This elegant study by Scott et al. takes advantage of primary neuron cultures from α-synuclein-GFP-transgenic mice to examine the effects of modest α-synuclein overexpression on presynaptic proteins. They find convincing evidence that α-synuclein can diminish levels of several critical presynaptic proteins involved in exocytosis and endocytosis. The authors also detect significant reductions in miniEPSC frequency, diminished presynaptic exocytosis, and altered vesicle size by EM in α-synuclein-overexpressing neurons. Thus, physiologically relevant increases in α-synuclein produce robust functional consequences that closely mimic those observed in animal models of endocytic protein deficiency.

The authors point out that similar effects on presynaptic proteins have recently been shown following Aβ oligomer exposure (Parodi et al., 2010), suggesting a possible common mechanism of synaptic dysfunction between AD and synucleinopathies. It is intriguing to speculate that this potential shared mechanism of synaptic dysfunction may play...  Read more

This elegant study by Scott et al. takes advantage of primary neuron cultures from α-synuclein-GFP-transgenic mice to examine the effects of modest α-synuclein overexpression on presynaptic proteins. They find convincing evidence that α-synuclein can diminish levels of several critical presynaptic proteins involved in exocytosis and endocytosis. The authors also detect significant reductions in miniEPSC frequency, diminished presynaptic exocytosis, and altered vesicle size by EM in α-synuclein-overexpressing neurons. Thus, physiologically relevant increases in α-synuclein produce robust functional consequences that closely mimic those observed in animal models of endocytic protein deficiency.

The authors point out that similar effects on presynaptic proteins have recently been shown following Aβ oligomer exposure (Parodi et al., 2010), suggesting a possible common mechanism of synaptic dysfunction between AD and synucleinopathies. It is intriguing to speculate that this potential shared mechanism of synaptic dysfunction may play a role in the acceleration of cognitive decline and aggressive disease course in patients and transgenic mice that co-exhibit both AD and Lewy body pathologies (Olchney et al., 1998; Clinton et al., 2010).

View all comments by Mathew Blurton-Jones

Our goal in this study was to try connecting the dots between two key pathologic events: modestly elevated α-synuclein levels within the neuron and the ultimate synaptic dysfunction. We used a cell-biological approach that allowed us to analyze and quantify thousands of α-synuclein overexpressing boutons. Based on the data, we suggest a cascade of pathologic events initiated by modest elevations of α-synuclein and culminating in synaptic damage. Studies by Nemani et al. focus on the effects of elevated α-synuclein on specific steps within the synaptic release/recycling machinery by directly imaging the synaptic cycle in α-synuclein transfected neurons.

First, it is important to emphasize that using a variety of methods, both studies show at a single-neuron level that the overall synaptic defect induced by modestly elevated α-synuclein is an inhibition of neurotransmitter release. Thus, collectively, these studies provide a firm pathologic role that can be attributed to α-synuclein overexpression. The studies by Nemani et al. also show a dose-dependent effect of excessive...  Read more

Our goal in this study was to try connecting the dots between two key pathologic events: modestly elevated α-synuclein levels within the neuron and the ultimate synaptic dysfunction. We used a cell-biological approach that allowed us to analyze and quantify thousands of α-synuclein overexpressing boutons. Based on the data, we suggest a cascade of pathologic events initiated by modest elevations of α-synuclein and culminating in synaptic damage. Studies by Nemani et al. focus on the effects of elevated α-synuclein on specific steps within the synaptic release/recycling machinery by directly imaging the synaptic cycle in α-synuclein transfected neurons.

First, it is important to emphasize that using a variety of methods, both studies show at a single-neuron level that the overall synaptic defect induced by modestly elevated α-synuclein is an inhibition of neurotransmitter release. Thus, collectively, these studies provide a firm pathologic role that can be attributed to α-synuclein overexpression. The studies by Nemani et al. also show a dose-dependent effect of excessive α-synuclein, and strongly implicate the N-terminus of α-synuclein in the pathogenesis. However, while Nemani et al. posit an exclusive impairment of “reclustering” of synaptic vesicles as the solitary defect induced by excessive α-synuclein upon the presynaptic apparatus, our studies suggest that that the effects of elevated α-synuclein on the synaptic apparatus are diverse, including defects in pathways involved in both endo- and exocytosis.

