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Still Early Days for α-synuclein Fluid Marker
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This is Part 6 of a nine-part series. See also Part 1, Part 2, Part 3, Part 4, Part 5, Part 7, Part 8, Part 9.
4 June 2009. In Alzheimer disease research, well over a decade of intense research into fluid biomarker candidates has reached a point where a so-called “pathological signature” of amyloid-β and tau proteins is beginning to emerge from the 59-center Alzheimer’s Disease Neuroimaging Study (Shaw et al., 2009). This signature validates on a larger scale a number of earlier studies that had shown essentially the same thing (Fagan et al., 2007; Fagan et al., 2006; Li et al., 2007; Hansson et al., 2006; Welge et al., 2009). Coming, as it did, with ups and downs along the way, this search for a fluid test is guiding researchers who are working to develop similar markers for α-synuclein and progranulin, two major proteins involved in many of the overlapping forms of dementia at issue in earlier parts of this news series. The 9th International Conference AD/PD, held last March in the Czech capital city of Prague, as well as an immediately preceding workshop on dementia with Lewy bodies (DLB) and Parkinson disease dementia (PDD) in Germany, showcased a rapidly growing field of groups who are racing to broaden the field. Here is a selection.
First, α-synuclein (for progranulin, see Part 7). The three investigators who started fluid-based markers on this intraneuronal protein are Michael Schlossmacher, now at the University of Ottawa, Canada, Brit Mollenhauer, now at Paracelsus-Elena-Klinik in Kassel, who worked in Schlossmacher’s former lab at Brigham and Women’s Hospital in Boston, and Omar El-Agnaf, now at United Arab Emirates University in Al Ain, UAE. All three collaborated extensively, first to build ELISA assays and to show that these assays can quantify α-synuclein in normal human cerebrospinal fluid (CSF), then to show that the CSF concentration of this protein normally declines with age and declines even further with Parkinson disease (Tokuda et al., 2006). A first cross-sectional study compared CSF α-synuclein concentration in various patient groups, i.e., AD, DLB, PD, multiple system atrophy (MSA), and Creutzfeldt-Jakob disease (CJD) with controls. These studies found the lowest levels in PD and MSA, whereas AD and controls had similar and higher levels, and DLB lay in between. In CJD, α-synuclein was curiously elevated, perhaps because the rapid cell death in this condition dumps this protein into the CSF so that it serves as a marker of degeneration in this situation, much as tau is viewed in AD (Mollenhauer et al., 2008).
However, the same difficulty that has dogged CSF measurements of Aβ since the beginning (e.g., Seubert et al., 1992) quickly caught up with α-synuclein, too. Its concentration varies greatly from person to person, creating enough overlap that the test in its original form is unable to distinguish which group a given person falls into. While as a group, the values of people with PD always cluster at the bottom, any given person with PD might have a value higher than a control or an AD patient. Moreover, other research groups, using their own, different ELISAs, have been unable so far to replicate Mollenhauer and colleagues’ result, calling into question whether α-synuclein can serve as a robust diagnostic marker to distinguish between overlapping diseases (Ohrfelt et al., 2009; Spies et al. 2009; Noguchi-Shinohara et al., 2009). In Prague, debate centered on the different assays and antibodies different groups are using to measure α-synuclein. Schlossmacher noted that his and collaborators’ ELISA is extensively validated. Other scientists agreed that before a final word can be spoken, more tests in additional patient cohorts, independent replication, a comparison of methods, and exchange of antibodies are needed.
“Right now, many groups are trying to measure CSF α-synuclein and are having a hard time seeing good separation between the groups. We, too, see a very narrow range of values,” said James Galvin of Washington University, St. Louis, Missouri. “But that’s no reason to be discouraged. It may just take more standardization of the steps and the right tools to get it down.”
In Kassel, Mollenhauer presented new data on total CSF α-synuclein measured in a separate cross-sectional cohort of clinically diagnosed patients. Again, PD and MSA lay at the bottom, AD and controls at the top, DLB in between. The overlap remained extensive, though expressing α-synuclein concentration relative to total protein teased the groups apart somewhat. In Prague, Mollenhauer’s poster of a separate series of 41 autopsy-confirmed cases showed that their CSF measurement matched the working diagnosis they had received during life.
On balance, then, the early days of α-synuclein biomarker research have made clear that this protein can be directly measured in the CSF (and peripheral blood; see ARF related news story), and, least with one assay, trends downward from controls to DLB and PD, Schlossmacher said. But besides technical collaboration to streamline protocols, much more scientific work remains to be done, he added. Challenges include understanding where exactly the CSF α-synuclein comes from (the brain, the periphery, the choroid plexus could all contribute), what different species of α-synuclein occur in CSF (truncated, full-length, or modified), and which one of those best indicates disease. To see how these species change in the same person over time, Schlossmacher’s and Mollenhauer’s groups have begun longitudinal studies.
