Exocytosis and Spreading of Normal and Aberrant α‐Synuclein

Abstract

It is now established that α‐synuclein can be physiologically secreted to the extracellular space. In this sense, mechanisms that govern the secretion of the protein may be of importance in the initiation and progress of synucleinopathies. It is possible that increased secretion may aid the formation of toxic seeds extracellularly. Alternatively, reduced presence of extracellular α‐synuclein due to impaired secretion may increase the intracellular load and trigger intracellular seeding. Once outside, α‐synuclein can exert various paracrine actions on neighboring cells again by mechanisms that have not been fully elucidated. It has been demonstrated that, when applied extracellularly, α‐synuclein species can induce multiple neurotoxic and inflammatory responses, and aid the transmission of pathology between neurons. Still, the exact mechanism(s) by which secreted α‐synuclein affects the homeostasis of other neurons is still not well understood. A portion of α‐synuclein has been shown to be associated with the surface and lumen of exosomes which can transfer it to the surrounding cells, and potentially trigger seeding. Interestingly, increased exosome release has been linked to pathological situations of lysosomal dysfunction as observed in Parkinson's disease (PD). However, the possibility that the observed α‐synuclein pathology spread is attributable to the passive diffusion of the initial injected α‐synuclein strains cannot be excluded. Importantly, most of the studies that have so far addressed the role of extracellular α‐synuclein have not employed naturally secreted forms of the protein. It is plausible that deregulation in the normal processing of secreted α‐synuclein may aid the formation of “toxic” species and as such it may also be a causative risk factor for PD. In this capacity, elucidation of the underlying mechanisms that regulate the protein‐levels of extracellular α‐synuclein becomes essential. Such mechanisms could involve its proteolytic clearance from the extracellular milieu.

Exocytosis of α‐Synuclein: Insights from Cell Culture and Animal Models

Despite the significance of α‐synuclein in the pathogenesis of PD and related synucleinopathies, the normal function(s) of the protein has not been elucidated yet 6. α‐Synuclein is localized predominately in the nerve terminals with low levels of expression in the cell body, dendrites or extrasynaptic sites along the axon 31. The topology of the protein in combination with its preferential binding to lipid membranes suggested that the potential physiological function(s) of the protein may be related to the synaptic vesicle (SV) cycle. Indeed, previous studies using in vitro and in vivo models demonstrated that α‐synuclein plays a role in endocytosis, mobilization and refiling of the readily releasable SV pool and vesicle trafficking 5, 12, 44, 57. In accordance with a functional role in the regulation of the SV cycle, α‐synuclein was found to be associated with the SV membrane where it interacts with the v‐SNARE protein, synaptobrevin‐2, promoting SNARE‐complex assembly both in vitro and in vivo 10, 11. Even though the association of α‐synuclein with SVs has been reported, it is still unclear how the protein reaches and is incorporated in the membrane of SVs. Most importantly, recent data indicated that SVs do not seem to be involved in the secretion of α‐synuclein 20.

The first evidence of the secretory mechanism of α‐synuclein was provided through work in two cellular models overexpressing α‐synuclein, yeast and SHSY5Y cells 16, 35. These two first studies showed controversial results indicating that intracellular overload of α‐synuclein may lead to differential intracellular localization and utilization of different cellular routes of release. In yeast, α‐synuclein was delivered to the plasma membrane through the classical ER to Golgi secretory pathway including the intra‐Golgi and secretory vesicle trafficking, vesicle docking and plasma membrane fusion 16. Even though all studies indicated vesicular exocytosis as the most possible mechanism for α‐synuclein release, the observations in yeast were not verified in neuronal cells in culture such as the SHSY5Y or the MES cells 29, 35. In these cellular systems, α‐synuclein release was mediated by a calcium‐dependent, nonconventional pathway. This pathway was dependent on the integrity of the endosomal compartments as treatment of the cells with compounds that interfere with the normal function of endosomes dramatically altered the levels of extracellular α‐synuclein 2, 18. In fact, when expressed, α‐synuclein could be found in all three endosomal compartments, the early endosomes, the late endosomes and the recycling endosomes despite their different morphological, biochemical and physicochemical properties 38.

