Sorting Out Release, Uptake and Processing of Alpha-Synuclein During Prion-Like Spread of Pathology

Abstract

Parkinson’s disease is a progressive neurological disorder that is characterized by the formation of intracellular protein inclusion bodies composed primarily of a misfolded and aggregated form of the protein α-synuclein. There is growing evidence that supports the prion-like hypothesis of α-synuclein progression. This hypothesis postulates that α-synuclein is a prion-like pathological agent and is responsible for the progression of Parkinson pathology in the brain. Potential misfolding or aggregation of α-synuclein that might occur in the peripheral nervous system as a result of some insult, environmental or genetic (or more likely a combination of both) that might spread into the midbrain, eventually causing degeneration of the neurons in the substantia nigra. As the diseases progresses further it is likely that α-synuclein pathology continues to spread throughout the brain, including the cortex, leading to deterioration of cognition and higher brain functions. While it is unknown why α-synuclein initially misfolds and aggregates, a great deal has been learnt about how the cell handles aberrant α-synuclein assemblies. In this review we focus on these mechanisms and discuss them in an attempt to define the role that they might play in the propagation of misfolded α-synuclein from cell-to-cell.

Graphical abstract

The prion-like hypothesis of α-synuclein pathology suggests a method for the transmission of misfolded α-synuclein from one neuron to another. This hypothesis postulates that misfolded α-synuclein becomes aggregation prone and when released and taken up by neighboring cells, seeds further misfolding and aggregation. In this review we examine the cellular mechanisms that are involved in the processing of α-synuclein and how these may contribute to the prion-like propagation of α-syn.

Introduction

Parkinson’s disease (PD) is a progressive neurological disorder that is characterized by the formation of intracellular protein inclusion bodies; Lewy bodies (LB) and Lewy neurites (LN). The main component of these inclusion bodies is a misfolded form of the protein, α-synuclein (α-syn) (Spillantini et al. 1998). In PD patients, these inclusions are found in autonomic nerves and throughout the brain, however it is the loss of dopaminergic neurons from the substantia nigra pars compacta that causes the most identifiable symptoms and signs of PD. The loss of nigrostriatal dopamine signaling is thought to underpin the motor symptoms, e.g. bradykinesia, resting tremor, rigidity and postural instability. These symptoms do not occur until other features of the PD neuropathology, including intraneuronal accumulation of α-syn aggregates, are already relatively advanced. Non-motor symptoms of PD such as depression, constipation, dementia, anosmia and sleep disturbances (Fahn 2003) frequently manifest before motor symptoms and are generally less responsive to dopamine replacement therapy (Chaudhuri et al. 2005), suggesting that they are more due to α-syn pathology than nigrostriatal degeneration. Indeed, it is thought that several early non-motor symptoms are caused due to emergence of Lewy pathology in the olfactory system, enteric nerves and brainstem, and that these α-syn eventually spread to other areas of the brain (Braak et al. 2003; Braak et al. 2004; Braak and del Tredici 2008). Non-motor symptoms were previously not recognized as important as they are considered today, when it is clear that they underlie a highly significant portion of the total disease burden for patients and their families (Martinez-Martin et al. 2011). Observance of Lewy pathology in several parts of the nervous system is, therefore, gaining increased attention. With this realization, future treatments that successfully target α-syn aggregation are likely to be very important additions to the therapeutic arsenal in PD.

Braak and his colleagues first uncovered tentative evidence that Lewy pathology propagates along neural pathways by post-mortem histopathological studies (Braak et al. 2003). They revealed that α-syn-positive LB and LN are most abundant in olfactory structures and the dorsal motor nucleus of the vagal nerve in early disease, and that they then appear in interconnected brain regions as the disease progresses (Braak et al. 2004). Initially, it was suggested that the “spreading agent” might be a neurotropic virus (Hawkes et al. 2007). When it was found that Lewy pathology can develop in immature neurons grafted to the brains of PD patients more than one decade earlier (Kordower et al 2008, Li et al 2007), the idea that the spreading agent is a misfolded variant of α-syn was launched (Li et al 2008; Brundin et al 2008). These observations led to the formulation the prion-like hypothesis of α-syn, which postulates that misfolded α-syn is transferred between interconnected neurons and inside the new neuron it acts as a template to seed further aggregation in a prion-like manner. The hypothesis further claims that the misfolded α-syn is transported intra-axonally between brain regions where the process is repeated (George et al. 2013; Dunning et al. 2011; Lema Tomé et al. 2012).

The objectives of the current review are to examine factors that influence this cell-to-cell spreading of α-syn. We will explore the evidence for spreading of α-syn in vivo, and the mechanisms that are known to be involved in the release of α-syn from cells and its subsequent uptake into neighboring cells. We also discuss the role that cellular homeostasis plays in α-syn processing and how disruption to this delicate balance may lead to acceleration of pathology.

Basic α-synuclein biology

α-Syn is a highly expressed neuronal protein. Both its exact function and its structure are under debate. This 140 amino acid protein is enriched in pre-synaptic nerve terminals (Iwai et al. 1995), yet it is also found within the nucleus (Mori et al. 2002). Because the protein is small enough to traverse the nuclear pore this may be a result of simple diffusion. Nevertheless, the roles of α-syn in the nucleus and the cytoplasm are not fully understood. The presence of α-syn in intraneuronal inclusions found in PD and other neurodegenerative conditions has implicated misfolded α-syn directly in neurotoxicity. It has also been suggested that the aggregates might be neuroprotective by sequestering toxic α-syn assemblies in the cell. However, it is not known whether the aggregates are toxic, truly protective, or simply an epiphenomenon that is related to progressive failure of cellular clearance mechanisms for aggregated proteins. There have been putative functions assigned to normal forms of α-syn including stabilization and biogenesis of membranes, lipid transport and packing, regulation of monoamines (including dopamine) and as a molecular chaperone (Burré, 2015). Studies in zebra finch suggest that α-syn may be involved in synaptic plasticity (George et al. 1995), while it has also been linked to regulation of phospholipids through the inhibition of the phospholipase D2 (PLD2) (Jenco et al. 1998). Besides these tenuous roles of α-syn, there is overwhelming evidence that suggests that α-syn acts as a chaperone during neurotransmitter release and SNARE complex formation and serves to protect the nerve terminals against degeneration (Chandra et al. 2005; Burré et al. 2010; Ninkina et al. 2012; Burré et al. 2014). However the exact role that α-syn plays in these mechanisms remains uncertain.