The experiments by Nemani et al. are direct and convincing, and it is possible that a “reclustering” defect is a major pathology induced by excessive α-synuclein, with other minor defects on other aspects of the synaptic machinery as well. As relative newcomers to the field, we have no favorite hypothesis on how α-synuclein causes inhibition of neurotransmitter release. However, given our data and other α-synuclein studies in yeast and mice, and the well-known pleiotropic effects of other well-studied proteins implicated in neurodegeneration (tau, amyloid), we favor the view that α-synuclein has diverse effects on the synaptic release apparatus.

View all comments by Subhojit Roy

The background for our work is that α-synuclein normally localizes to the axon terminal of essentially all neurons, but its role, if any, in neurotransmitter release has remained very unclear. In general, knockout mice have shown either no effect or conflicting effects on synaptic transmission. Increased expression of α-synuclein causes Parkinson disease (PD)—duplication and triplication of the wild-type gene cause severe familial PD, and the protein accumulates in all sporadic PD. In light of this, we wondered what overexpression might do to synaptic transmission. This seemed particularly interesting because overexpression of wild-type α-synuclein in mice actually fails to produce degeneration, and an effect on transmitter release would be easier to interpret in the absence of toxicity.

To understand how α-synuclein affects neurotransmitter release, we used a combination of primary neuronal culture and genetic manipulation in mice. The reason is that, although culture is very powerful to dissect molecular mechanism, it suffers from greater variability and has more potential...  Read more

The background for our work is that α-synuclein normally localizes to the axon terminal of essentially all neurons, but its role, if any, in neurotransmitter release has remained very unclear. In general, knockout mice have shown either no effect or conflicting effects on synaptic transmission. Increased expression of α-synuclein causes Parkinson disease (PD)—duplication and triplication of the wild-type gene cause severe familial PD, and the protein accumulates in all sporadic PD. In light of this, we wondered what overexpression might do to synaptic transmission. This seemed particularly interesting because overexpression of wild-type α-synuclein in mice actually fails to produce degeneration, and an effect on transmitter release would be easier to interpret in the absence of toxicity.

To understand how α-synuclein affects neurotransmitter release, we used a combination of primary neuronal culture and genetic manipulation in mice. The reason is that, although culture is very powerful to dissect molecular mechanism, it suffers from greater variability and has more potential for artifact than analysis in vivo. By optical imaging, we found that α-synuclein specifically inhibits synaptic vesicle exocytosis, in particular, the extent of exocytosis rather than the rate, with no effect on synaptic vesicle endocytosis. We used a variety of experimental approaches (FM dyes, pHluorin reporter) to document a selective reduction in the size of the synaptic vesicle recycling pool, with no change in the total number of synaptic vesicles. Since previous work has suggested differences between dopamine and other neurons, we also used the primary culture to compare midbrain dopamine neurons with hippocampal glutamate neurons, and found that α-synuclein inhibits transmitter release in both populations.

In collaboration with our lab neighbor Roger Nicoll, we found that hippocampal slices prepared acutely from transgenic mice also show a reduction in transmitter release, establishing the physiological relevance of our findings in culture. Further, we used the culture system to show that the effect on release requires the N-terminal membrane-binding domain of α-synuclein.

The paper by Roy and colleagues also shows that α-synuclein inhibits transmitter release. The effect on synaptic vesicle exocytosis appears different from what we observed, and they also find a variety of other changes that we did not. We observed no effect of α-synuclein on the rate of synaptic vesicle exocytosis; the Roy paper uses FM dyes to suggest an effect on the rate but does not corroborate this with other methods. Surprisingly, Roy et al. use transgenic mice to produce the cultures that serve as the basis for their experiments, but perform little analysis using the mice themselves to corroborate their in-vitro findings in vivo. Rather, they rely on the cultures to pursue biochemical and ultrastructural analysis, finding 1) a major reduction in all synaptic vesicle proteins, including some synaptic boutons without synaptic vesicle proteins that they term “vacant boutons,” although it is a bit difficult to call anything a bouton if there are no synaptic vesicles in it; and 2) gross changes in presynaptic ultrastructure by EM.