There is now growing evidence to support that extracellular α‐synuclein species can be uptaken by recipient cells although the exact mechanism of internalization is still under investigation. It is logical to assume that the intracellularly localized (either internalized or cell‐produced) α‐synuclein would initially be transferred to the early endosomes. On translocation of α‐synuclein into the early endosomal compartment, it could be sorted into the recycling endosomes which can subsequently fuse with the plasma membrane in the form of large secretory vesicles. An interaction between α‐synuclein and the Rab GTPase, Rab11a, seems to regulate this sorting process 37. Alternatively, endosome‐residing α‐synuclein can be targeted to multivesicular bodies (MVBs), which are continuously produced by the gradual invagination and scission of the endosomal membrane. MVBs are predominately used by the cell as carriers for proteins destined for degradation. These proteins are packaged into small intraluminal vesicles (40–100 nm in diameter) that are generated by inward budding from the limiting membrane of MVBs 38. Fusion of MVBs with lysosomes results in the degradation of their protein cargo. Following a different fate, MVBs can fuse with the plasma membrane releasing the intraluminal vesicles to the extracellular space as exosomes. Importantly, α‐synuclein has been shown to be associated with exosomes both in vitro and in vivo suggesting that this alternative route of secretion can be used by the cell to facilitate α‐synuclein export 2, 18, 34. In addition to MVBs, recent data indicated that fusion of amphisomes with the plasma membrane, a process termed “exophagy,” could be an alternative way of α‐synuclein export, part of which could also be mediated by exosomes 17. The implication of autophagy in α‐synuclein secretion was initially implied by an earlier study where treatment with the autophagy inhibitor, bafilomycin, resulted in a robust increase in the release of exosome‐associated α‐synuclein oligomers 14. It should be stressed, however, that the exosome‐associated α‐synuclein still represents a minor fraction of secreted α‐synuclein 17, 18 suggesting that exosomes are not the main route of α‐synuclein release.

Exosomes have the exceptional property to interact with target cell membranes in a cell type‐specific manner thereby acting as nanocarriers of multilevel molecular information. Their biochemical composition has been proven fairly heterogeneous containing, however, elements from the cytoplasm, the endosome and the plasma membrane 4. The functional role of exosomes has emerged from carriers of unwanted protein material to mediators of intercellular communication transferring proteins, mRNAs and microRNAs to recipient cells 9, 29, 49. The fact that α‐synuclein is included in the lumen and membrane of exosomes 18 not only indicates a possible route of export, but also reveals a yet undiscussed potential role of α‐synuclein as intercellular messenger. Oligomeric species can also be trapped into exosomes 18. Given the neuron‐to‐neuron transmissibility of α‐synuclein species, exosomes may provide an attractive mechanism for the spreading of aberrant α‐synuclein that could account for the progression of pathology throughout different, axonally linked brain areas. Interestingly, this “Trojan horse” hypothesis has also been proposed for other PD‐related proteins such as LRRK2 50.

What is the exact trigger for the incorporation of α‐synuclein into exosomes is currently not known. Previous work highlighted the AAA (ATPases Associated with diverse cellular Activities)‐ATPase, VPS4, as an important regulator of α‐synuclein sorting into MVBs 2. Recent data suggested that SUMOylation, the covalent conjugation of SUMO (Small Ubiquitin like Modifier) molecule to proteins, is responsible for the recruitment of α‐synuclein to exosomes via a mechanism that involves the interaction of SUMO with phospholipids and also requires VPS4, Alix and TSG101 38. Importantly, α‐synuclein release was found to be a SUMO‐dependent process.