Biophysical chemistry of α-synuclein

Structurally, α-syn in its monomeric form is natively unfolded (Spillantini et al. 1997; Weinreb et al. 1996). Under certain conditions it can form oligomers, and fibrils (Meier and Böckmann 2015). Whether or not the monomeric form of α-syn is the predominant physiological state has been brought into question. Multimeric forms of α-syn, principally tetrameric, have been isolated from neuronal and non-neuronal cell lines, brain tissue and living human cells (Bartels et al. 2011; Wang et al. 2011; Burré et al. 2014). One proposed model suggests that the stable, folded tetrameric form of the protein is the physiological and functional form that exists in equilibrium with the monomeric form. When a destabilizing force, such as misfolding of the monomeric α-syn, damages this equilibrium, it has been reported that it causes a shift leading to aggregation (Dettmer et al. 2015). This dynamic potentially has important implications for PD and other synucleinopathies as many cellular stresses or other factors that influence cellular homeostasis might interfere with this equilibrium. Regardless of why α-syn equilibrium may shift to a more aggregate prone state, it is clear that α-syn, in its misfolded aggregated state or otherwise, can move from cell-to-cell.

In vivo evidence supporting a prion-like role of α-synuclein

The first tentative evidence for the cell-to-cell transfer of α-syn was seen in the autopsy of PD patients where embryonic neural tissue was grafted to the striatum to replace degenerated dopaminergic neurons. When the brains of these patients were examined post-mortem, over a decade after injection of the graft, pathological α-syn inclusions were found in the cells derived from the transplanted young embryonic tissue (Kordower et al. 2008; Li et al. 2008; Li et al. 2010; Kurowska et al. 2011). One possible explanation for this is, is that misfolded α-syn transferred from the PD host brain into the young neuronal tissue, inducing the characteristic PD pathology by recruiting further α-syn from the recipient cell and generating cytoplasmic LB and LN. In order to show that this series of events is indeed feasible, many in vitro and in vivo models have been generated subsequently and collectively demonstrate that α-syn can be excreted, taken up and seed aggregation in a recipient cell (Dehay 2014; Vermilyea and Emborg 2015; George et al. 2013).

Some of the recent evidence comes from the injection of mice with brain homogenates or recombinant strains of α-syn. Brain extracts from old, symptomatic mice overexpressing A53T α-syn injected into young mice (lacking α-syn aggregates at the time of surgery) of the same transgenic line caused accelerated α-syn pathology in the younger mice (Mougenot et al. 2012). Similarly, transgenic α-syn overexpressing mice that develop α-syn inclusions over time showed an accelerated pathology when injected with central nervous system (CNS) homogenate from sick animals into the striatum (Luk et al. 2012a). The use of preformed α-syn fibrils (PFFs), which are recombinant α-syn monomers that are induced to aggregate in vitro then sonicated to produce a mixture of α-syn seeds, can also be taken up by neurons and induce toxicity (Volpicelli-Daley et al. 2011). Wild-type mice injected with α-syn PFFs exhibited a nearly identical distribution of pathology seen in transgenic mice (Luk et al. 2012b). In a similar experiment, recombinant monomeric and oligomeric α-syn injected into the olfactory bulb of wild-type mice spread to interconnected brain regions resulting in the formation of α-syn inclusions (Rey et al. 2013). Additional evidence that α-syn can travel to the brain from the peripheral nervous system was demonstrated by the intramuscular injection of α-syn PFFs into mice hind limbs. These mice, either expressing wild type α-syn or A53T mutated α-syn, developed pathology rapidly in the CNS (Sacino et al. 2014).

There is also evidence that α-syn may be pathogenic, as α-syn isolated from the brains of Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA) patients can induce α-syn pathology in vitro. Hemizygous transgenic A53T α-syn mice (TgM83^+/−) generally remain healthy compared to their homozygous (TgM83^+/+) counterparts. However, when TgM83^+/− mice were inoculated with brain homogenates derived from sick, TgM83^+/+ mice, they developed widespread deposits of phosphorylated α-syn inclusions. Furthermore, inoculation of brain homogenates from patients clinically diagnosed with MSA resulted in neurodegeneration to TgM83+/− mice after incubation periods of around 120 days, which was accompanied by deposition of α-syn within neuronal cell bodies and axons. Interestingly extracts from PD patients did not induce aggregation of α-syn in cultured cells expressing A53T α-syn whereas extracts from MSA patients did, suggesting that PD and MSA are caused by different strains of α-syn (Watts et al. 2013; Prusiner et al. 2015). A similar phenomenon was seen in WT mice injected with recombinant α-syn that developed distinct patterns of pathology in brain regions with direct neural connections to the injection site. When sarkosyl-insoluble fractions were isolated from WT mouse brains injected with recombinant α-syn fibrils at 9 months post-injection and intracerebrally injected into 4-month-old WT mice, these mice developed a similar α-syn pathology (Masuda-Suzukake et al. 2014). Lewy bodies extracted from PD patients and injected into the substantia nigra or striatum of wild-type mice and macaque monkeys triggered neurodegeneration and the accumulation of host α-syn within nigral neurons and anatomically interconnected regions (Recasens et al. 2013). Further complicating this story is the recent finding that different conformations of α-syn fibrils, or ‘strains’, may cause different types of neuropathology. Indeed, injections of ‘fibrils’ and ‘ribbons’ (two distinct forms of α-syn aggregates) do lead to distinct histopathological and behavioral phenotypes when injected into the brains of rats (Peelaerts et al. 2015). Taken together, these studies clearly demonstrate that α-syn is capable of transferring between cells and seeding aggregation in vivo. The molecular events that trigger the initial misfolding of α-syn are not known, although environmental toxins and tissue inflammation have been suggested (Lema Tomé et al. 2012; Manning-Bog et al. 2002; George et al. 2010). The precise location of the first α-syn misfolding events is also not known with certainty, or necessarily identical in different patients, but Braak and coworkers would argue that the olfactory system and enteric nerves are preferred sites (Hawkes et al. 2007). Furthermore, it is not known in detail how the cell-to-cell transfer of α-syn occurs, but it seems clear that there is a complex harmony between proteostasis and propagation. The remainder of this review will focus on the mechanisms underpinning cellular release and uptake of α-syn and the molecular pathways involved in intracellular α-syn processing.

