao PJ. Zhu M. Pyun EI. Brooks AI. Therianos S. Meyers VE. Coleman PD. Defects in expression of genes related to synaptic vesicle trafficking in frontal cortex of Alzheimer's disease. Neurobiology of Disease. 12(2):97-109, 2003. Abstract

View all comments by Paul Coleman

This article by Koffie et al. contributes importantly to elucidating the contribution of amyloid plaque pathology to synapse loss in Alzheimer’s disease. Heretofore, studies examining the effects of Aβ on synapse morphology have been performed primarily in ex vivo paradigms; however, this work sheds light on spine dynamics at the plaque interface in vivo.

Decreased synapse density has been well documented in human brain affected by AD (1). Importantly, the extent of synapse loss correlates with the severity of dementia, a finding also applicable to individuals with mild cognitive impairment (2, 3). Aβ is most commonly implicated as the pathogenic species responsible for the initial insidious loss of synapse density (4-6). While biochemical and genetic evidence suggests that accumulation of parenchymal Aβ is a critical initiator, a finding requiring reconciliation is that amyloid plaque burden does not correlate strongly with the severity of disease (7,8). Soluble Aβ, on the other hand, correlates strongly with disease severity, and specifically oligomeric...  Read more

This article by Koffie et al. contributes importantly to elucidating the contribution of amyloid plaque pathology to synapse loss in Alzheimer’s disease. Heretofore, studies examining the effects of Aβ on synapse morphology have been performed primarily in ex vivo paradigms; however, this work sheds light on spine dynamics at the plaque interface in vivo.

Decreased synapse density has been well documented in human brain affected by AD (1). Importantly, the extent of synapse loss correlates with the severity of dementia, a finding also applicable to individuals with mild cognitive impairment (2, 3). Aβ is most commonly implicated as the pathogenic species responsible for the initial insidious loss of synapse density (4-6). While biochemical and genetic evidence suggests that accumulation of parenchymal Aβ is a critical initiator, a finding requiring reconciliation is that amyloid plaque burden does not correlate strongly with the severity of disease (7,8). Soluble Aβ, on the other hand, correlates strongly with disease severity, and specifically oligomeric assembly forms are the ones to demonstrate robust effects on synapse physiology. It is within this context that Koffie et al. examines how Aβ oligomers loosely associated at the periphery of neuritic plaques affects synapse density. While prior work by Spires-Jones et al. (9) demonstrated that synapse loss was most pronounced within 30 μm of plaques, the identity of the toxic species contained within this complex β-sheet rich structure remained unidentified. By using array tomography and an antibody characterized as Aβ oligomer-specific (10), the authors here present compelling evidence that implicates oligomeric Aβ surrounding insoluble plaques as the likely culprit inducing synapse loss.

The authors first demonstrate using in vivo multiphoton imaging and postmortem sectioning that the vast majority of compact plaques contain a penumbra that is positive for Aβ oligomers as judged by NAB61 immunostaining. Array tomography is subsequently used to illustrate that synapse density is reduced within the compact portion of plaques as well as in the oligomer-rich halo region. The magnitude of synapse loss is blunted with increasing distance, with this significant difference extending up to 20 μm radially from the halo margins. The authors also describe that the NAB61 signal co-localizes with PSD-95 puncta, suggesting an interaction of Aβ oligomers with post-synaptic components. Of physiologic relevance, spines associated with Aβ oligomers were smaller, suggestive of a morphologic manifestation of synapse depression. Taken together, these findings are all highly suggestive that soluble Aβ oligomers are enriched at amyloid plaques and can ultimately initiate the synapse loss observed in AD brain.

This powerful combination of in vivo multiphoton imaging, postmortem analysis with array tomography, and Aβ oligomer-specific reagents opens up a new avenue for understanding AD pathophysiology. With these tools in hand and other ones now emerging, the field is now poised to visualize answers that have been elusive in the past. For instance, it may be of interest to address whether spine density is similarly affected in regions surrounding diffuse plaques that are concentrated in Aβ oligomers. Given that compact plaques contain a panoply of non-Aβ factors, the relatively more simple composition of diffuse plaques may be able to provide additional direct evidence that Aβ oligomers are sufficient to induce synapse loss. Second, is this synapse loss reversible? Given the recent clinical trials examining the therapeutic advantage of passive immunization, NAB61 (or other oligomer-specific antibodies) may provide insight as to whether clearance of soluble higher order Aβ assembly forms can prevent or reverse the dramatic spine loss surrounding amyloid plaques. Third, application of more recently developed higher resolution imaging techniques, such as stimulation emission depletion (STED) microscopy, may provide clearer visualization of the more subtle changes in synapse structure that take place near plaques as described in this study or elsewhere in the parenchyma. Lastly, the authors suggest a physiologic role for Aβ oligomers in regulating synapse function. A cross of APP transgenic mice with channel rhodopsin-2 (ChR2), halorhodopsin (NpHR) mice (11) may be useful to further examine the general role of network dysfunction (12,13) in Aβ oligomer rich parenchyma, such as the penumbra surrounding compact plaques described by Koffie et al. More specifically, by expressing these optically activated ChR2/NpHR ion channels, neuronal activity can be focally modulated revealing perturbations in the circuitry surrounding amyloid plaques.