Collectively, the evidence that have been so far obtained from cell culture models point toward an endosome‐dependent mechanism for α‐synuclein secretion that also involves, at least in part, participation of exosomes (Figure 1). It is important to note that, even though there have been several studies on the elucidation of α‐synuclein secretory pathway in vitro, the characteristics of the in vivo mechanism of release have not been addressed yet. To assess the presence of extracellular α‐synuclein in real time, we have previously developed an in vivo microdialysis approach in conjunction with an ultra‐sensitive ELISA for α‐synuclein detection in the interstitial fluid (ISF) of the striatum of living, freely‐moving mice 18. This work demonstrated that extracellular α‐synuclein can readily be detected in stable levels in the brain parenchyma of Wt and A53T transgenic mice under experimental conditions that maintained the integrity of the striatal cells and fibers. Importantly, the excess of α‐synuclein measured in the ISF from the transgenic mouse mirrored the increase in the expression levels of the protein in the striatum. Further confirming the presence of secreted α‐synuclein in the brain parenchyma, α‐synuclein was also measured in human microdialysis samples obtained from patients admitted to the ICU after severe head injury. In this case, microdialysis was performed as part of the patients’ routine monitoring in a brain area that was unaffected from the initial head trauma 18. This was the first study to imply the presence of a regulatory mechanism that would maintain the balance between intracellular and extracellular α‐synuclein levels. Given the fact α‐synuclein is expressed in almost all areas of the brain, we would expect that different types of neurons would be involved in the secretory mechanism for α‐synuclein in vivo. In the molecular level, we could hypothesize that regulation of the release would be the outcome from the mobilization of intracellular molecular cascades within the neuron. To this end, our recent work 20 highlights the fact that α‐synuclein secretion not only results from the co‐operation of several molecular players within the secretory neuron but also its regulation indeed requires the crosstalk of different neurons and terminals present in a certain brain region, in this case the striatum. Such a regulatory cascade would be expected given the high organization complexity of the specific neuronal networks. Of note, since neuronal communication is orchestrated by neurotransmitter release, this work also suggested that neurotransmitters could play a significant role in α‐synuclein secretion in vivo 20. This would be of critical importance in neurodegenerative diseases, such as Parkinson's disease, where loss of dopamine dramatically alters the firing pattern of neurons that receive dopamine input in other brain areas including the striatum and the cortex 27.

Figure 1.

Proposed pathways for α‐synuclein exocytosis. α‐synuclein (red line) is associated with synaptic vesicles promoting the SNARE complex assembly and facilitating neurotransmitter release 1. The synaptic vesicle‐free can enter the early endosomal compartment via Golgi or clathrin‐mediated endocytosis 2. Endosome‐residing α‐synuclein can be then secreted by two separate pathways. It can be incorporated in MVBs 12 with the assistance of VSP4 and SUMO proteins and be externalized as exosome cargo on fusion of the MVB with the plasma membrane. Alternatively, it can be sorted into recycling endosomes 5 and be released possibly via secretory granules in a Rab11a‐dependent fashion. In all cases, α‐synuclein release is regulated by intracellular calcium concentration.

Other studies have also provided indirect proof of α‐synuclein secretion through monitoring the propagation capacity of the protein in different brain areas. Aggregation‐prone α‐synuclein material or cell transplants containing α‐synuclein species were injected in the mouse striatum or cortex and shown to spread to distal areas of the host brain, which requires export of α‐synuclein from the carrier cells 3, 15, 39. These studies will be discussed in detail in the next session of this review.