Galvin foresees a future where academic centers interested in earlier-stage clinical trials use a CSF assay for α-synuclein to distinguish preclinical AD from preclinical DLB. An α-synuclein imaging ligand is not on the horizon (see Part 5 of this series), but amyloid imaging is available and it shows a large fraction of non-demented elderly people who have brain amyloid and may turn out to be presymptomatic for either AD or DLB. Most DLB cases share Aβ and α-synuclein pathology; hence, an α-synuclein fluid assay could conceivably flag amyloid-positive people who are at high risk for future DLB, much like combining amyloid imaging with CSF Aβ/tau measurement is predicting who will develop AD symptoms. Other groups are drilling deeper with Aβ biochemistry, measuring some of its truncated and oxidized forms to distinguish between AD and DLB (Bibl et al., 2006; Bibl et al., 2007).
“In our studies, we already have a number of people who are PIB positive and are not demented, but when you look at them with some of the biomarkers we are developing, some of these people are clustering with the DLB group. The idea is to be able to diagnose preclinical DLB,” Galvin said. The Kassel meeting ended with the designation of a working group to hammer out a research path toward that goal, Mollenhauer wrote to ARF.
For his part, El-Agnaf has focused on measuring oligomers of α-synuclein, initially in plasma (El-Agnaf et al., 2006) and more recently in brain extracts. In Prague, he showed the results of a study looking for such oligomers in lysates prepared from postmortem brains of people who had suffered from DLB. As measured by a sandwich ELISA El-Agnaf developed with a commercial antibody that recognizes α-synuclein aggregates but not monomers, these brains contained far higher concentrations of α-synuclein oligomers than control or AD brains. The data showed less overlap between the groups, but no clear separation, either (Paleologou et al., 2009). Since then, the researchers used their ELISA on CSF samples and again found high levels. A final cohort of 60 samples from people with PD and controls displayed, again, a group difference but also a spread of the individual data points and overlap between the groups. Calculating the ratio of oligomeric α-synuclein to total α-synuclein improved the separation, El-Agnaf noted. “This is the first time we have been able to detect soluble oligomers from CSF in humans,” El-Agnaf said in Prague, and here, too, the work of replication and broadening the effort is only just beginning.
Meanwhile, research underpinning the rationale for going after oligomeric α-synuclein in body fluids is advancing in parallel. Here, too, Prague offered some news. For example, in the last talk of the AD/PD conference, Kostas Vekrellis of the Biomedical Research Foundation Academy of Athens, Greece, reported that secreted α-synuclein oligomers are up to no good. Even though α-synuclein is primarily a cytosolic protein, scientists know that cultured cells can release it. Cells also can take up external α-synuclein, usually at their peril as they tend to die soon after, Vekrellis said.
Vekrellis investigated this apparent toxicity with lines of human neuroblastoma cells that can be induced to express wild-type α-synuclein. Soluble monomeric and oligomeric α-synuclein showed up in the conditioned medium from these cells. Using liquid chromatography-mass spectrometry proteomics and electron microscopy, Vekrellis and colleagues showed that the cells actively export α-synuclein via an exosome pathway that itself depends on intracellular calcium. These cells sustained no harm from the α-synuclein. But their conditioned medium, when squirted onto primary rat cortical neurons, killed those cells. A high-molecular-weight fraction of α-synuclein species proved toxic. Medium depleted of α-synuclein, or medium subjected to oligomer-busting compounds such as scylloinositol did not, pointing to oligomers as the active component. This new data invoke parallels with amyloid-β oligomers, which have been shown to impair synaptic activity and to damage cells, and whose sensitivity to scylloinositol has led to a Phase 2 trial. For more on oligomers in mixed disease, see Part 4 of this series.—Gabrielle Strobel.
This is Part 6 of a nine-part series. See also Part 1, Part 2, Part 3, Part 4, Part 5, Part 7, Part 8, Part 9.
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Comments on Related Papers |
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Related Paper: Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival.
Comment by: Lary Walker, ARF Advisor
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Submitted 21 May 2010
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Posted 21 May 2010
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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...
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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 |
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Related Paper: Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival.
Comment by: Lawrence Rajendran
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Submitted 3 June 2010
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Posted 3 June 2010
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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...
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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
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Related Paper: Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival.