References:
1. Davies, C.A. et al. A quantitative morphometric analysis of the neuronal and synaptic content of the frontal and temporal cortex in patients with Alzheimer's disease. J Neurol Sci 78:151-164 (1987). Abstract

2. Masliah, E. et al. Altered expression of synaptic proteins occurs early during progression of Alzheimer's disease. Neurology 56:127-129 (2001). Abstract

3. Scheff, S.W. et al. Synaptic alterations in CA1 in mild Alzheimer disease and mild cognitive impairment. Neurology 68:1501-1508 (2007). Abstract

4. Hsieh, H. et al. AMPAR removal underlies Abeta-induced synaptic depression and dendritic spine loss. Neuron 52, 831-43 (2006). Abstract

5. Lacor, P.N. et al. Abeta oligomer-induced aberrations in synapse composition, shape, and density provide a molecular basis for loss of connectivity in Alzheimer's disease. J Neurosci 27, 796-807 (2007). Abstract

6. Shankar, G.M. et al. Natural oligomers of the Alzheimer amyloid-beta protein induce reversible synapse loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway. J Neurosci 27, 2866-75 (2007). Abstract

7. Terry, R.D. et al. Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment. Ann Neurol 30, 572-80 (1991). Abstract

8. McLean, C.A. et al. Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease. Ann Neurol 46, 860-6 (1999). Abstract

9. Spires, T.L. et al. Dendritic spine abnormalities in amyloid precursor protein transgenic mice demonstrated by gene transfer and intravital multiphoton microscopy. J Neurosci 25, 7278-87 (2005). Abstract

10. Lee, E.B. et al. Targeting amyloid-beta peptide (Abeta) oligomers by passive immunization with a conformation-selective monoclonal antibody improves learning and memory in Abeta precursor protein (APP) transgenic mice. J Biol Chem 281(7):4292-9 (2006). Abstract

11. Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446(7136):633-9 (2007). Abstract

12. Palop, J.J. et al. Aberrant excitatory neuronal activity and compensatory remodeling of inhibitory hippocampal circuits in mouse models of Alzheimer's disease. Neuron 55(5):697-711 (2007). Abstract

13. Busche, M.A. et al. Clusters of Hyperactive Neurons Near Amyloid Plaques in a Mouse Model of Alzheimer’s Disease. Science 321:1686-9 (2008). Abstract

View all comments by Ganesh M Shankar

This paper confirms, in vivo, a role for soluble Aβ oligomers in the disassembly of synapses surrounding plaques. The authors for the first time apply array tomography to quantitatively assess the interaction between postsynaptic densities/spines with microdeposits of oligomeric Aβ present in a halo extending from the edge of the dense core of plaques. Interestingly, they find that the reduction in the density but not in the size of postsynaptic densities is inversely correlated to the distance from the plaques. Overall, this paper suggests that in vivo plaques act as a source of toxic soluble oligomeric Aβ, which directly interacts with dendritic spines, causing their disappearance. However, these data don’t explain why 60 percent of postsynaptic densities and dendritic spines resist the toxic effects of Aβ, or why plaques in elderly individuals are not always associated with cognitive decline. Maybe the answer for the latter point can be found in a recent paper (Lesne et al., 2008) where the authors studied plaque-bearing mice with reduced levels of...  Read more

This paper confirms, in vivo, a role for soluble Aβ oligomers in the disassembly of synapses surrounding plaques. The authors for the first time apply array tomography to quantitatively assess the interaction between postsynaptic densities/spines with microdeposits of oligomeric Aβ present in a halo extending from the edge of the dense core of plaques. Interestingly, they find that the reduction in the density but not in the size of postsynaptic densities is inversely correlated to the distance from the plaques. Overall, this paper suggests that in vivo plaques act as a source of toxic soluble oligomeric Aβ, which directly interacts with dendritic spines, causing their disappearance. However, these data don’t explain why 60 percent of postsynaptic densities and dendritic spines resist the toxic effects of Aβ, or why plaques in elderly individuals are not always associated with cognitive decline. Maybe the answer for the latter point can be found in a recent paper (Lesne et al., 2008) where the authors studied plaque-bearing mice with reduced levels of oligomeric Aβ assemblies and find that they have intact memory function. Finally Koffie et al. describe oligomeric Aβ puncta either juxtaposed to PSD clusters or on the extracellular surface of dendritic spines (see Fig. 4C) and occasionally of presynaptic compartments. However, due to limitation in their technique they cannot exclude the possibility that the Aβ puncta, even though they appear on the extracellular surface of dendritic spines, could be in reality on or within the glia processes surrounding individual synapses.