Cellular release of α-synuclein

Although there is the possibility that α-syn might move between cells by the means of tunneling nanotubes, in a similar manner suggested to apply to prions (Gousset et al. 2009), this has not yet been reported. Otherwise, prion-like transfer and seeding of α-syn aggregates requires that, at some point, this normally cytoplasmic protein is present in the extracellular space. Specific processes can lead to extracellular availability of intracellular proteins. In this section we discuss how and when α-syn can gain access to the extracellular space, if the process is regulated and whether its aggregation state also affects its localization.

The first report highlighting that α-syn can exist in the extracellular space preceded the notion that it could act as a prion-like protein and was based on observations in cultured neuroblastoma cells, and human cerebrospinal fluid (CSF) and plasma (El-Agnaf et al. 2003). If α-syn is secreted from cells in the brain, is the secretion regulated or stochastic? Evidence exists for both of these possibilities. Multiple molecular species of α-syn are found in CSF and they are present both in healthy and PD individuals (El-Agnaf et al. 2003). The α-syn in CSF originates largely from CNS cells (Mollenhauer et al. 2012). It is not yet clear if there are major differences in α-syn secretion in diseased versus healthy conditions (Simonsen et al. 2015). These results imply that, in humans, α-syn is regularly available in the extracellular space of the CNS and that that availability may occur regardless of disease state.

Alternately, several studies on cultured cells indicate that α-syn can be actively secreted and that the secretion can be constitutive and regulated. There is also a correlation between the levels of α-syn in the cell and the amount of α-syn that is released to the extracellular space. In iPSC-derived neurons expressing a triplication of the gene that encodes α-syn, high levels of secreted α-syn were detected (Reyes et al. 2015). This finding suggests that increased cytoplasmic α-syn levels lead to more release, which may also be true during aging in PD brains where it is presumed that there are higher neuronal α-syn levels and then maybe more release of α-syn. Lee and colleagues first confirmed that α-syn is released from rat primary cortical cultures and both monomeric and aggregated α-syn is released constitutively. However, these studies were performed on SH-SY-5Y differentiated neuroblastoma cells overexpressing α-syn and therefore the relevance to physiological conditions is debatable (Lee et al. 2005). As α-syn release was attenuated by low temperature, but not blocked by brefeldin A-mediated inhibition of endoplasmic reticulum (ER)-Golgi-dependent release, the authors identified non-classical/ER-Golgi-independent exocytosis as the mechanism of secretion. Additionally, Jang and colleagues described other cellular stressors that alter exocytotic α-syn release from cells (Jang et al. 2010). They inhibited mitochondrial complex I, as well as proteasomal and lysosomal activities, and demonstrated that vesicular release of α-syn is altered in conditions of cell stress.

Another way that α-syn can end up in the extracellular space is necrotic cell death, during which cells expel their contents into the extracellular space. There is, as yet, little evidence to suggest that the α-syn released from dying neurons contributes significantly to the extracellular pool of α-syn. In fact, some experiments suggest that most extracellular monomeric α-syn does not appear after passive release due to cell death, but instead from active release (Ulusoy et al. 2015).

Overall, cellular stress appears to drive α-syn secretion from neural cells, which supports the idea that neurodegenerative disease may lead to more exogenous α-syn release, uptake, and later processing that could contribute to disease progression.

Cellular uptake of α-synuclein

Whichever mechanisms dictate α-syn release, in order to achieve pathological intracellular actions including seeding and prion-like spreading, α-syn must enter the cell from the extracellular space.

One of the simplest mechanisms for cellular entry of substances is diffusion across the plasma membrane. Monomeric α-syn readily diffuses into cells, as its entry is not inhibited by cold temperature or blockade of typical entry pathways such as endocytosis (Lee et al. 2008a). Larger assemblies appear to utilize specific pathways in order to access intracellular environments including dynamin-dependent endocytosis. Lee and colleagues demonstrated that oligomeric and fibrillar α-syn species are taken up into cells via low temperature- and dynamin K44A-sensitive endocytosis (Lee et al. 2008a).

Sung and colleagues had previously identified another mechanism of α-syn uptake (Sung et al. 2001). In an immortalized hippocampal cell line and in primary cortical cultures, the authors determined that monomeric α-syn was taken up and caused cell degeneration. Subsequently, they showed that the uptake of α-syn was dependent on Rab5A-dependent endocytosis, as when a GTPase-deficient Rab5A was introduced to the system, the level of α-syn uptake and cellular dysfunction decreased. Later, pharmacological inhibitors allowed non-genetic studies aimed at discerning the role of endocytosis in α-syn uptake. In studies aimed at modeling α-syn pathology transfer from diseased host cells to grafted cells, we created animal models of in vivo intercellular transfer. When we injected monomeric α-syn into the cortex of mice, MAP-2-immunoreactive neurons internalized the proteins. When Dynasore, a small molecule inhibitor of dynamin, was co-injected, the internalization decreased (Hansen et al. 2011). Finally, unconventional forms of endocytosis have also been shown to traffic α-syn. Heparan sulfate proteoglycans have been shown to mediate macropinocytosis of not only α-syn but also other aggregation-prone proteins such as tau (Holmes et al. 2013). Effort has clearly been spent attempting to identify a receptor for α-syn, as many classical forms of endocytosis utilize receptor binding to trigger internalization of the receptor upon ligand binding. Thus far, no specific receptor has been shown to mediate the endocytosis of α-syn.