Comment by: Evangelia Emmanouilidou
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Submitted 9 June 2010
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Posted 10 June 2010
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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...
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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 |
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Related Paper: Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival.
Comment by: Felix Hernandez
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Submitted 11 June 2010
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Posted 13 June 2010
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 I recommend this paper
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 |
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Related Paper: Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival.
Comment by: Tim West
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Submitted 14 June 2010
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Posted 15 June 2010
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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...
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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 |
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Related Paper: Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival.
Comment by: Kostas Vekrellis
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Submitted 16 June 2010
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Posted 16 June 2010
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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...
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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
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Related Paper: Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival.
Comment by: Rudolf Bloechl
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Submitted 16 June 2010
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Posted 16 June 2010
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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 |
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Related News: SfN: Early Steps toward Parkinson Disease Blood Test
Comment by: Omar El-Agnaf
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Submitted 29 December 2005
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Posted 30 December 2005
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α-Synuclein (α-syn) is mainly expressed by neuronal cells, and is generally considered to exist as a cytoplasmic protein. However, our recent studies have shown that neuronal cells in culture constitutively secrete α-syn into the culture medium, and that α-syn is normally present in CSF and peripheral plasma (El-Agnaf et al., 2003a). Our findings suggest that cells normally secrete α-syn into their surrounding media, both in vitro and in vivo. Future cell biological research will have to address the mechanism by which α-syn protein can be found in the extracellular space under physiological conditions (El-Agnaf et al., 2003a; Lee et al., 2005), since no alternative splice variant has been published to date that would direct the nascent α-syn protein into and through the secretary pathway, and no such transcript of the SNCA gene could be found in an extensive investigation of primate brain specimens (M.G. Schlossmacher, unpublished data). We have suggested that the detection of extracellular α-syn and/or its modified forms in body...
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α-Synuclein (α-syn) is mainly expressed by neuronal cells, and is generally considered to exist as a cytoplasmic protein. However, our recent studies have shown that neuronal cells in culture constitutively secrete α-syn into the culture medium, and that α-syn is normally present in CSF and peripheral plasma (El-Agnaf et al., 2003a). Our findings suggest that cells normally secrete α-syn into their surrounding media, both in vitro and in vivo. Future cell biological research will have to address the mechanism by which α-syn protein can be found in the extracellular space under physiological conditions (El-Agnaf et al., 2003a; Lee et al., 2005), since no alternative splice variant has been published to date that would direct the nascent α-syn protein into and through the secretary pathway, and no such transcript of the SNCA gene could be found in an extensive investigation of primate brain specimens (M.G. Schlossmacher, unpublished data). We have suggested that the detection of extracellular α-syn and/or its modified forms in body fluids, particularly in peripheral plasma, offers new opportunities for the development of diagnostic tests for PD and related diseases (El-Agnaf et al., 2003a).
The mounting evidence for soluble oligomers being the pathogenetic species that drive neurodegeneration and neuronal cell death, led us to hypothesize that the detection of soluble oligomers of α-syn in biological fluids could have potential use as a biomarker for PD and related diseases (El-Agnaf et al., 2003b). Therefore, we developed a simple and novel ELISA method that specifically recognizes only oligomeric species of α-syn. We employed this method to probe for oligomeric forms of α-syn in human CSF and in plasma. Interestingly, based on our preliminary results, the ELISA was able to detect α-syn oligomers in post mortem CSF from some PD and DLB patients, but, in contrast, only a very low signal was obtained from all control samples tested. These data suggested a higher amount of α-syn oligomer production in PD patients either in vivo or during post-mortem autolysis (El-Agnaf et al., 2005). These promising results led us to carry out a more extensive study on the more accessible peripheral blood plasma. We found that there was a highly statistically significant difference between PD samples and controls, with most of the PD samples giving high signals, whereas only a few control samples gave a high signal (El-Agnaf et al., 2005).
If α-syn oligomerization occurs before the death of nigral neurons in PD, then our ELISA could potentially provide a diagnostic tool for the detection of oligomers in the early stages of the disease. This could lead to earlier detection and neuroprotective treatment intervention for high-risk subjects in the future. More extensive clinical studies will be required to confirm and extend our results and to validate the ELISA as a potential diagnostic test for disease state. As a matter of fact, in collaboration with M.G. Schlossmacher we recently began enrollment of subjects in a prospective, case-control study of 300 persons to be conducted over two years to monitor their oligomeric and total α-syn load in peripheral plasma, this study is funded by Michael J. Fox Foundation. Furthermore, it will be interesting to determine if there is any correlation between the detection of α-syn oligomers and the severity and/or the stage of the disease and/or the rate of its progression. Studies performed on blood samples from familial PD cases will be useful to further validate the ELISA as an early diagnostic method. We also recognize that any medication including dopaminergic replacement therapy, taken by previously diagnosed PD patients could influence the ELISA results. Our novel ELISA can also be used for high-throughput screening for modulators of α-syn oligomerization as potential novel drugs for PD and related disorders during preclinical validation studies, prior to their evaluation in rodent or nonhuman primate models of PD (El-Agnaf et al., 2004). Moreover, the principle of our ELISA could be applied for the development of similar sensitive diagnostic tests for the presence of other forms of oligomeric protein aggregates, such as those found in AD (Aβ and tau) and the TSEs (PrP).