References:
Lesné S, Kotilinek L, Ashe KH. Plaque-bearing mice with reduced levels of oligomeric amyloid-beta assemblies have intact memory function. Neuroscience. 2008 Feb 6;151(3):745-9. Epub 2007 Nov 17. Abstract

View all comments by Barbara Calabrese

1. Before comparing structural studies of Aβ fibrils from different laboratories, it is crucially important to compare the conditions under which the fibrils were grown, as our own solid-state NMR and electron microscopy studies have shown that Aβ fibril structures depend strongly on growth conditions. In the recent cryoEM studies by Zhang et al., the fibrils were grown at 37 C in 10 mM HCl. In our solid-state NMR studies, fibrils were grown at room temperature in pH 7.4 buffer.

2. The Aβ1-40 and Aβ1-42 peptides apparently adopt quite similar molecular conformations in amyloid fibrils, and both form parallel β-sheets, based on solid-state NMR, H/D exchange, and other data. But other aspects of the fibril structures may be somewhat different. Structures of Aβ1-42 fibrils have not yet been characterized completely by solid-state NMR.

3. The most surprising aspect of the cryoEM reconstruction reported by Zhang et al. is the central pore in the Aβ1-42 fibril structure. Structural models for Aβ1-40 fibrils based on solid-state NMR and...  Read more

1. Before comparing structural studies of Aβ fibrils from different laboratories, it is crucially important to compare the conditions under which the fibrils were grown, as our own solid-state NMR and electron microscopy studies have shown that Aβ fibril structures depend strongly on growth conditions. In the recent cryoEM studies by Zhang et al., the fibrils were grown at 37 C in 10 mM HCl. In our solid-state NMR studies, fibrils were grown at room temperature in pH 7.4 buffer.

2. The Aβ1-40 and Aβ1-42 peptides apparently adopt quite similar molecular conformations in amyloid fibrils, and both form parallel β-sheets, based on solid-state NMR, H/D exchange, and other data. But other aspects of the fibril structures may be somewhat different. Structures of Aβ1-42 fibrils have not yet been characterized completely by solid-state NMR.

3. The most surprising aspect of the cryoEM reconstruction reported by Zhang et al. is the central pore in the Aβ1-42 fibril structure. Structural models for Aβ1-40 fibrils based on solid-state NMR and electron microscopy (especially scanning transmission electron microscopy) do not contain such a large pore. Solid-state NMR data for Aβ1-40 fibrils indicate intermolecular contacts that would be inconsistent with the cryoEM results of Zhang et al. But again, the experiments of Zhang et al. were performed on Aβ1-42, rather than Aβ1-40, and the pH and temperature during fibril growth were quite different.

4. Finally, the Aβ1-40 peptide (and possibly also the Aβ1-42 peptide) can probably form five or six different fibril structures. It will be interesting to identify the structure or structures that develop in the human brain. This is one of the goals of our own current work.

View all comments by Robert Tycko

Zhang at al. report a three-dimensional reconstruction of an Aβ1-42 amyloid fibril based on cryoelectron microscopy data. The obtained structure varies very significantly from the fibril structure that our groups have published for Aβ1-40 peptide. This does not only hold for the Aβ1-40 structure quoted by the authors (Sachse et al., 2006; Sachse et al., 2008). It is also true for a very recently published analysis of the structure of 12 Aβ1-40 amyloid fibrils (Meinhardt et al., 2009) None of them are similar to the Aβ(1-42) fibril structure reported here.

The now published Aβ1-42 fibrils were obtained by in-vitro incubation of pure peptide at pH 2.0 for four weeks. Incubation at strongly acidic conditions and for a prolonged time is generally known to lead to peptide fragmentation or other covalent modifications. Furthermore, different pH values can lead to dramatically different fibril structures. Therefore,...  Read more

Zhang at al. report a three-dimensional reconstruction of an Aβ1-42 amyloid fibril based on cryoelectron microscopy data. The obtained structure varies very significantly from the fibril structure that our groups have published for Aβ1-40 peptide. This does not only hold for the Aβ1-40 structure quoted by the authors (Sachse et al., 2006; Sachse et al., 2008). It is also true for a very recently published analysis of the structure of 12 Aβ1-40 amyloid fibrils (Meinhardt et al., 2009) None of them are similar to the Aβ(1-42) fibril structure reported here.

The now published Aβ1-42 fibrils were obtained by in-vitro incubation of pure peptide at pH 2.0 for four weeks. Incubation at strongly acidic conditions and for a prolonged time is generally known to lead to peptide fragmentation or other covalent modifications. Furthermore, different pH values can lead to dramatically different fibril structures. Therefore, it is possible that the analyzed fibrils differ quite substantially from the ones that are present in Alzheimer patients and that are formed, of course, under physiologically relevant pH conditions.