Alternative mechanisms to endocytosis have been proposed to contribute to uptake of α-syn. Tran and colleagues recently reported a role for immunotherapy in in vivo α-syn uptake (Tran et al. 2014). When antibodies directed against misfolded α-syn were intraperitoneally injected into mice that had received intrastriatal injections of preformed α-syn fibrils, the authors found less pathology and motor dysfunction. This result was attributed to a direct blockade of uptake of preformed α-syn fibrils into cells as well as a diminishment of intercellular transfer.

α-Syn found in exosomes, vesicles, or other structure, can present to cells in a protected manner, whereas uptake of α-syn from extracellular space incurs the risk of proteolysis prior to uptake (Steiner et al. 2011). A more detailed discussion of exosomal activity is present in the clearance section later in this review. The question then turns to how encapsulated α-syn could escape its membrane-bound state and access the cytosol.

Processing of α-synuclein

The misfolding and aggregation of α-syn is a key pathological event in the etiology of PD. The removal of these aberrant proteins is crucial for the survival and normal functioning of neuronal cells. Two distinct, but complementary systems; the Autophagy/Lysosomal Pathway (ALP) and the Ubiquitin Proteasome System (UPS) generally handle this process and monomeric α-syn can be degraded by both systems (Cuervo et al. 2004; Dargemont and Ossareh-Nazari 2012). However, larger species of α-syn, such as higher molecular oligomers, are directed to the lysosome for degradation by autophagy (Vogiatzi et al. 2008; Yu et al. 2009).

Post-translational modifications of α-synuclein

Several post-translational modifications may be responsible for directing α-syn towards one path of degradation or another. The most widely studied form of α-syn post-translational modification is phosphorylation, which can occur at several serine (S129 and S78) and tyrosine (Y125, Y133 and Y135) residues on the protein (Okochi et al. 2000). Phosphorylation at S129 is arguably the most abundant of these sites as 90% of α-syn found in LB is phosphorylated at this amino acid residue (Fujiwara et al. 2002), suggesting a link between phosphorylation and aggregation. However, studies that have attempted to correlate aggregation or toxicity of α-syn with phosphorylation have met with conflicting results; these experiments are beyond the scope of this review but are summarized excellently elsewhere (Tenreiro et al. 2014a).

Many proteins use phosphorylation to signal for degradation, by either the autophagy or proteosomal machinery, and there is emerging evidence that the phosphorylation of α-syn can also act as a signal for degradation. Increasing pS129 in a rat model by overexpressing PLK2 (Polo-like kinase 2, one of the kinases that is responsible for phosphorylating α-syn at S129 (Inglis et al. 2009; Mbefo 2010)) promoted α-syn clearance by autophagy, resulting in reduced dopaminergic neurodegeneration and motor deficits in this model (Oueslati et al. 2013). A similar effect was seen in yeast models where blocking S129 phosphorylation resulted in the failure of the cells to clear α-syn inclusions through the autophagy pathway (Tenreiro et al. 2014b). It appears that phosphorylation on sites other than S129 also signal that α-syn should be degraded. For example, the c-Abl kinase phosphorylates α-syn at Y39 and Y125 and blocking the activity of c-Abl in cortical neurons prevented α-syn-induced degeneration via the autophagy and proteasome pathways (Mahul-Mellier et al. 2014).

α-Syn is also prone to ubiquitination and sumoylation. Monomeric α-syn is degraded by the proteasome in a ubiquitin-independent process (Tofaris et al. 2001), yet disease-associated forms of α-syn and α-syn oligomers found in LBs are highly ubiquitinated (Spillantini et al. 1998; Goedert et al. 2001; Shimura et al. 2001; Tofaris et al. 2003). In fact, ubiquitination of α-syn has been linked to the aggregation of α-syn in dopaminergic cells (Rott et al. 2008). In a cellular model, the levels of ubiquitination correlate with that of α-syn oligomers and UPS dysfunction (Martins-Branco et al. 2012), shifting the burden of α-syn degradation to the ALP. Further studies have also shown that ubiquitination of α-syn by the Co-chaperone carboxyl-terminus of Hsp-70-Interacting Protein (CHIP) can act as a molecular switch where the E3 ubiquitin ligase activity of CHIP drives α-syn towards lysosomal degradation (Shin 2005; Tetzlaff et al. 2008). The small ubiquitin-related modifier (SUMO) has also been shown to modify α-syn in both cultured cells and mammalian brains in vivo (Dorval and Fraser 2006; Krumova et al. 2011). Sumoylation of α-syn can prevent aggregation and fibrillation of the protein and, interestingly, inhibition of the UPS leads to higher levels of sumoylated α-syn aggregates (Kim et al. 2011; Oh et al. 2011), suggesting that sumoylation of α-syn aggregates targets the protein for lysosomal degradation. Studies using yeast support this hypothesis and show that inhibition of sumoylation causes a decline in α-syn degradation. However, reduced sumoylation is compensated for by enhanced α-syn phosphorylation, which promotes α-syn clearance by both the UPS and autophagy (Shahpasandzadeh et al. 2014).