References: El-Agnaf, O. M. A., Salem, S. A., Paleologou, K. E., Cooper, L. J., Fullwood ,N. J., Gibson, M. J., Curran, M. D., Court, J. A., Mann, D. M. A., Ikeda, S. I., Cookson, M. R., Hardy, J., and Allsop, D. (2003a). α-Synuclein implicated in Parkinson’s disease is present in extracellular biological fluids, including human plasma. FASEB J 17, 1315-1317.
El-Agnaf, O. M. A., Walsh, D. M., and Allsop, D. (2003b). Soluble oligomers for the diagnosis of neurodegenerative diseases. Lancet (Neurol.); 2, 461-462.
El-Agnaf, O. M. A., Paleologou, K. E., Greer, B., Abogrein, A. M., King, J. E., Salem, S. A., Fullwood, N. J., Benson, F. E., Hewitt, R., Ford, K. J., Martin, F. L., Harriott, P., Cookson, M. R. and Allsop, D. (2004). A strategy for designing inhibitors of α-synuclein aggregation and toxicity as a novel treatment for Parkinson's disease and related disorders. FASEB J. 18, 315-317.
El-Agnaf, O. M. A., Salem, S. A., Paleologou, K. E., Curran, M.D., Gibson, M. J., Court, J. A., Michael, S. G. and Allsop, D. (2005). Detection of oligomeric forms of α-synuclein protein in cerebrospinal fluid and plasma as a potential biomarker for Parkinson’s disease. FASEB J. (In press).
Lee, H. J., Patel, S. And Lee, S. J. (2005). Intravesicular localization and exocytosis of alpha-synuclein and its aggregates. J Neurosci; 25:6016-24. View all comments by Omar El-Agnaf |
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Related News: More Than Gaucher’s—GBA Throws Its Weight Around Lewy Body Disease
Comment by: J. Lucy Boyd
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Submitted 9 June 2009
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Posted 9 June 2009
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Related News: Neither Fish Nor Fowl—Dementia With Lewy Bodies Often Missed
Comment by: david Gardiner
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Submitted 10 June 2009
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Posted 11 June 2009
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Six months after my wife's first symptoms of cognitive impairment were noted in late September 2005, she was diagnosed with DLB in March 2006. The process was orderly and took time mainly to go from her primary physician to his referral to a neurologist to her referral to a neuropsychiatrist for a second, more knowledgable diagnosis. We first heard the term Lewy Body Disease from the third doctor at our second visit to her.
For the first two years her progression was gradual and three medications seemed to help control it: alprazolam for her anxiety, then namenda for confusion, then depakote for memory and confusion. In the last year, the disease has progressed more rapidly. She has become bedridden within the last three months, her hallucinations have returned, her confusion about where she is and who I am have become more persistent. We can still talk, but her vision is very poor, exacerbated by her keratoconus which has led to five corneal transplants since 1978. Her ability to manage her body is very limited, though her vital signs are strong and her appetite, her...
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Six months after my wife's first symptoms of cognitive impairment were noted in late September 2005, she was diagnosed with DLB in March 2006. The process was orderly and took time mainly to go from her primary physician to his referral to a neurologist to her referral to a neuropsychiatrist for a second, more knowledgable diagnosis. We first heard the term Lewy Body Disease from the third doctor at our second visit to her.
For the first two years her progression was gradual and three medications seemed to help control it: alprazolam for her anxiety, then namenda for confusion, then depakote for memory and confusion. In the last year, the disease has progressed more rapidly. She has become bedridden within the last three months, her hallucinations have returned, her confusion about where she is and who I am have become more persistent. We can still talk, but her vision is very poor, exacerbated by her keratoconus which has led to five corneal transplants since 1978. Her ability to manage her body is very limited, though her vital signs are strong and her appetite, her weight, and digestive system are fairly consistent. I want to know more and eventually to help publicize DLB.
View all comments by david Gardiner |
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