It would be helpful if the manuscript provided more of the technical information that a reader would like to know for judging the reliability of this new Aβ1-42 structure and whether it truly reflects the structure of the analyzed fibrils. For example, comparisons between the raw images obtained in the electron microscope and projections of the structure are not included. No statistical analysis of the different observed fibril symmetries is shown. No mass-per-length measurements were carried out to support the interpretation of the structure with two peptides in cross-section. It is not clear to us why the published structure does not show more structural detail despite its resolution of 10 angstroms. In the light of these concerns, the presented structural model remains speculative at this point, and its relevance for Alzheimer disease remains to be further clarified.

View all comments by Marcus Fandrich
View all comments by Niko Grigorieff

Aβ40 and Aβ42 are 40- and 42-residue peptides produced by the sequential cleavage of amyloid precursor protein by β-secretase and γ-secretase. The peptides have a strong tendency to self-aggregate, initially into soluble oligomers, and eventually into insoluble fibrils and large neuronal deposits. Although the soluble oligomers are considered the major culprit of neuronal toxicity, there is nevertheless strong interest in the structure of the Aβ fibrils. Aβ fibrils have been a longstanding subject of various biophysical studies, including cryoEM. Nevertheless, the cryoEM structure of Aβ42 fiber at 10-angstrom resolution as reported by Lee and colleagues represents a significant step forward in our pursuit of the structural basis of Aβ peptide fibrillization. The new structure reveals the expected two protofilaments twisted along the fiber axis. The novelty of the new structure is that the β-sheets are arranged at the periphery surrounding a hollow core, thus forming a long tube-like structure. This architecture is drastically different...  Read more

Aβ40 and Aβ42 are 40- and 42-residue peptides produced by the sequential cleavage of amyloid precursor protein by β-secretase and γ-secretase. The peptides have a strong tendency to self-aggregate, initially into soluble oligomers, and eventually into insoluble fibrils and large neuronal deposits. Although the soluble oligomers are considered the major culprit of neuronal toxicity, there is nevertheless strong interest in the structure of the Aβ fibrils. Aβ fibrils have been a longstanding subject of various biophysical studies, including cryoEM. Nevertheless, the cryoEM structure of Aβ42 fiber at 10-angstrom resolution as reported by Lee and colleagues represents a significant step forward in our pursuit of the structural basis of Aβ peptide fibrillization. The new structure reveals the expected two protofilaments twisted along the fiber axis. The novelty of the new structure is that the β-sheets are arranged at the periphery surrounding a hollow core, thus forming a long tube-like structure. This architecture is drastically different from the fiber structure formed by Aβ40 peptide, also determined by cryoEM, in Niko Grigorieff’s lab at Brandeis University, Waltham, Massachusetts, and reported previously (Meinhardt et al., 2009; Sachse et al., 2008). In the Aβ40 fiber, the β-sheets are arranged radially, twisting along the helical axis to form the long fiber with a solid core. As Aβ fibers are highly heterogeneous and polymorphic, it will be interesting to find out whether the structural differences observed in these studies merely reflects the peptide constituents (i.e., Aβ40 versus Aβ42) of the particular species of fibers selected for 3D reconstruction, or whether the structural differences represent a true defining feature of two functionally different peptides (i.e., Aβ40 is significantly less toxic than Aβ42).

The new cryoEM map by Zhang et al. fits the cryoEM micrograph well and appears solid. Furthermore, the structure model derived from the cryoEM map is supported by their extensive proteolysis data. Nevertheless, the interpretative model shall be taken with a grain of salt. Since the accurate mass per unit length of Aβ42 fiber is not known in this case, the display threshold for surface-rendering of the cryoEM map has to be somewhat artificial. The choice of threshold would thus have implications in building the structural model. I also want to point out that the proteolysis data, although supportive of their model, is not in conflict with a previous model that involves inter-β-sheets interaction (Sato et al., 2006). In summary, my impression is that there is a need for understanding the structural basis of Aβ peptide fibrillization. The current work might not be the final elucidation of such mechanism, but is a significant step forward in the long quest.

View all comments by Huilin Li

CryoEM-determined structures of Alzheimer’s peptide Aβ1-42 reveal significant differences between the fibrils of this peptide and the other most-studied Alzheimer’s peptide, Aβ1-40. Thus, they extend the known differences in kinetic, thermodynamic, and dynamic properties of these two peptides observed in solution to the supramolecular architecture of fibrils formed by them.

One of the significant points of this study is that fibrils formed by Aβ1-42 have a hollow core in contrast to those formed by Aβ1-40. At a cross-sectional plane, each protofilament accommodates a single molecule of Aβ1-42 in a hairpin-like conformation while two Aβ1-40 peptides are present in extended conformation in their respective fibrils. Structures of both fibrils were determined to the same resolution (~10 angstrom vs. ~8 angstrom); therefore, the differences can’t be attributed to the differences in experimental data collection.