Propagation of α‐Synuclein Pathology: Mechanisms of Cell‐to‐Cell Transmission

Braak et al proposed that Lewy pathology begins in non‐dopaminergic structures of the lower brainstem or in the olfactory bulb prior to the appearance of classic motor symptoms in PD and then gradually spreads rostrally throughout the brain to involve eventually large parts of the central nervous system 8. This staging concept gained support from graft studies following the discovery that fetal dopaminergic neurons that had been implanted into PD patients 10–14 years earlier developed α‐synuclein pathology 33, 36. One plausible explanation is that toxic α‐synuclein species were transmitted from the already affected host neurons to the healthy implanted fetal neurons, causing the endogenous α‐synuclein to misfold. This spreading mechanism that was first described for prion protein and later on for amyloid‐beta, provided an explanation for the conversion of soluble proteins to pathologic amyloid fibrils in vitro 13, 32. Such an “infectious” process mechanism for α‐synuclein was initially supported by the Desplats et al. data 15 and could be an explanation of the step‐wise progression of the disease pathology and the involvement of specific neural pathways as suggested by the Braak staging of PD progression 8. It has been speculated that the initiation step of α‐synuclein propagation consists of the formation of pathogenic species, which are able to self‐amplify by recruiting other proteins. A number of in vivo studies have also provided evidence for the cell‐to‐cell transmissibility of specific extracellular α‐synuclein fibrlillar strains that are sufficient to cause all the major pathological changes observed in PD, including aggregate deposition, neurodegeneration and neuroinflammation 7, 45. In addition, using multiple microfluidics‐chamber systems, investigators have demonstrated that the formed α‐synuclein inclusions are able to propagate through transynaptic passage from one neuron to another 53, 58. In vivo injection of pathogenic α‐synuclein revealed that a single inoculation of a small amount of α‐synuclein preformed fibrils (Pffs) is sufficient to initiate the conversion of normal endogenous protein to pathogenic aggregated forms in the brain of adult 39, 41 and newborn mice 51 including hyper‐phosphorylation at Ser129. In fact, transmission of α‐synuclein pathology is experimentally verified by the presence of these phosphorylated species in interconnected brain regions. These paradigms also involved direct injections of more natural α‐synuclein containing preparations into the mouse brain such as issue homogenates from PD patients and brain lysates from transgenic mice containing oligomerized α‐synuclein 30, 43, 45, 47, 48, 54. α‐Synuclein spreading in these models appeared to depend on the expression of endogenous α‐synuclein and interactions between endogenous α‐synuclein and deleterious forms of the protein present in the inoculates. Similar to a prion like‐mechanism of spreading no diffusion of pathology was observed if α‐synuclein‐containing preparations were injected into the brain of α‐synuclein‐deficient mice 40. These studies collectively suggest that the propagating material is the endogenous α‐synuclein seeds formed by the exogenously added fibrils. Other animal models of synuclein propagation have been generated by injections of adeno‐associated viral vectors carrying human α‐synuclein into the rat vagus nerve. In this model, enhanced intracellular levels of α‐synuclein were sufficient to trigger its diffusion to pontine, midbrain and finally forebrain regions in a time dependent fashion 54, 55. These experiments suggested that the pathogenic α‐synuclein species can travel long distances and can propagate from the peripheral to the central nervous system. The role of the endogenous α‐synuclein on disease transmission was not, however, examined in these studies.

Importantly, in a recent study 45 injection of lysate from PD brains into the gut of rats resulted in the appearance of α‐synuclein inclusions in the nucleus of the vagus nerve supporting not only the retrograde transport of pathologic α‐synuclein but also the potential role of intestine α‐synuclein in the progression of PD pathology. In another in vivo study, Rey et al 48 inoculated monomeric and oligomeric forms of α‐synuclein in the olfactory bulb of mice and showed that transmission of the exogenous protein occurred very fast to brain regions synaptically connected to the olfactory bulb. Collectively, these observations demonstrate that the seeding is an important step in α‐synuclein propagation and represents a rate‐limiting factor for the initiation of this process (Figure 2). Such propagation studies clearly debate the relevance and nature of α‐synuclein species in the templating and seeding progress. It is obvious that not only the preparation method but also the origin of the synuclein species (recombinant, intracellular or secreted) is critical for its capacity to seed the protein and spread the disease.

Figure 2.

Possible mechanisms for uptake and propagation of α‐synuclein. Exogenous α‐synuclein (red line) can enter neuronal cells via various, yet unidentified, pathways such as translocation through a membrane pore or protein complex 2, exosome fusion with the plasma membrane 12 or endocytosis 5. The internalized protein can then act as a template to assist the production of higher order α‐synuclein species during a process where the endogenous α‐synuclein (red cube) is also incorporated. Alternatively, exogenous α‐synuclein species can interact with a receptor protein in the plasma membrane 1 signaling the seeding of endogenous α‐synuclein and generating aggregated α‐synuclein material. In all cases, aggregated α‐synuclein can be externalized and affect neighboring neurons.