It is true that culture can be very helpful to elucidate changes at the level of individual boutons. Yet our transgenic mice express α-synuclein in essentially all neurons, so we should also have seen a reduction in synaptic vesicle proteins if there were any. Furthermore, we did observe a reduction in the synapsins, but not other synaptic vesicle proteins, demonstrating that we could detect a biochemical effect of the transgene. We also looked at the transgenic mice by electron microscopy and found a much more specific effect of α-synuclein overexpression—a dispersion of synaptic vesicles away from the release site.

Overall, the Roy paper attributes the effect of α-synuclein on synaptic transmission to “upstream events,” but also states that the effects are pleiotropic and hence suggestive of toxicity. In contrast, we found very selective effects on the transmitter release mechanism and little evidence of toxicity. So the debate comes down to a basic question: Does α-synuclein overexpression produce changes through a gain in the normal function of the protein (as we suggest) or through the gain of an abnormal function (as suggested by Roy et al.)? This is a profound question with direct relevance for the pathogenesis of PD. It is always difficult to tell whether the toxicity observed is relevant to the disease, or just some other kind of injury. That is why we prefer to study a system with selective rather than general effects.

In the end, the question of normal function will be settled through the analysis of α-synuclein knockout mice. At this point, only double-knockout mice have been reported, but triple-knockouts lacking all the isoforms exist and are now being analyzed. If these animals show an increase in transmitter release, it would strongly suggest that the effects we observe involve a gain in the normal function of α-synuclein. Indeed, recent work using double-knockout mice has suggested an increase in dopamine release (Senior et al. 2008). Speaking in more practical terms, it will be difficult to elucidate the toxicity observed by Roy et al. since it is not clear where to begin. In contrast, we think that the selectivity of the defect we observe provides a clear starting point for future investigation.

In summary, both our and Roy's studies observe an effect of α-synuclein on synaptic vesicle exocytosis, but the details and the interpretation are quite different. The extent of our analysis in multiple experimental systems with a range of complementary methods suggests that the effect of α-synuclein is highly specific, and perhaps related to its normal role. However, this conclusion awaits further analysis of α-synuclein knockout mice, and identification of the mechanism by which α-synuclein acts to inhibit synaptic vesicle exocytosis. The identification of a specific effect also provides an entry point for future study of its role in disease as well as normal physiology.

View all comments by Robert Edwards

The work by Scott and colleagues is of great interest as it is trying to pinpoint the molecular details in the synaptic pathology caused by a modest transgenic overexpression of α-synuclein. The authors found that PK-resistant and abnormally phosphorylated α-synuclein tends to accumulate in dysfunctional boutons. They also elegantly demonstrated that such boutons display a gradual reduction in levels of certain endogenous presynaptic proteins. In an attempt to extend their findings to human disease, they looked for and confirmed similar alterations on a DLB brain section.

I think another transgenic model that moderately overexpresses another neuronal protein (e.g., APP) should have been looked at in parallel (to exclude that the effects seen are merely an effect of protein overproduction). Also, more human cases should have been included to verify that the observed differences are truly relevant to disease. Even so, the findings are intriguing, and the described model would be very useful to test effects of heat-shock proteins and other putative rescuing molecules. Moreover,...  Read more

The work by Scott and colleagues is of great interest as it is trying to pinpoint the molecular details in the synaptic pathology caused by a modest transgenic overexpression of α-synuclein. The authors found that PK-resistant and abnormally phosphorylated α-synuclein tends to accumulate in dysfunctional boutons. They also elegantly demonstrated that such boutons display a gradual reduction in levels of certain endogenous presynaptic proteins. In an attempt to extend their findings to human disease, they looked for and confirmed similar alterations on a DLB brain section.

I think another transgenic model that moderately overexpresses another neuronal protein (e.g., APP) should have been looked at in parallel (to exclude that the effects seen are merely an effect of protein overproduction). Also, more human cases should have been included to verify that the observed differences are truly relevant to disease. Even so, the findings are intriguing, and the described model would be very useful to test effects of heat-shock proteins and other putative rescuing molecules. Moreover, with the emergence of novel antibodies and other detection tools, it will be interesting to relate cellular effects and the distribution of monomeric, oligomeric, and fibrillar α-synuclein accumulating presynaptically and in other parts of the affected neuron.