However, fibril morphology is highly dependent on growth conditions. Under a variety of growth conditions, a different conformation from...  Read more

CryoEM-determined structures of Alzheimer’s peptide Aβ1-42 reveal significant differences between the fibrils of this peptide and the other most-studied Alzheimer’s peptide, Aβ1-40. Thus, they extend the known differences in kinetic, thermodynamic, and dynamic properties of these two peptides observed in solution to the supramolecular architecture of fibrils formed by them.

One of the significant points of this study is that fibrils formed by Aβ1-42 have a hollow core in contrast to those formed by Aβ1-40. At a cross-sectional plane, each protofilament accommodates a single molecule of Aβ1-42 in a hairpin-like conformation while two Aβ1-40 peptides are present in extended conformation in their respective fibrils. Structures of both fibrils were determined to the same resolution (~10 angstrom vs. ~8 angstrom); therefore, the differences can’t be attributed to the differences in experimental data collection.

However, fibril morphology is highly dependent on growth conditions. Under a variety of growth conditions, a different conformation from an ensemble of conformations may prevail under a given set of conditions for each peptide. Nevertheless, data shown in this work are consistent with the differences observed between the two peptides in solution studies. They remind us that we still don’t know the nature of molecular interactions that affect these two similar peptides such that they can behave so differently in solution leading to significantly different consequences.

One of the common points between the EM structures of both peptides is that both structures suggest protofilaments are joined through the flexible N-terminal residues of both peptides, which also agree with the solution studies. Thus, it is very likely that dynamic properties of these peptides (i.e., switching between various conformations and their thermodynamic consequences) play a significant role in determining how the individual peptides form the initial complex and extend it to a protofilament and fibril level. This would allow small differences to be amplified, yielding significant kinetic and structural differences in fibrils of the same peptide or between the fibrils of the two peptides.

View all comments by Engin Serpersu

The study by Pigino et al. study elegantly highlights a possible mechanism by which Aβ oligomers can influence axonal transport. Though the validity of intracellular Aβ is debatable in the context of human AD pathology, Pigino et al. convincingly show that in a simple model-system of axonal transport, nanomolar levels of Aβ can influence transport; they also provide convincing evidence for the involvement of a specific signaling cascade in this process. The paper is a must-read!

View all comments by Subhojit Roy

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

The work on murine models of tauopathies conducted by Clavaguera et al. has brought an intriguing view that fibrillar tau pathologies are intracranially transmittable from a single site affected by injected and possibly endogenous tau aggregates. The spreading of Gallyas-positive tau depositions was seemingly consequent to a chain reaction of fibrillogenesis consisting of either transgenically overproduced or endogenously expressed wild-type tau proteins, while the injected brain extracts from transgenic mice expressing the FTDP-17 P301S mutant tau only gave the initial seed for this surge of tangles. Since a PBS-soluble fraction of the extract did not induce overt changes in tau pathology, it is unlikely that monomeric foreign tau proteins convert the conformation of resident tau molecules from a flexible mode to a rigid, more amyloidogenic type, but insoluble tau assemblies preformed in the donor mice acted as seeds of massive inclusions. Pieces of these protein chunks might be axonally transported, and could be the secondary seeds at remote regions. Mechanisms by which alien...  Read more

The work on murine models of tauopathies conducted by Clavaguera et al. has brought an intriguing view that fibrillar tau pathologies are intracranially transmittable from a single site affected by injected and possibly endogenous tau aggregates. The spreading of Gallyas-positive tau depositions was seemingly consequent to a chain reaction of fibrillogenesis consisting of either transgenically overproduced or endogenously expressed wild-type tau proteins, while the injected brain extracts from transgenic mice expressing the FTDP-17 P301S mutant tau only gave the initial seed for this surge of tangles. Since a PBS-soluble fraction of the extract did not induce overt changes in tau pathology, it is unlikely that monomeric foreign tau proteins convert the conformation of resident tau molecules from a flexible mode to a rigid, more amyloidogenic type, but insoluble tau assemblies preformed in the donor mice acted as seeds of massive inclusions. Pieces of these protein chunks might be axonally transported, and could be the secondary seeds at remote regions. Mechanisms by which alien tau aggregates enter neuronal and glial cells to contact intracellular tau species are so far unknown, but one speculative possibility is that the injectants are endocytosed and attract autophagically sequestered resident tau proteins in endosomes or lysosomes. Such compartmentalization might explain the lack of neurodegenerative alterations in the recipient animals, despite the high abundance of abnormal fibrils. It is yet to be clarified whether such endosomal/lysosomal tau assemblies could grow into neurofibrillary tangles (NFTs) and coiled bodies without structural disruptions of these vesicles.