Other forms of α-syn post-translational modifications also occur, namely nitration and truncation. Several tyrosine residues of the α-syn protein are prone to nitration and nitrated α-syn is found within LBs (Giasson et al. 2000). Nitrated α-syn is extremely toxic when administered to cell culture or to the susbstantia nigra of rats (Yu et al. 2010), therefore timely clearance of this modified form of α-syn is essential. However, it has been shown that while nitration of α-syn can block fibril formation it also reduces the rate of degradation by the proteasome (Hodara et al. 2004), meaning that the ALP must compensate to avoid intracellular accumulation, or potentially increased excretion, of α-syn.

Truncation of α-syn occurs at the C-terminal and is a normal cellular process that occurs more frequently in patients with familial PD-linked mutations (Li et al. 2005). The enzyme Calpain1 facilitates truncation. Calpain1 can cleave soluble α-syn in several locations, including the NAC region, therefore preventing aggregation (Mishizen-Eberz et al. 2003). However, Calpain1 activity on aggregated α-syn causes truncation at the C-terminus which promotes further aggregation of α-syn (Murray et al. 2003; Ulusoy et al. 2010; Baba et al. 1998), increasing the burden on the autophagy/lysosomal degradation pathway. Calpain1 is increased in the brains of PD patients (Dufty et al. 2007), and therefore it is easy to see this as a response mechanism to unwanted α-syn that backfires once the protein begins to aggregate, effectively making the problem worse. Moreover, Calpain1 might also act in the extracellular space, as it is secreted along with α-syn (Games et al. 2014). Acting on α-syn extracellularly would create more aggregate prone species that are susceptible to uptake from neighboring cells and potentially would promote propagation of α-syn neuropathology.

The role of the autophagy-lysosomal pathway in α-synuclein clearance

As discussed above, higher order oligomeric assemblies of α-syn are generally cleared from the cell by the autophagy/lysosomal pathway. While autophagy is required for the removal of misfolded or harmful intracellular components it is also required to maintain homeostasis by degradation and recycling of redundant proteins and organelles to maintain proteostasis (Tanaka and Matsuda 2014). Therefore, it is not unexpected that under conditions of suppressed autophagy the cell resorts to other mechanisms to maintain proteostasis, such as the release of the unwanted protein to the extracellular space. As autophagy is the dominant mechanism of α-syn clearance, it is becoming clear that there is a complex relationship between α-syn turnover and propagation.

Autophagy is a process that manifests itself in several different forms, Chaperone Mediated Autophagy (CMA), macroautophagy and microautophagy. Degradation of α-syn has only been shown, so far, to occur through CMA and macroautophagy (Cuervo et al. 2010). CMA is a selective form of autophagy that relies on the recognition of specific amino acid motif (KFERQ) (Dice 1990), which binds to cytosolic Hsc70 (cHsc-70) (Chiang et al. 1989). This complex interacts with the Lysosome-Associated Membrane Protein type 2A (LAMP2A) on the lysosomal membrane forming a translocation complex where the substrate protein is directed into the lysosomal lumen assisted by lysosomal-hsc70 for degradation (Agarraberes and Dice 2001; Cuervo and Dice 1996). Importantly, this KFERQ recognition motif for CMA is found in α-syn, suggesting that this pathway can degrade α-syn. Indeed, wild-type, monomeric and dimeric forms of α-syn are degraded by this mechanism (Mak et al. 2010; Martinez-Vicente et al. 2008). However, familial mutations of α-syn; A30P and A53T, inhibit CMA by causing irreversible binding of α-syn-c-Hsc70 complex to LAMP2A on the surface of the lysosome, effectively blocking the system (Cuervo et al. 2004). Further studies have shown that A53T α-syn not only blocks CMA, but also leads to the activation of macroautophagy as a compensatory mechanism (Xilouri et al. 2009).

While CMA is an actively selective process, macroautophagy is a constitutively expressed mechanism that is generally non-selective and degrades cargo that has undergone sorting through the endosomal pathway. During macroautophagy, a double-membrane compartment, the autophagosome, engulfs and sequesters cellular components and eventually fuses with the lysosome (Kraft and Martens 2012). Several studies have demonstrated that macroautophagy is involved in degrading α-syn (Spencer et al. 2009; Tofaris et al. 2011; Paxinou et al. 2001). However, it also seems that α-syn can interfere with this pathway, as overexpression of α-syn, familial mutations of α-syn and LB-like α-syn inclusions have all been shown to inhibit macroautophagy (Winslow et al. 2010; Tanik et al. 2013; Yan et al. 2014; Stefanis et al. 2001). This has been suggested to result in a feedback loop where α-syn can no longer be degraded, leading to larger aggregates and eventually cell death (El-Agnaf et al. 1998). In an attempt to maintain cellular homeostasis and delay this fate the cell excretes misfolded α-syn using various pathways.

One such mechanism is increased exocytosis. Cells have been shown to excrete α-syn in exosomes, which was strongly influenced by autophagic activity (Danzer et al. 2012). Induced autophagic failure by pharmacological and genetic methods leads to a marked increase in exocytosis of α-syn from α-syn-expressing donor cells in a mixed culture with recipient cells, resulting in increased cell death in the recipient cells (Lee et al. 2013). In another cellular study, inhibition of autophagy by bafilomycin A led to increased secretion of α-syn oligomers, which was exacerbated by additional stresses such as inflammatory mediators, uptake of α-syn and cellular damage (Poehler et al. 2015). Interestingly, this same study suggested alternative secretion pathways for different α-syn species. They observed lower molecular weight aggregates being secreted by exosomes and higher molecular weight aggregates being released via membrane shredding.

The exocytosis and endocytosis pathways are intrinsically intertwined, as both endosomes (which normally transport their cargo to lysosomes for degradation) and exosomes (which are released from the cell) are specialized multivesicular bodies (MVBs) (Piper and Katzmann 2007). PD-related genes ATP13A2, HDAC6, and VPS35 are thought to influence the ability of MVBs to sort cargo, possibly determining the cellular fate of aggregated α-syn; degradation or release (Kong et al. 2014; Leyk et al. 2015; Follett et al. 2014). Also, induction of autophagy promotes the fusion of MVBs with autophagic vacuoles and inhibits exosomal release (Fader et al. 2008).