Effects on neurotransmission by α-synuclein were the subject of study in the paper by Nemani et al. Also here, a modest increase in the intraneuronal levels of α-synuclein was sufficient to cause aberration. More specifically, the N-terminal domain of α-synuclein seems to be critical in mediating an impairment in transmitter release. However, contrary to the study by Roy, this paper did not find any corresponding changes in vesicle numbers or presence of presynaptic proteins (although the experimental set-up was different and the two groups did not study the same proteins).

Taken together, the two studies demonstrate new and interesting aspects of synaptic pathology induced by deposition of pathological α-synuclein. Applying the insights from the study by Nemani et al. on the model presented by Scott et al., it would be of interest to see if the N-terminal part of α-synuclein is responsible for all the aspects described.

View all comments by Martin Ingelsson

Both papers present evidence that the pathophysiological mechanism in synucleinopathies is not neuronal cell death but a synaptic dysfunction; that is very interesting. With regard to the clinical symptoms in PD, (also PDD and DLB), the synaptic pathology is due to a decrease in neurotransmitter release. The two publications provide us with a link between α-synuclein overexpression and an impairment of vesicle turnover. With this approach, it might be possible to explain the clinical symptoms of PD. Both papers show that α-synuclein-related pathology is not restricted to dopaminergic neurons.

The conclusion to be drawn from the results of these papers is that PD and DLB research should move away from models of α-synuclein-related toxicity or cell death that can be achieved only by unphysiologically high overexpression of α-synuclein. Rather, research should concentrate on synaptic failure associated with moderately altered α-synuclein levels. The link to α-synuclein aggregation was only drawn in the Scott et al. paper.

View all comments by Walter J. Schulz-Schaeffer

α-synuclein and Synaptic Failure in PD
α-synuclein has been biochemically and genetically linked to sporadic and familial PD. Mutations or multiplications of the α-synuclein gene cause familial forms of PD (Polymeropoulos et al., 1997; Krüger et al., 1998; Singleton et al., 2003; Zarranz et al., 2004). The aberrant function of α-synuclein is not understood, although there is evidence that abnormal folding and aggregation may play a role and that the toxic α-synuclein species may be oligomeric intermediates. It has been shown that α-synuclein is highly enriched in presynaptic terminals. At this site, it could be acting as a modifier of synaptic vesicle recycling, dopamine storage, and release at nerve terminals. Recent work has also suggested a role for α-synuclein in SNARE-mediated exocytosis at the synapse. In this respect, the synaptic role of α-synuclein is based primarily on the...  Read more

α-synuclein and Synaptic Failure in PD
α-synuclein has been biochemically and genetically linked to sporadic and familial PD. Mutations or multiplications of the α-synuclein gene cause familial forms of PD (Polymeropoulos et al., 1997; Krüger et al., 1998; Singleton et al., 2003; Zarranz et al., 2004). The aberrant function of α-synuclein is not understood, although there is evidence that abnormal folding and aggregation may play a role and that the toxic α-synuclein species may be oligomeric intermediates. It has been shown that α-synuclein is highly enriched in presynaptic terminals. At this site, it could be acting as a modifier of synaptic vesicle recycling, dopamine storage, and release at nerve terminals. Recent work has also suggested a role for α-synuclein in SNARE-mediated exocytosis at the synapse. In this respect, the synaptic role of α-synuclein is based primarily on the results of knockout studies (Chandra et al., 2005; Larsen et al., 2006). Still, the effects of α-synuclein overexpression on synaptic function had not received much attention. The recent papers by Nemani et al., 2010, and Scott et al., 2010, clearly show that overexpression of full-length α-synuclein at mild levels leads to defects in the synapse, including altered exocytosis, suggesting that this is an early event in the pathogenesis of PD.

In the Nemani et al. study, the authors use a battery of elegant optical and physiological in vitro and in vivo experiments to show, for the first time, that a relatively short time of overexpression of α-synuclein leads to the fast appearance of presynaptic defects in glutamatergic hippocampal neurons and mesencephalic dopaminergic neurons.