It should also be noted that all tau molecules seen in this experiment, mutant and wild-type, transgenic and endogenous, were four-repeat tau isoforms (4RTs) bearing exon 10, as only 4RTs are endogenously expressed in adult rodents. Although 4RT filaments could be a seed of exclusive 4RT inclusions via a selective incorporation of 4RT into fibrils, this notion needs to be examined in the same transgenic strain receiving an injection of postmortem brain extracts from tauopathy patients with 4RT-dominant (e.g., progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease), or three-repeat tau isoform- (3RT-) dominant (e.g., Pick disease) pathologies. Alternatively, transgenic mice generated with the use of the genomic human tau DNA, which expresses all six human tau isoforms besides endogenous 4RTs (1,2), could be treated with these extracts to assess whether exogenous insoluble 4RT and 3RT yield their replicas in living brains. Likewise, the formation of NFTs constituted of all six tau isoforms would be experimentally generated by injecting homogenates of Alzheimer disease brains into the genomic tau transgenic mice. An additional assumption to be tested in these analyses is that oligodendrocytes prefer 4RT aggregation to 3RT+4RT fibrils, resulting in the lack of prominent glial tau pathologies as in Alzheimer disease and several other tauopathies with six insolubilized tau isoforms (FTDP-17s with the G272V, V337M, and R406W tau mutations, diffuse neurofibrillary tangles with calcification, etc.).

The 4RT and 3RT aggregates are ultrastructurally identified as straight filaments (SFs) and/or twisted ribbons, and are distinguishable with paired helical filaments (PHFs) characteristic of Alzheimer disease type 3RT+4RT polymers. Moreover, many β-sheet-binding imaging agents show high affinities for Aβ plaques and/or NFTs in Alzheimer disease, but only a small subset of these compounds, as exemplified by X-34 analogs (3-5), are capable of binding to 4RT and 3RT inclusions. This might also support differential accessibilities of SFs and PHFs to intracellular components responsible for tau processing, eventually producing heterogeneous neuropathological phenotypes in tauopathies. X-34 and its derivatives are not optimal for in-vivo positron emission tomographic (PET) imaging, while the development of agents with faster kinetics more suitable for PET assays is ongoing. Longitudinal PET scans with these emerging radiotracers would provide four-dimensional maps of fibrillar tau pathologies spreading in the brain, which might be distinctive depending on the isoform composition of tau deposits. This technology, in conjunction with currently available high-resolution scanners (6), or more advanced future systems, would help researchers and clinicians to spot the site of the primal seeding. If tauopathies are not of multifocal origin, there would be a chance for local injections of genetic, immunological, and pharmacological erasers of the seeds and incipient pathologies. The therapeutic effects and “local recurrence” of the tau deposition after the treatment could also be monitored by the in-vivo imaging techniques.

References:
1. Duff K, Knight H, Refolo LM, et al. Characterization of pathology in transgenic mice over-expressing human genomic and cDNA tau transgenes. Neurobiol Dis 2000;7:87-98. Abstract

2. McMillan P, Korvatska E, Poorkaj P, et al. Tau isoform regulation is region- and cell-specific in mouse brain. J Comp Neurol 2008;511:788-803. Abstract

3. Schmidt ML, Schuck T, Sheridan S, et al. The fluorescent Congo red derivative, (trans, trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB), labels diverse beta-pleated sheet structures in postmortem human neurodegenerative disease brains. Am J Pathol 2001;159:937-943. Abstract

4. Velasco A, Fraser G, Delobel P, et al. Detection of filamentous tau inclusions by the fluorescent Congo red derivative FSB [(trans,trans)-1-fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene]. FEBS Lett 2008;582:901-906. Abstract

5. Higuchi M. Visualization of brain amyloid and microglial activation in mouse models of Alzheimer's disease. Curr Alzheimer Res 2009;6:137-143. Abstract

6. de Jong HW, van Velden FH, Kloet RW, et al. Performance evaluation of the ECAT HRRT: an LSO-LYSO double layer high resolution, high sensitivity scanner. Phys Med Biol 2007;52:1505-1526. Abstract

View all comments by Makoto Higuchi

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

Clavaguera, Tolnay, Goedert, and colleagues present a compelling argument for the exogenous induction and endogenous spread of tauopathy in rodent models. In these experiments, tauopathy was seeded de novo both in a transgenic mouse strain that normally does not generate filamentous tau, and even (to a lesser degree) in non-transgenic mice. Insoluble tau was the most potent seed, and in both murine host strains the tau filaments that developed consisted of host tau protein. Three key findings are that 1) tauopathy can be seeded within neurons in the living brain by an exogenous seed; 2) once initiated, tauopathy spreads from one brain region to another, possibly via a chain reaction of molecular corruption along with intracellular and intercellular trafficking; and 3) aggregated tau (like prions, Aβ, and probably other pathogenic proteins) may exist as polymorphic and polyfunctional strains, the pathogenicity of which is governed by the characteristics of the corruptive seed and of the host. The findings add to the evidence that disorders of protein...  Read more