There are less widely studied autophagy/lysosomal-related mechanisms of extracellular protein release that may be involved in α-syn propagation. One such process that is regulated by macroautophagy via an unconventional ER-Golgi–independent mechanism releases proteins via a novel “compartment for unconventional protein secretion” (CUPS), derived from a subdomain of the endoplasmic reticulum (Hayashi-Nishino et al. 2009; Bruns et al. 2011). Although no studies have so far linked this CUPS to PD, it has been coupled to the accumulation of Alzheimer’s-related amyloid-β peptide upon inhibition of macroautophagy (Nilsson et al. 2013). It is possible that α-syn could also be secreted via, or interfere with, CUPS. Lysosomes can also undergo exocytosis in a process that is similar to synaptic vesicle release (Andrews 2000). This is regulated by transcription factor EB (TFEB), which also regulates autophagy and release of α-syn from dopaminergic neurons in vivo (Decressac et al. 2013). As α-syn is involved in SNARE complex formation (Burré et al. 2014) and has been implicated in the regulation of neurotransmitter release (Liu et al. 2004; Nemani et al. 2010), this is also a possible route for α-syn secretion.

Autophagic and lysosomal dysfunctions also play crucial roles in α-syn propagation. In the absence of a system to regulate misfolding and aggregation of α-syn and other aggregation prone proteins, these build up in the cell unchecked. Inhibition of lysosomal function in α-syn-overexpressing SH-SY-5Y cells led to increased α-syn in exosomes and cell-to-cell transfer via exosomes (Alvarez-Erviti et al. 2011). Thus, α-syn release from an overexpressing neural cell line can be regulated by lysosomal dysfunction, which suggests that in humans with lysosomal deficits, more α-syn might be released from neurons and be available for uptake by neighboring cells. Indeed, Lee and colleagues connected autophagic dysfunction, exocytosis, and cell-to-cell transfer of α-syn (Lee et al, 2013). Lysosomal dysfunction, or its inability to digest certain protein assemblies, can be caused by a deficiency in key enzymes. One of the most studied causes of lysosomal impairment occurs in Gaucher’s disease, a genetic disorder that causes a deficiency in the lysosomal hydrolase glucocerebrosidase (GCase) (Hruska et al. 2008). This leads to a buildup of the glycolipid glucocerebrosidase within lysosomes, which is believed to impair their ability to degrade α-syn (Ginns et al. 2014). Bae and colleagues demonstrated that GCase deficiency leads to lysosomal dysfunction, greater release of aggregated α-syn into the extracellular space, and more cell-to-cell transfer and seeding of α-syn aggregates in the host (Bae et al. 2014). Thus, α-syn release from overexpressing neural cell lines can be regulated by lysosomal dysfunction, which suggests that in humans with GCase deficiency or lysosomal deficits, more α-syn may be released from neurons and be available for uptake into and processing in recipient cells.

There is emerging evidence that deficiencies of the retromer can also cause lysosomal dysfunction. The retromer is a protein complex that is responsible for the sorting of endosomal compartments, which depending on its cargo and their interactions with other complexes, directs endosomes to the Golgi apparatus for recycling or to the lysosome for degradation (Klinger et al. 2015). As one of the known substrates of the retromer complex, M6PR (mannose 6-phosphate receptor), is responsible for the delivery of essential enzyme precursors to the lysosome, it is clear how retromer dysfunction can lead to lysosomal deficiency (Arighi et al. 2004; Bonifacino and Rojas 2006). The retromer is a pentameric complex consisting of the vacuole sorting proteins VPS26, VPS29 and VPS35 and two sorting nexins, which can be two of SNX1, SNX2, SNX5 or SNX6 (Seaman 2012). A mutation in one of these genes, VPS35 (D620N), has been linked to late-onset familial PD (Vilariño-Güell et al. 2011; Zimprich et al. 2011). This mutation results in reduced affinity to the WASH complex which is responsible for the sorting of cargo into at least two distinct endosomal recycling pathways, one of which is the endosomal to golgi retrieval of M6PR (Gomez and Billadeau 2009). Experiments in cells expressing the VPS35 D620N mutation or lacking the WASH complex revealed impaired autophagy (Zavodszky et al. 2014). Furthermore, mutations in the most common familial PD-related gene, LRRK2, are linked to endosomal dysfunction. Defects in the PD-associated RAB7L1-LRRK2 pathway result in abnormal lysosomal structures and defective retromer complex function (Bonifacino and Hurley 2008). PD-associated defects in RAB7L1 or LRRK2 result in endolysosomal and Golgi apparatus sorting deficits and deficiency of VPS35. This deficiency is rescued by wild-type VPS35 but not the mutated form of the protein (MacLeod et al. 2013).

Deficiency of cathepsin D (CTSD), the main enzyme present in the lysosome that is responsible for α-syn degradation (Sevlever et al. 2008) unsurprisingly impairs α-syn degradation in animal PD models (Qiao et al. 2008; Cullen et al. 2009). There is also post-mortem evidence that PD patients have reduced CTSD in neurons that contain α-syn inclusions (Chu et al. 2009). In the context of α-synucleinopathies, CTSD deficiency alone would prevent clearance of α-syn, promoting its aggregation, yet in response to a CTSD deficiency, levels of CTSB are increased. This might seem like a reasonable response, as CTSB is known to cleave α-syn and circumvent fibril formation (McGlinchey and Lee 2015). However, CTSB has been found to actually increase the aggregate forming activity of endogenous α-syn fibrils that have already assembled (Tsujimura et al. 2014). This potentially leads to a dangerous situation within the cell as these aggregates and other α-syn species taken up by the cell can cause the lysosome to rupture leading to cell death (Freeman et al. 2013). Therefore, autophagic failure might eventually trigger a cascade of events resulting in a dying cell filled with α-syn aggregates that will be released into the extracellular space in conjunction with apoptosis. These aggregates can then be taken up by neighboring cells and act as seeds for further aggregation as a step in the prion-like propagation of Lewy pathology.