What is exciting about this manuscript is that the reduction of neurotransmitter release occurs under only modest overexpression of α-synuclein and in the absence of any aggregation and toxicity. What is more, the effects appear to be dose-dependent. This suggests that the observed effects might reflect early pathologic events in the disease pathway. Detailed kinetic examination further demonstrated that the inhibition did not involve defects in vesicle fusion or a reduction in the number of transmitter-containing vesicles, but rather a specific defect in the synaptic vesicle recycling pathway by preventing the reclustering of vesicles after endocytosis. Interestingly, the effect was specific to membrane-associated mutations of α-synuclein and therefore to the N-terminus of the molecule.

Although the molecular pathway by which overexpression of α-synuclein inhibits synaptic vesicle reclustering was not deciphered in the study of Nemani et al., it is possible that synapsin downregulation observed in α-synuclein overexpressing cells may play a role. Still, a direct physical (inter)action of the proteins was not identified.

To address the question of α-synuclein expression and synapse physiology, and to understand the mechanisms by which α-synuclein expression affects synapse function, Scott et al. set out a series of rigorous quantitative cell biological experiments using a transgenic mouse model in which human α-synuclein was tagged at the C-terminus to GFP (Rockenstein et al., 2005). Unlike the study by Nemani et al., the model of α-synuclein overexpression used by Scott et al. exhibited post-translationally modified and pathologically altered α-synuclein despite the modest levels of expression. Using electrophysiological assessment, the authors elegantly demonstrated a significant reduction in neurotransmitter release and a failure of the presynaptic exocytotic machinery in the synaptic boutons of transgenic mice neurons. Importantly, styryl dye uptake experiments further suggested that the recycling machinery in the transgenic cultures was inoperable, since a significant number of boutons overexpressing α-synuclein had failed to endocytose the dye. Electron microscopy examination of such synapses further revealed variation in the size of synaptic vesicles, including vesicles enlarged in size. Since this phenotype resembled previously reported animal models that lacked presynaptic proteins (Schoch et al., 2001; Nonet et al., 1998; Deitcher et al., 1998), the authors went on to quantitatively evaluate the levels of a number of endocytic and exocytic proteins (including synapsin) in transgenic synapses and found that they were either absent or diminished.

The authors concluded (and rightly so), that multiple exocytic and endocytic pathways are involved in α-synuclein pathogenesis. Significantly, such changes were also observed in human autopsy samples of synucleinopathies (DLB). In this respect, it would have been interesting to see whether and how the distribution of such proteins changes in the vulnerable brain areas during aging. What is important in this study of Scott et al. is the sustained expression of α-synuclein in the model used, and the observation that the protein is pathologically altered. It is very likely that misfolded and/or aggregated α-synuclein have different effects on the synaptic function and morphology. One might also conclude that a long-lasting expression of α-synuclein, as in the case of multiplications of the gene, has different effects on the physiology and morphology of the synapse, to a transient/shorter one as the two studies suggest. Again, the identification of species generated in the two scenarios is in need of further examination.

Another question that arises is whether “aggregated” α-synuclein species have a direct interaction with presynaptic proteins. What would the effect of “oligomeric” modifiers be on the morphology of the vesicles and function of the synapse? Collectively, the findings presented in these studies suggest that PD may result from a “dying back” process. This would be an early event initiated at the site of synapse, stemming from the accumulation and misfolding of α-synuclein and leading to degeneration of basal ganglia axons followed by more widespread denervation. However, it will take many more studies to pinpoint which of the cellular actions of overexpressed α-synuclein lie at the center of synaptic physiology. A critical question that remains open is how a reduction in neurotransmitter release as an initiating event can lead to inclusions pathology and neuronal demise. An answer to this question may provide insights regarding the early steps that lead to neurodegeneration, and that could further identify targets for preventative intervention. For example, development of drugs that inhibit α-synuclein actions at the synapse could be a potentially effective treatment for Parkinson disease.