Clavaguera, Tolnay, Goedert, and colleagues present a compelling argument for the exogenous induction and endogenous spread of tauopathy in rodent models. In these experiments, tauopathy was seeded de novo both in a transgenic mouse strain that normally does not generate filamentous tau, and even (to a lesser degree) in non-transgenic mice. Insoluble tau was the most potent seed, and in both murine host strains the tau filaments that developed consisted of host tau protein. Three key findings are that 1) tauopathy can be seeded within neurons in the living brain by an exogenous seed; 2) once initiated, tauopathy spreads from one brain region to another, possibly via a chain reaction of molecular corruption along with intracellular and intercellular trafficking; and 3) aggregated tau (like prions, Aβ, and probably other pathogenic proteins) may exist as polymorphic and polyfunctional strains, the pathogenicity of which is governed by the characteristics of the corruptive seed and of the host. The findings add to the evidence that disorders of protein aggregation may originate and amplify by a similar molecular mechanism, i.e., by the seeded corruption of endogenously produced molecules. While the various proteopathies may not be communicable in exactly the same sense as are the prionoses, the mechanistic similarities can be instructive, and argue yet again for a more integrative dialogue among researchers studying a wide spectrum of deceptively dissimilar diseases.

View all comments by Lary Walker

Propagation and Prion-like Spreading of Proteins in Common Neurodegenerative Disorders; New Perspectives Emerging From Tau and Synuclein
Many major neurodegenerative diseases are characterized by protein aggregation and deposition in specific regions of brain. This protein pathology generally occurs in discrete regions of brain but eventually spreads into much larger areas (1,2). Several recent studies propose a prion-like, templated aggregation hypothesis regarding the mechanism underlying this propagation of disease-specific protein aggregation (3-5). The most recent report supporting this hypothesis has come from the work by Goedert, Tolnay, and their colleagues, who studied the propagation of tauopathy in transgenic mouse brain (6). In this study, they injected the brain extract of P301S tau transgenic mouse, which has filamentous tau aggregates, into the hippocampus and cerebral cortex of ALZ17, a transgenic line overexpressing wild type tau protein, and examined the spread of tau pathology over time. They found the spread of tauopathy not only within the...  Read more

Propagation and Prion-like Spreading of Proteins in Common Neurodegenerative Disorders; New Perspectives Emerging From Tau and Synuclein
Many major neurodegenerative diseases are characterized by protein aggregation and deposition in specific regions of brain. This protein pathology generally occurs in discrete regions of brain but eventually spreads into much larger areas (1,2). Several recent studies propose a prion-like, templated aggregation hypothesis regarding the mechanism underlying this propagation of disease-specific protein aggregation (3-5). The most recent report supporting this hypothesis has come from the work by Goedert, Tolnay, and their colleagues, who studied the propagation of tauopathy in transgenic mouse brain (6). In this study, they injected the brain extract of P301S tau transgenic mouse, which has filamentous tau aggregates, into the hippocampus and cerebral cortex of ALZ17, a transgenic line overexpressing wild type tau protein, and examined the spread of tau pathology over time. They found the spread of tauopathy not only within the injection sites but also in neighboring brain regions, with the severity diminishing as the distance from the injection site increases. Both neuropil threads and neurofibrillary tangles were found, as well as oligodendroglial coiled bodies, and the lesions increased with time up to 15 months post injection. When the P301S brain extract was injected to non-transgenic mouse, the aggregation of mouse endogenous tau was induced, but this mouse tau aggregation was confined near the injection site and temporal progression was not observed between six and 12 months. Whether this is due to the species difference or related more to expression levels remains to be determined.

This prion-like spread of protein pathology has also been shown for amyloid-beta aggregates in a transgenic mouse model (5). However, the tau study is particularly striking as this protein and its aggregates have been believed to reside within the cytoplasm. Earlier this year, tissue culture studies with mutant huntingtin fragments and tau proposed aggregate spreading through cell-to-cell transmission of misfolded proteins (7,8). More recently, our laboratories demonstrated the propagation of a-synuclein aggregates through cell-to-cell transmission in cultured cells and transgenic mouse models (9). This transmission apparently involves the release of a-synuclein aggregates from neuronal cells under stresses and subsequent endocytosis by neighboring cells. This interneuronal transmission of a-synuclein may account for the recent reports showing the spread of Lewy inclusions from host tissues to long-term fetal cell grafts in Parkinson’s patients and may be the underlying mechanism for the sequential progression of the brain stem-originated Lewy pathology in PD, proposed by Braak and colleagues. Therefore, this prion-like propagation of pathological aggregation may be the general underlying principle for the progressive deterioration of myriad neurodegenerative diseases associated with protein misfolding.