Clearance of α-synuclein from the extracellular space

Once misfolded α-syn finds its way into the extracellular space via one of the mechanisms discussed above, it is prone to be taken up by neighboring neurons, initiating propagation of the aggregate pathology. However, there are several putative clearance mechanisms that may operate in the extracellular space. Microglia are the most likely cells to clear extracellular α-syn and microglial activation has been recorded in PD patients (Kraft and Martens 2012; Imamura et al. 2003; George and Brundin 2015). Cultured microglia can take up α-syn, becoming activated (Klegeris et al. 2008) and actively degrade the protein (Lee et al. 2008b). This suggests that microglia can clear extracellular α-syn and interrupt the prion-like spreading of pathology; however, this system may be α-syn species-dependent. The toll-like receptor (TLR) family, in particular the members TLR2 and TLR4, are essential for microglial phagocytosis of α-syn (Fellner et al. 2013). Interestingly, it has been shown that TLR2 specifically mediates phagocytosis of oligomeric α-syn (Kim et al. 2013), and aggregated species of α-syn inhibit microglial phagocytosis while monomeric α-syn accelerates this process (Park et al. 2008). These microglial findings concur with the claims that other α-syn clearance mechanisms effectively handle monomeric α-syn, but fail to process aggregated α-syn. Indeed, microglia themselves are most likely not insensitive to autophagic/lysosomal failure caused by genetic defects and considering their ability to internalize α-syn species may provide a pool of α-syn aggregates that become a source for α-syn seeds. Much more study of microglial processing of α-syn is required in order to elucidate their role in α-syn transfer.

Astrocytes and oligodendrocytes are potentially capable of clearing α-syn from the extracellular space and α-syn inclusions have been found in both these types of glial cells in PD patients (Wakabayashi et al. 2000; Hishikawa et al. 2001). In fact, the topographical distribution of astrocytic α-synuclein-immunoreactive inclusions in post-mortem PD cortex closely parallels that of intraneuronal LN and LB (Braak et al. 2007). This suggests that astrocytes might partake in the propagation of α-syn. Moreover, cell culture studies have shown that α-syn released from neurons can be taken up by astrocytes, and trigger their transformation into a reactive state (Lee et al. 2010). Oligodendrocytes, on the other hand, both express low levels of endogenous α-syn (Djelloul et al. 2015) as well as take up α-syn from the extracellular space. Additionally, imported α-syn can promote the formation of cytoplasmic inclusions (Reyes et al. 2014; Radford et al. 2015).

Extracellular α-syn can also be degraded by proteolyitc cleavage. Several cell types, including neurons, oligodendrocytes and astrocytes, secrete proteases that can degrade α-syn. Two of these, Calpain1 and CTSD were discussed earlier in this review, but several others exist. Neurosin is a serine protease capable of cleaving α-syn and preventing further oligomerization, although it is not as efficient at degrading A53T α-syn (Iwata et al. 2003). It appears that neurosin is active in PD and AD brains as it has been associated with LB and amyloid plaques (Ogawa et al. 2000). Additionally, in PD and DLB patients, reduced levels of neurosin expression have been reported (Spencer et al. 2013). Mouse models overexpressing neurosin exhibit reduced accumulation of α-syn and mitigated neurodegenerative deficits in wild type (but not A53T mutant) α-syn transgenic mice. In cell culture models, neurosin is secreted. Extracellular neurosin is proteolytically active while intraneuronal neurosin is not (Tatebe et al. 2010). Interestingly, neurosin precursors localize to the ER before secretion, meaning that disruptions in the endosomal-sorting pathway could interfere with this process and contribute to PD pathogenesis.

Plasmin is another serine protease that is expressed in neurons and astrocytes that is known to degrade oligomeric and monomeric extracellular α-syn. This protease cleaves extracellular α-syn at the N-terminus, inhibiting glial activation and interneuronal spreading of α-syn (Kim et al. 2012). Considering that plasmin is also induced by and degrades Alzheimer disease-related Aβ (Van Nostrand and Porter 1999; Tucker et al. 2000), it could be speculated that the plasmin system is a general defense mechanism against extracellular protein aggregates and may be critical for other neurological proteinopathies. However, monomeric extracellular α-syn reduces the expression of tPA (Type Plasminogen Activator, the enzyme essential for the conversion of plasminogen to plasmin) in astrocytes and microglia in a dose-dependent manner (Joo et al. 2010), showing that high concentrations of α-syn can actually inhibit this system. Furthermore, extracellular α-syn increases PAI-1 (Plasminogen Activator Inhibitor) expression in neurons and glial cells (Kim et al. 2012). Therefore, it is possible that the plasmin system is down regulated in PD.

Finally, members of the matrix metalloproteinase (MMP) family can also degrade extracellular α-syn. MMP3 degrades α-syn more efficiently than other MMPs, and its expression levels are elevated by oxidative stress. This protease cleaves α-syn at the C-terminal end leaving behind truncated α-syn fragments that are even more prone to aggregation once taken up by a nearby cell (Sung et al. 2005). It is more than likely that several of the aforementioned proteases work together to degrade α-syn, but the deficiency of one of them potentially destabilizes this delicately balanced system, leading to further aggregation and propagation of α-syn.