Although the central role of the seeding process on the initiation of α‐synuclein cell‐to‐cell propagation has been demonstrated, the nature of the cell‐produced seed(s) that aid the formation of pathogenic α‐synuclein species in synucleinopathies remains unknown. To this end, work by Ronald Melki's group 7 specifically showed that high molecular weight strains generated from the same α‐synuclein precursor have different structures, levels of toxicity, and in vitro and in vivo seeding and propagation properties. The group further demonstrated that each α‐synuclein strain imprints its intrinsic architecture to endogenous reporter α‐synuclein on its recruitment in vivo suggesting a prion‐like behavior. Although a sequence‐specific templating and a typical cross β‐sheet conformation is suggested to be a prerequisite for seeding of amyloidogenic proteins including α‐synuclein 26, 52 a recent study by Sacino et al using non‐amyloidogenic α‐synuclein species that lacked the NAC region demonstrated that it was equally potent in spreading disease pathology in vivo 51. These results challenged the notion that the sequence–specific templating is essential for α‐synuclein seeding in vivo. Still, the use of naturally secreted α‐synuclein in such studies would have provided a much better insight into the capacity of α‐synuclein to initiate seeding and disease propagation.

A Possible Role of Exosomes in the Transmission of α‐Synuclein

Even though the fraction of exosome‐associated α‐synuclein represents a minor part of secreted α‐synuclein, a number of observations suggest that the cell‐to‐cell spreading of some forms of α‐synuclein may occur via exosomes. Importantly it has been shown that under certain conditions, exosomes can be biologically active entities, important for intercellular communication 56 and key players in significant biological processes. Our group and others 18, 24 have shown that α‐synuclein species (monomeric and oligomeric) can be associated with the surface and lumen of exosomes. Similarly, Fevrier and Raposo demonstrated association of Prion Protein with exosomes 23, 24. Release of exosomes from a variety of different neuronal cell lines has been described 22, 23 and there is evidence that exosomes may be involved in the pathogenesis of AD 46. Upregulation of exosome secretion is correlated with conditions of impaired lysosomal function 21, hence, increase cytosolic cargo of a misfolded protein 1. In this sense, exosomes are the central component of the “Trojan horse” theory according to which, toxic proteins packed into exosomes, are transferred to the extracellular milieu and enter recipient cells 25. Exosomal cargo is released intracellularly and causes spread of pathology. In this context, it is feasible that exosomes may contain α‐synuclein seeds or strains that could aid pathology. Although this hypothesis has not been proven yet, it has been shown that cells treated with oligomeric α‐synuclein carrying exosomes exhibit increased caspase‐3 cleavage 24. However, so far, no study has convincingly shown by which mechanisms exosome‐associated α‐synuclein enters neuronal cells. It is still unclear whether exosomes spill their toxic cargo by fusion with the membranes of the recipient cells, or whether they are taken up as a whole by the neighboring neurons. It would be interesting to know whether exosomes derived from different synucleinopathy patients have the same spreading capacity and importantly if such exosomes also facilitate the nucleation of endogenous α‐synuclein. One major culprit in our understanding of synuclein spreading via exosomes is the amount of α‐synuclein carried in them. One might argue that the amount is so small that it probably has no significant effect on disease transmission. However, it is also feasible that the “toxic quality” of α‐synuclein species associated with exosomes is more crucial for disease transmission even if present at much lower concentrations. Perhaps, exosome‐associated α‐synuclein may serve as a better template for disease transmission (Figure 2). This is also a speculation that has not been tested yet. In addition, one cannot rule out the possibility that increased exosome number may also be critical in the transmission of toxic α‐synuclein species. However, work by Melachroinou et al 42 has shown that exosomes from wild type α‐synuclein expressing cell lines do not cause aberrant effects on neuronal cultures, arguing for a role of the exosomal cargo in synuclein transmission. Another mechanism by which exosomes could initiate and propagate disease pathology in PD was recently suggested by Grey et al 28. This group demonstrated that exosomes accelerate the oligomerization of recombinant monomeric α‐synuclein in vitro and as such can increase the “toxic” protein load that is transferred to recipient cells. The study also showed that this acceleration of α‐synuclein oligomerization was partly attributed to the lipid content of the exosomal membrane. A demonstration that exosomes allow exchange of proteins within the nervous system in vivo would also give an explanation of how pathologies like Alzheimer's Creuzfeld Jacob or Parkinson's diseases, which begin in discrete regions spread overtime to connected regions of the central nervous system. This concept suggests that drugs directed toward reducing the formation and/or facilitating the clearance of misfolded α‐synuclein, so as to arrest or reverse the self‐propagation process, might represent a novel therapeutic interventions for the treatment of PD. In addition, understanding how the neuropathology spreads throughout the nervous system in Parkinson's disease would open up avenues for new treatments.