View all comments by Kostas Vekrellis

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

The pathologies occurring in Alzheimer disease (AD) are curious because of their overlap with other disorders. Although accumulation of Aβ is most commonly associated with AD, neuritic plaques are also observed in Parkinson dementia, diffuse Lewy body diseases, and other less common disorders. Similarly, tau inclusions occur in AD and frontotemporal dementia (as well as other less common diseases), and tau haplotypes are implicated in Parkinson disease. α-synuclein inclusions also occur in multiple diseases including Parkinson disease, diffuse Lewy body disease, and even AD. Prior work by Eliezer Masliah’s group produced a double-transgenic cross expressing both APP and α-synuclein, and showed enhanced accumulation of α-synuclein inclusions (Masliah et al., 2001). Similarly, John Trojanowski, Virginia Lee, and colleagues showed enhanced neurodegeneration in tau mice expressing mutant human α-synuclein (Giasson et al., 2003). The current manuscript from Frank LaFerla’s group, by Clinton et al., now pushes this idea a step further by combining his triple-transgenic model (3xTg-AD),...  Read more

The pathologies occurring in Alzheimer disease (AD) are curious because of their overlap with other disorders. Although accumulation of Aβ is most commonly associated with AD, neuritic plaques are also observed in Parkinson dementia, diffuse Lewy body diseases, and other less common disorders. Similarly, tau inclusions occur in AD and frontotemporal dementia (as well as other less common diseases), and tau haplotypes are implicated in Parkinson disease. α-synuclein inclusions also occur in multiple diseases including Parkinson disease, diffuse Lewy body disease, and even AD. Prior work by Eliezer Masliah’s group produced a double-transgenic cross expressing both APP and α-synuclein, and showed enhanced accumulation of α-synuclein inclusions (Masliah et al., 2001). Similarly, John Trojanowski, Virginia Lee, and colleagues showed enhanced neurodegeneration in tau mice expressing mutant human α-synuclein (Giasson et al., 2003). The current manuscript from Frank LaFerla’s group, by Clinton et al., now pushes this idea a step further by combining his triple-transgenic model (3xTg-AD), which develops Aβ and tau inclusions, with an α-synuclein model.

The quadruple-transgenic mice show a striking increase in accumulation of all three types of inclusions—Aβ, tau, and α-synuclein. The cognitive deficits also develop at an accelerated pace. One of the more striking aspects of the work is the location of the inclusions that form. The primary location of the α-synuclein inclusions shifts from brain stem in the mono-transgenic α-synuclein mouse to cortex and subiculum in the DLB-AD mouse. The shift in gross localization of the α-synuclein combined with the acceleration of cognitive decline highlights the interactions among these different types of inclusions. The accelerated neurodegeneration also emphasizes the additive damage associated with increasing burden of inclusions. Whether the additive damage reflects a direct mechanistic interaction of neurodegenerative pathways contributed by each type of inclusion or simply the additive burden of increased cumulative damage remains to be determined.

Although the combined effects of the inclusions is to produce additive degenerative effects, one of the surprising observations is that the tau and α-synuclein inclusions don’t show strong colocalization within neurons. Both form distinct and separate inclusions. This observation is reminiscent of observations by Giasson and colleagues, and by my group (Frasier et al., 2005), showing the presence of phosphorylated tau in brains of α-synuclein overexpressing mice, but also showing that the phospho-tau and α-synuclein accumulated in a different set of neurons (Frasier et al., 2005; Giasson et al., 2003). These observations raise a classic theme in neurodegenerative research—that of selective neuronal vulnerability. It remains unclear why specific inclusions form in particular sets of neurons, and in the case of LaFerla’s quadruple-transgenic mice, why one set of neurons might develop tau pathology while another develops α-synuclein pathology. Regardless of the ultimate answers, the model put forth by Clinton et al. will go a long way toward providing tools allowing us to investigate these questions.

References:
Frasier M, Walzer M, McCarthy L, Magnuson D, Lee JM, Haas C, Kahle P, Wolozin B. Tau phosphorylation increases in symptomatic mice overexpressing A30P alpha-synuclein. Exp Neurol. 2005 Apr;192(2):274-87. Abstract

Giasson BI, Forman MS, Higuchi M, Golbe LI, Graves CL, Kotzbauer PT, Trojanowski JQ, Lee VM. Initiation and synergistic fibrillization of tau and alpha-synuclein. Science. 2003 Apr 25;300(5619):636-40. Abstract