Conclusions

The clearance of misfolded and aggregated proteins is crucial for the health of neuronal cells. In PD and other α-synucleinopathies, aberrant α-syn assemblies accumulate in the cell, triggering several pathways that degrade this protein (Figure 1). These mechanisms appear to be dependent on the protein species, as well as the physiological state of the cell; as in relatively healthy cells, α-syn that incidentally misfolds is likely quickly degraded. However, in compromised cells or in cells expressing familial forms of α-syn that are more prone to aggregation, the evidence suggests that these degradation systems may become overwhelmed causing a cascade of pathological events that lead to cellular dysfunction and death. Compounding these detrimental effects, it appears that, at least in some cases, protein degradation systems could backfire, cleaving aggregated α-syn in such a manner as to create smaller oligomers of α-syn, some of which may be more aggregation-prone or become templates for further seeding. These species eventually are expelled from the cell, either in an attempt to maintain homeostasis via exocytosis, or upon cell death, in which case a pool of potential α-syn seeds are released into the extracellular space where they are available to be taken up by neighboring cells resulting in propagation of the pathology.

Figure 1.

Mechanisms affecting release and uptake of α-syn: (1) Native α-syn is degraded by CMA and the proteasome (not depicted). (2) Aggregated α-syn is processed by the autophagosome, where it is degraded after the autophagosome fuses to the lysosome. (3) When the ALP is compromised or overwhelmed, α-syn is released from the cell by exocytosis or by (4) lysosomal secretion. (5) Alternatively, α-syn can be recycled from the endosomal pathway or (6) diffuse across the cellular membrane via membrane shredding or through leaky membranes from dying cells. (7) α-Syn may also be secreted by CUPS, derived by a subdomain of the ER. (8) α-Syn from the extracellular space is taken up via endocytosis or (9) maybe internalized via an as yet unidentified receptor mediated mechanism. (10) Lysosomal dysfunction can be caused by binding of misfolded α-syn to receptors on the lysosomal membrane or by failure of the endosomal sorting pathway to deliver key enzymes to the lysosome, resulting in a build-up of aberrant α-syn species.

Although there is compelling evidence that α-syn can propagate in vivo in a prion-like manner, it is still unclear if this process occurs in PD patients and, if it does so, it is not known how this process is initiated. The Braak staging of PD has been challenged on several occasions (Jellinger 2008; Parkkinen et al. 2008; Zaccai et al. 2008; Beach et al. 2009), with some studies finding that approximately 50% of cases exhibit Lewy pathology outside of the Braak schema. However, these findings may be explained by considering that Braak pre-selected cases based on the presence of inclusions in the dorsal motor nucleus of the vagus nerve and the assumption that this is the starting point of the disease (Braak et al. 2003). It still remains possible that initial point of insult occurs at a different location (and could vary from one patient to another) and that α-syn can propagate bi-directionally (Lerner and Bagic 2008). Indeed, it is more than likely that the pathology of PD is widely diverse and that the disease manifests itself differently from one individual to another depending on genetic predispositions and environmental factors. Indeed, such subtypes of PD have been suggested previously (Halliday et al. 2008). Another thing to consider is whether LB are actually harmful to the cell or merely an artifact of the cell’s attempt to sequester toxic protein species as has been suggested by some studies (Olanow et al. 2004; Kramer and Schulz-Schaeffer 2007; Tanaka et al. 2004). However, neither possibility precludes the prion-like hypothesis.

It is also important to consider the caveats involved in modelling PD in animal systems where α-syn is over-expressed to unnaturally high levels or injected locally at high concentrations in order to generate a PD-like phenotype. One might argue that this is an unavoidable necessity when using short-lived animals to model a chronic disease that takes decades to develop in humans. While these experiments give us invaluable information on how α-syn pathology may propagate from neuron-to-neuron in vivo, how this process is initiated in idiopathic PD remains to be seen.

Nevertheless, the prion-like hypothesis for PD can be a convincing one with an abundance of circumstantial evidence to support it. Also, as it is dependent upon aberrant α-syn species being present in the extracellular space, it is one that presents a practical focus for disease intervention. Therefore, immunotherapy targeting extracellular α-syn might be a valid strategy for halting the progression of α-synucleinopathies (George and Brundin 2015). Experiments in rat models of PD demonstrated that active vaccination against recombinant α-syn results in protection against progression of the phenotype (Sanchez-Guajardo et al. 2013). Similar results were found in a mouse model that used a short peptide carrier conjugate created for improved safety (Schneeberger et al. 2012). A passive immunity approach has also generated positive results. In the first study of this kind directed against α-syn in α-syn transgenic mice the treatment resulted in a reduction of calpain1-cleaved α-syn in neurons and a reduction of motor and cognitive impairment compared to control mice (Masliah et al. 2011). However, these treatments are dependent on identifying the correct species of α-syn for intervention and a deeper understanding of the interplay between inflammation, microglia and α-syn degradation. Ultimately, in order to prevent the spread of α-syn we need to understand how all the processes that affect α-syn clearance and propagation interact. Further research into these mechanisms and the underlying factors that influence them are required in order to identify the best targets for intervention and development of therapies for PD.

Abbreviations

α-syn

Alpha Synuclein

ALP

Autophagy/Lysosomal Pathway

CHIP

Co-chaperone carboxyl-terminus of Hsp-70-Interacting Protein

cHsc-70

Cytosolic Hsc70

CMA

Chaperone Mediated Autophagy

CNS

Central Nervous System

CSF

Cerebrospinal Fluid

CTSB

Cathepsin D

CTSD

Cathepsin B

CUPS

Compartment for Unconventional Protein Secretion

DLB

Dementia with Lewy Bodies

ER

Endoplasmic Reticulum

GCase

Glucocerebrosidase

LAMP2a

Lysosome-Associated Membrane Protein type 2A

LB

Lewy Body

LN

Lewy Neurite

MMP

Matrix Metalloproteinase

MSA

Multiple System Atrophy

MVB

Multivesicular Body

PAI-1

Plasminogen Activator Inhibitor

PD

Parkinson's Disease

PLD2

Phospholipase D2

SUMO

Small Ubiquitin-related Modifier

TFEB

Transcription Factor EB

TLR

Toll-like Receptor

tPA

Type Plasminogen Activator

UPS

Ubiquitin Proteasome System