There are several outstanding questions. First, given the cytosolic localization of tau and other disease-linked proteins, mechanisms must exist by which these proteins are released from neurons and gain access to the cytoplasm of neighboring cells. Second, the propagation of tau aggregates was not accompanied by other pathological changes, such as neuronal loss, gliosis, inflammation, and axonal damage: The relationship between aggregate spreading and other degenerative changes has to be elucidated. Third, it needs to be addressed which molecular species are responsible for aggregate propagation and whether these species are identical to the species responsible for neurotoxicity. A related issue is whether the aggregate propagation mechanism involves co-factors cooperating with the aggregate itself. Resolution of these questions may open up opportunities for novel therapeutic and diagnostic strategies for major neurodegenerative diseases.

References:
1. Braak H and Braak E (1991) Neuropathological staging of Alzheimer-related changes. (Translated from eng) Acta Neuropathol 82(4):239-259. Abstract

2. Braak H, et al. (2003) Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging 24(2):197-211. Abstract

3. Kordower JH, Chu Y, Hauser RA, Freeman TB, and Olanow CW (2008) Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease. Nat Med 14(5):504-506. Abstract

4. Li JY, et al. (2008) Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation. Nat Med 14(5):501-503. Abstract

5. Meyer-Luehmann M, et al. (2006) Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. Science 313(5794):1781-1784. Abstract

6. Clavaguera F, et al. (2009) Transmission and spreading of tauopathy in transgenic mouse brain. (Translated from Eng) Nat Cell Biol. Abstract

7. Frost B, Jacks RL, and Diamond MI (2009) Propagation of tau misfolding from the outside to the inside of a cell. (Translated from eng) J Biol Chem 284(19):12845-12852. Abstract

8. Ren PH, et al. (2009) Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates. (Translated from eng) Nat Cell Biol 11(2):219-225. Abstract

9. Desplats P, et al. (2009) Inclusion formation and neuronal cell death through neuron-to-neuron transmission of a-synuclein. Proc. Nat. Acad. Sci. USA, in press.

View all comments by Seung-Jae Lee
View all comments by Eliezer Masliah

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

This paper seems to be interesting, revealing an absolute requirement for intracellular delivery of the fibrillated alpha-synuclein to induce Lewy-body like inclusions. The cell-to-cell communication requires intracellular seeding, which is, however, revealing a pattern similar to prion proteins. Hence the question arises whether alpha-synuclein acts like a prion.

View all comments by Bharathi Mahadevaiah

This may be a naive question, but if amyloid deposition in the brain is a critical factor in AD-related behavioral sequelae, why is it so difficult to induce a behavioral modification of statistical relevance following Aβ vaccination, since reports show a strong amyloid plaque clearance effect?

View all comments by Elliott Mufson

I think another model for this kind of study (after parabiotics and vampires) could be pregnant mice. The placental barrier between mother and fetus highly leaky, allowing the passage of, for instance, maternal antibodies (mainly IgG). It seems to me that there is a general observation that the maternal organism appears 'rejuvenated' during pregnancy.

View all comments by Ivan Goussakov

It's of interest that mRNA levels of the calcineurin inhibitor, DSCR1, are also much higher in AD brain (1). The recent study be Lee and colleagues finds that DSCR1 interacts with Tollip and positively modulates IL-1R signalling (2). Tollip is an IRAK-1 inhibitor. This would seem to suggest problems with TLR2/TLR4 signalling in AD. This is supported by the Landreth study finding that CD14 and TLR2 and TLR4 bind Aβ to stimulate microglial activation (3). The KEGG link is below for the TOLL RECEPTOR signaling pathway (4).

References:
1. Ermak G, Morgan TE, Davies KJ. Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer's disease. J Biol Chem. 2001 Oct 19;276(42):38787-94. Abstract

2. Lee JY, Lee HJ, Lee EJ, Jang SH, Kim H, Yoon JH, Chung KC. Down syndrome candidate region-1 protein interacts with Tollip and positively modulates interleukin-1 receptor-mediated signaling. Biochim Biophys Acta. 2009 Dec;1790(12):1673-80. Abstract

3. Reed-Geaghan EG, Savage JC, Hise AG, Landreth GE. CD14 and toll-like receptors 2 and 4 are required for fibrillar A{beta}-stimulated microglial activation. J Neurosci. 2009 Sep 23;29(38):11982-92. Abstract

4. Toll-like receptor signaling pathway—Homo sapiens (human)

View all comments by Mary Reid

I'd like to post a correction to a mistake in this article: the difference in amino acid sequence of alpha-synuclein between human and rat/mouse is 7, not 5. These include A53T, S87N, L100M, N103G, A107Y, D121S/G, and N122S. The sequences were from GenBank: human, NP000336.1; rat, NP062042.1; mice, NP033247.1

View all comments by Junchao Tong

I have never found the concept of mimicking phosphoserine with glutamate convincing. Glutamate lacks an oxygen and cannot have a doubly negative charge, unlike phosphoserine. Aspartate, which is often also used to replace phosphoserine, is even worse, as it is one bond shorter. If the protein or peptide is short enough, it can be synthesized using a phosphorylated amino acid.

View all comments by Andrew Doig