Masliah E, Rockenstein E, Veinbergs I, Sagara Y, Mallory M, Hashimoto M, Mucke L. beta-amyloid peptides enhance alpha-synuclein accumulation and neuronal deficits in a transgenic mouse model linking Alzheimer's disease and Parkinson's disease. Proc Natl Acad Sci U S A. 2001 Oct 9;98(21):12245-50. Abstract

View all comments by Benjamin Wolozin

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...  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 agents that...  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 isoforms are largely similar, so I would expect...  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 de novo sequencing...  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

By directly examining SNARE assembly, Burre et al. present good evidence that α-synuclein plays a role in facilitating the assembly of SNARE complexes. They also show that this facilitating process depends on synaptic activity and propose that the physical binding of α-synuclein to VAMP may ultimately mediate the process. However, there were no changes in synaptic transmission in acute brain slices from WT, synuclein overexpressing mice, or mice lacking all synucleins.

We recently reported that excessive α-synuclein induces a series of pathologic changes including deficits in neurotransmitter release (Scott et al., 2010), in general agreement with a recent study from Robert Edwards's group (Nemani et al., 2010), as well as other reports on cellular and cell-free systems (Larsen et al., 2006; Darios et al., 2010). More recently, we have performed additional electrophysiologic experiments in WT and α-synuclein -/- neurons, in collaboration with Iustin Tabarean, an electrophysiologist at Scripps.

Though we do not find significant neurotransmitter release deficits in...  Read more

By directly examining SNARE assembly, Burre et al. present good evidence that α-synuclein plays a role in facilitating the assembly of SNARE complexes. They also show that this facilitating process depends on synaptic activity and propose that the physical binding of α-synuclein to VAMP may ultimately mediate the process. However, there were no changes in synaptic transmission in acute brain slices from WT, synuclein overexpressing mice, or mice lacking all synucleins.

We recently reported that excessive α-synuclein induces a series of pathologic changes including deficits in neurotransmitter release (Scott et al., 2010), in general agreement with a recent study from Robert Edwards's group (Nemani et al., 2010), as well as other reports on cellular and cell-free systems (Larsen et al., 2006; Darios et al., 2010). More recently, we have performed additional electrophysiologic experiments in WT and α-synuclein -/- neurons, in collaboration with Iustin Tabarean, an electrophysiologist at Scripps.

Though we do not find significant neurotransmitter release deficits in α-synuclein -/- neurons, we consistently see such deficits in neurons overexpressing modest levels of α-synuclein. We cannot comment on the lack of electrophysiologic abnormalities in the α-synuclein overexpressing neurons as seen by Burre et al., but we are pretty sure that in our system, excessive α-synuclein leads to a reduction in neurotransmitter release.

The authors argue that overexpression (or lack thereof) of α-synuclein does not directly affect neurotransmitter release. However, given the seemingly dramatic effects on SNARE assembly as well as the profound motor/behavioral deficits in the synuclein triple-knockout mice, one question is, then, What is the eventual physiologic defect in the mice described by Burre et al., if not a decrease in neurotransmitter release?

References:
Scott DA, Tabarean I, Tang Y, Cartier A, Masliah E, Roy S. A pathologic cascade leading to synaptic dysfunction in alpha-synuclein-induced neurodegeneration. J Neurosci 30:8083-8095. Abstract

Nemani VM, Lu W, Berge V, Nakamura K, Onoa B, Lee MK, Chaudhry FA, Nicoll RA, Edwards RH. Increased expression of alpha-synuclein reduces neurotransmitter release by inhibiting synaptic vesicle reclustering after endocytosis. Neuron 65:66-79. Abstract

Larsen KE, Schmitz Y, Troyer MD, Mosharov E, Dietrich P, Quazi AZ, Savalle M, Nemani V, Chaudhry FA, Edwards RH, Stefanis L, Sulzer D. (2006) Alpha-synuclein overexpression in PC12 and chromaffin cells impairs catecholamine release by interfering with a late step in exocytosis. J Neurosci 26:11915-11922. Abstract

Darios F, Ruiperez V, Lopez I, Villanueva J, Gutierrez LM, Davletov B. Alpha-synuclein sequesters arachidonic acid to modulate SNARE-mediated exocytosis. EMBO Rep 11:528-533. Abstract

View all comments by Subhojit Roy